TABLE OF CONTENTS - ULisboarepositorio.ul.pt/bitstream/10451/1147/2/18315_ulsd_re282_MJD_Tese... ·...

1

TABLE OF CONTENTS Abbreviations list 5

Abstract 9

Resumo 11

Figure index 13

1. Introduction 19 1.1 Neurotrophins 19

1.1.1Neurotrophin synthesis 19 1.1.2 Neurotrophin receptors 20 1.1.3 Pathophysiological implications of BDNF actions 28

1.2 Adenosine 31 1.2.1 Adenosine synthesis 31 1.2.2 Adenosine receptors 33 1.2.3 Pathophysiological implications of adenosine actions 37

1.3 Hippocampus

41

2. Aims 45

3. Methods 49 3.1 Biological sample preparations 49

3.1.1 Hippocampal slices 49 3.1.2 Hippocampal homogenates 52 3.1.3 Neuronal cell cultures 52 3.1.4 Cytosolic protein isolation 54

3.2 Drugs and antibodies 55 3.2.1 Drugs 55 3.2.2 Antibodies 56

3.3 Techniques 57 3.3.1 Microelectrophysiological recordings 57 3.3.2 Western blotting 60 3.3.3 Binding assays 61 3.3.4 Apoptosis detection 62

3.4 Statistics 64 4. Results 65 4.1 Interplay between TrkB and adenosine A2A receptors in infant rats 65

4.1.1 Rationale 65

2

4.1.2 Pre-depolarisation induced by high K+ facilitates BDNF excitatory action on hippocampal synaptic transmission through TrkB receptors

65

4.1.3 Theta Burst stimulation paired with BDNF can elicit synaptic potentiation

69

4.1.4 Adenosine A2A receptor activation facilitates BDNF excitatory action on synaptic transmission

69

4.1.5 The excitatory action of BDNF is facilitated by the selective adenosine kinase inhibitor 5-iodotubercidin

73

4.1.6 The activation of the cAMP–PKA transducing system is a critical step in the excitatory action of BDNF

74

4.1.7 Discussion 78 4.2 Influence of age on BDNF modulation of hippocampal synaptic transmission: interplay with adenosine A2A receptors

83

4.2.1 Rationale 83 4.2.2 BDNF facilitates synaptic transmission in young adult rats in a “LTP like” process

83

4.2.3 Adenosine A2A receptors are involved in the excitatory action of BDNF in the hippocampus of young adult rats and aged rats

89

4.2.4 The density of TrkB full length receptors and adenosine A2A receptors is altered in older rats

94

4.2.5 Discussion 97 4.3 Influence of age on the BDNF modulation of long term potentiation: interplay with adenosine A2A receptors

101

4.3.1 Rationale 101 4.3.2 BDNF effect on LTP induced by θ-Burst stimulation (3 Bursts, 100Hz, 3 stimuli, separated by 200 ms).

102

4.3.3 BDNF effect on LTP induced by a weak θ-Burst stimulation (2 Bursts, 100Hz, 3 stimuli, separated by 200 ms)

111

4.3.4 Effects of a BDNF scavenger on LTP in hippocampal slices 113 4.3.5 Discussion 116

4.4 Influence of adenosine A2A receptors on the protective action of BDNF against apoptosis induced by the amyloid beta peptide

120

4.4.1 Rationale 120 4.4.2 Apoptosis induced by amyloid beta in primary cultures of rat neurons is prevented by BDNF and this protection is potentiated by adenosine A2A receptors activation.

121

4.4.3 Amyloid beta induced fragmentation of Pro-caspase-3 is prevented by BDNF in the presence of adenosine A2A receptors activation

126

4.4.4 Amyloid beta induced increase in caspase-3 activity is prevented by BDNF in the presence of adenosine A2A receptors activation

130

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4.4.5 Discussion 132 5. Conclusions 135 6. Future perspectives 137 7. Acknowledgements 141 8. References 145

5

Abbreviations list Aβ – Amyloid beta peptide

AC – Adenylate cyclase

AD – Alzheimer’s disease

ADA – Adenosine deaminase

ADAC – Adenosine amine congenere

ADO – Adenosine

AK – Adenosine kinase

AMP – Adenosine 5’-monophosphate

AP5 – DL-2-amino-5-phosphonopentanoate

ATP – Adenosine 5’-triphosphate

BDNF – Brain-derived neurotrophic factor

Bmax – Maximal number of binding sites

cAMP – cyclic adenosine monophosphate

CGS 21680 – 4-[2-[[6-Amino-9-(N-ethyl-β-D-ribofuranuroanamidosyl)-9H-purin-2-

yl]amino]ethyl]benzenepropanoic acid

CNS – Central nervous system

CR – Cysteine-repeated

CREB – cAMP response element binding protein

DAG – Diacylglycerol

DbcAMP – Dibutyryl cyclic adenosine monophosphate

DG – Dentate Gyrus

DMEM – Dulbecco’s modified Eagle’s medium

DMSO – Dimethylsulphoxide

DPCPX- 1,3-Dipropyl-8- cyclopentylxanthine

DTT– 1,4-Dithiothreitol

6

EC – Entorhinal cortex

EDTA – Ethylenediaminetetraacetic acid

EGTA – Ethylene glycol-bis(2-aminoethyl ether)-N,N –tetraacetic acid

ER – Endoplasmic reticulum

ERK– extracellular signal-regulated protein kinases

FCT – Fundação para a ciência e a tecnologia

fEPSP – Field excitatory post-synaptic potential

GABA – γ-aminobutyric acid

GFAP – Glial fibrillary acidic protein

HBSS – Hank’s balanced salt solution

HD – Huntington’s disease

IP3 – Inositol 1,4,5-triphosphate

ITU – 5-Iodotubercidin

JNK – c-jun N-terminal kinases

KAc – Potassium acetate

Kd – Equilibrium dissociation constant

Ki – Equilibrium dissociation constant of the competitor

LDH – Lactate dehydrogenase

L-DOPA – (l-3,4-dihydroxyphenylalanine)

LRR– Leucine-rich repeats

LTP – Long-term potentiation

MAPK – mitogen-activated protein kinase

NF-KB – Nuclear factor KB

NGF – Nerve growth factor

NMDA– N-Methyl-D-aspartate

3-NP – 3-nitropropionic acid

NT – Neurotrophin

7

PBS – Phosphate buffered saline

PI3K – Phosphatydilinositol-3-kinase

PIP2 – Phosphatidylinositol-4,5-biphosphate

PKA – Protein kinase A

PKC – Protein kinase C

PLC – Phospholipase C

PLD – Phospholipase D

pNA – p-nitroanilide

SAH – S-adenosylhomocysteine

SAPK – Stress-activated protein kinases

SCH 58261 – 7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1 ,2,4-triazolo

[1 ,5-c]pyrimidine

SDS - PAGE –Sodium dodecyl sulphate-polyacrylamide-gel electrophoresis

SEM – Standard error of the mean

TGN – Trans-golgi network

Tris – Tris-hydroxymethyl-aminomethane

Trk – Tropomyosin-related kinase

XAC – 1,3-dipropyl-8-phenylxanthine amine congener

ZM 241385 – 4-(2-[7-Amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-

ylamino]ethyl)phenol

9

Abstract

The brain-derived neurotrophic factor (BDNF) belongs to a group of signalling factors

that are essential for neuronal survival and differentiation, inducing modulation of cell

death events such as apoptosis. This neurotrophin also has synaptic regulatory

actions on basal synaptic transmission, including effects on plasticity such as long-

term potentiation (LTP).

The hippocampus is under neuromodulatory control of adenosine, which

through activation of inhibitory (A1) and excitatory (A2A) receptors fine-tunes the action

of neurotransmitters and neuromodulators. The hippocampus is a brain area where

both A2A and TrkB receptors are expressed. Since activation of A2A receptors can

acutely induce transactivation of BDNF TrkB receptors in cell culture, in the present

work it was evaluated how the activation of adenosine A2A receptors could influence

the actions of BDNF. The first aim of the present study was to investigate the

influence of adenosine A2A receptors on BDNF effects on hippocampal synaptic

transmission and the possible mechanisms involved. Secondly, the influence of age

on BDNF modulation of hippocampal synaptic transmission, as well as the interplay

between adenosine A2A receptors and BDNF TrKB receptors, was examined. As a

third objective, the BDNF actions on LTP during aging and the influence of adenosine

A2A receptors on these actions was also evaluated. Finally, the influence of the A2A

receptor activation on the neuroprotective action of BDNF on apoptosis was

investigated.

The acute excitatory action of BDNF on synaptic transmission in the

hippocampus of infant rats was found to be inducible by a depolarisation that is

dependent on adenosine A2A receptor activation, through a mechanism that requires

cyclic adenosine monophosphate (cAMP) formation and protein kinase A (PKA)

10

activity. Subsequently, for the first time a relationship between age-related changes in

the density of TrkB and A2A receptors as it concerns BDNF-induced enhancement of

synaptic transmission in the hippocampus was elucidated. In LTP studies, it was

observed that BDNF increases the magnitude of θ-Burst stimuli-induced LTP and that

this excitatory action is also dependent on A2A adenosine receptor activation.

Moreover, it was found that BDNF loses the ability to increase LTP in aged animals.

Finally, BDNF protected neurons from apoptosis induced by amyloid beta peptide

(Aβ) 25-35 and this protection was more evident when adenosine A2A receptors are

activated.

In conclusion, the results now presented demonstrate that activation of

adenosine A2A receptors facilitates the synaptic and neuroprotective actions of BDNF.

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Resumo

O factor neurotrófico derivado do cérebro (BDNF, brain-derived neurotrophic factor)

pertence a um grupo de factores de sinalização essenciais para a sobrevivência e

diferenciação neuronal, modulando a morte celular por apoptose. Esta neurotrofina

regula quer a transmissão sináptica quer a plasticidade sináptica como é o caso da

potenciação de longa duração (LTP, long term potentiation).

O hipocampo encontra-se sob controlo da adenosina, que através da

activação dos seus receptores inibitórios (A1) e excitatórios (A2A) regula a acção de

neurotramissores e neuromoduladores. Uma vez que a activação dos receptores A2A

pode induzir transactivação dos receptores TrkB do BDNF em células em cultura, no

presente estudo avaliou-se se a activação dos receptores A2A poderia influenciar a

função do BDNF no hipocampo, área cerebral que expressa quer receptores A2A da

adenosina quer receptores TrkB para o BDNF. Primeiro avaliou-se a influência dos

receptores A2A na acção do BDNF sobre a transmissão sináptica no hipocampo e

possíveis mecanismos envolvidos. Seguidamente, foi avaliada a influência da idade

na modulação da transmissão sináptica induzida pelo BDNF, e a sua relação com os

receptores A2A da adenosina. Posteriormente estudaram-se as acções do BDNF na

LTP em função do envelhecimento e a influência exercida pelos receptors A2A da

adenosina. Finalmente, estudou-se a consequência da activação dos receptores A2A

da adenosina no efeito neuroprotector do BDNF, ou seja sobre a sua influência na

morte celular por apoptose.

Os resultados obtidos mostraram que a acção excitatória do BDNF aplicado

agudamente na transmissão sinaptica em hipocampo de ratos jovens pode ser

induzida por uma despolarização, e que este efeito é dependente da activação dos

receptores A2A da adenosina, através de um mecanismo que requer a formação de

monofosfato ciclico de adenosina (cAMP, cyclic adenosine monophosphate) e a

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activação da cinase de proteínas tipo A (PKA, protein kinase A). Observou-se pela

primeira vez que, ao longo da idade, existe uma relação entre a alteração do efeito

do BDNF na transmissão sináptica e modificações nas densidades dos receptores

A2A da adenosina e TrkB de BDNF no hipocampo. Verificou-se também que o BDNF

aumenta a magnitude da LTP induzida por um estimulo θ-Burst, e que perde esta

acção em animais idosos. Estes efeitos são, igualmente, dependentes da activação

dos receptores A2A da adenosina. Finalmente, demonstrou-se que o BDNF protege

os neurónios da morte celular induzida pelo péptido beta amiloide (Aβ, amyloid beta

peptide) 25-35 e que esta protecção é mais evidente quando os receptores A2A se

encontram activados, deixando de se observar quando estes receptores são

bloqueados.

Em conclusão, os resultados apresentados na presente dissertação

demonstram que a activação dos receptores A2A da adenosina facilita a acção

sináptica e neuroprotectora do BDNF quando administrado agudamente.

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Figure index

1. Background Figure 1.1.1 Structure of the rat BDNF gene and protein 21 Figure 1.1.2 Diagram illustrating the pathways of BDNF synthesis and

secretion

22 Figure 1.1.3 Representation of the domain structures of the Trk and

p75 receptors

24 Figure 1.1.4 TrK-mediated neurotrophin signalling 27 Figure 1.1.5 NGF administration directly to the brain 28 Figure 1.2.1 Adenosine chemical structure 31 Figure 1.2.2 Pathways of adenosine production, metabolism and

transport

32 Figure 1.2.3 Distribution of adenosine receptores (A1, A2A and human

A3) in brain regions.

35 Figure 1.2.4 Adenosine receptor-signalling pathways 36 Figure 1.3.1 Hippocampus in rat and human 43 Figure 1.3.2 Human hippocampus dissected free compared to a

specimen of Hippocampus leria

43 Figure 1.3.3 The hippocampal formation anatomy

44

3. Methods Figure 3.1.1 Hippocampal slices preparation 51 Figure 3.1.2 Resting chamber for hippocampal slices 52 Figure 3.3.1 Setup for extra-cellular microelectrophysiology recordings 57 Figure 3.3.2 Extracellular recordings in hippocampal slices

60

4. Results

Chapter 4.1 Figure 4.1.1 The averaged time course of changes in fEPSP slope

induced by application of BDNF alone 20 ng/ml or 100 ng/ml.

67 Figure 4.1.2 Pre-depolarisation induced by high K+ facilitates BDNF

excitatory action on hippocampal synaptic transmission through TrkB receptors.

68 Figure 4.1.3 Theta-Burst stimulation paired with BDNF can elicit

potentiation effect.

71

14

Figure 4.1.4 Adenosine A2A receptors activation facilitates BDNF excitatory action on synaptic transmission

72

Figure 4.1.5 The excitatory action of BDNF is facilitated by a selective adenosine kinase inhibitor, 5-iodotubercidin

75

Figure 4.1.6 The activation of the cAMP–PKA transducing system is a critical step for the excitatory action of BDNF

76

Figure 4.1.7 Presynaptic cAMP mimics the effect of high-K+, ITU, or CGS 21680

77

Figure 4.1.8 Mechanism of the facilitatory action of adenosine A2A receptor activation on the BDNF effects on hippocampal synaptic transmission in infant rats

80 Chapter 4.2

Figure 4.2.1 The action of BDNF on excitatory synaptic transmission varies in rats from different age groups

85

Figure 4.2.2 Comparison between the averaged effects of BDNF in rats from different age groups

86

Figure 4.2.3 A high concentration (100 ng/ml) of BDNF increases synaptic transmission in slices taken from old adult rats

87

Figure 4.2.4 BDNF facilitates synaptic transmission in young adult rats in a “LTP- like” process.

88

Figure 4.2.5 Involvement of adenosine A2A receptors in the excitatory action of BDNF in synaptic transmission

91

Figure 4.2.6 Involvement of adenosine A2A receptors in the excitatory action of BDNF in synaptic transmission

92

Figure 4.2.7 Pre-depolarisation induced by high K+ facilitates BDNF excitatory action on hippocampal synaptic transmission in slices taken from old adult (36-38 week-old) rats

93 Figure 4.2.8 Changes in the density of TrkB full length receptors in

different age groups

95 Figure 4.2.9 Specific binding of adenosine A2A receptors in the

hippocampus of rats from different age groups

96 Chapter 4.3

Figure 4.3.1 Effect of the BDNF (20 ng/ml) on θ-Burst (3x3) induced LTP in slices taken from 4 week-old rats

103

Figure 4.3.2 Effect of the BDNF (20 ng/ml) on θ-Burst (3x3) induced LTP in slices taken from 4 week-old rats is dependent on adenosine A2A receptors

104 Figure 4.3.3 Effect of the BDNF (20 ng/ml) on θ-Burst (3x3) induced

LTP in slices taken from 10-16 week-old rats

105 Figure 4.3.4 Effect of the BDNF (20 ng/ml) on θ-Burst (3x3) induced

LTP in slices taken from 10-16 week-old rats is dependent

15

LTP in slices taken from 10-16 week-old rats is dependent on adenosine A2A receptors

106

Figure 4.3.5

Absence of effect of the BDNF (20 ng/ml) on θ-Burst (3x3) induced LTP in slices taken from 36-38 week-old rats

108

Figure 4.3.6 Absence of effect of the BDNF (20 ng/ml) on θ-Burst (3x3) induced LTP in slices taken from 70-80 week-old rats

109

Figure 4.3.7 Age-related modulation of long-term potentiation (LTP) by BDNF

110

Figure 4.3.8 Absence of effect of the BDNF (20 ng/ml) on θ-Burst (2x3) induced LTP in slices taken from 36-38 week-old rats and 70-80 week-old rats

112 Figure 4.3.9 Effect of Trk-Fc (2 µg/ml) on θ-Burst (3x3) induced LTP in

slices taken from 38-38 week-old rats

114 Figure 4.3.10 Effect of the K252a (200 nM) on θ-Burst (3x3) induced

LTP in slices taken from 38-38 week-old rats

115 Chapter 4.4

Figure 4.4.1 Morphologic analysis of cells 123 Figure 4.4.2 Amyloid-beta (Aβ)-induced apoptosis in primary rat

neurons

123 Figure 4.4.3 Apoptosis induced by Amyloid-beta (Aβ) in primary rat

neurons is prevented by BDNF and CGS 21680

124 Figure 4.4.4 SCH 58261 reverts the neurprotection induced by BDNF 125 Figure 4.4.5 Processing of caspase-3 in primary rat neurons in the

absence of Amylod-beta

127 Figure 4.4.6 Amyloid-beta induced processing of caspase-3 in primary

rat neurons

128 Figure 4.4.7 Amyloid-beta induced processing of caspase-3 in primary

rat neurons, which is reduced by BDNF in the presence of adenosine A2A receptors activation

129 Figure 4.4.8 Activity of caspase-3 in primary rat neurons 130 Figure 4.4.9 Amyloid-beta induced enhancement of caspase-3 activity

in primary rat neurons is reduced by by BDNF in the presence of adenosine A2A receptors activation

131

5. Conclusions Figure 5.1 Cross talk between signalling pathways of adenosine A2A

receptors and TrkB receptors

139

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The scientific content of the present thesis has been included in the publication of the

following original articles:

I. Diógenes M.J., Fernandes C.C., Sebastião A.M., Ribeiro J.A. (2004)

Activation of adenosine A2A receptor facilitates BDNF modultion of synaptic

transmission in hippocampal slices. J. Neurosci. 24, 2905-2913.

II. Diógenes M.J., Assaife-Lopes N., Pinto-Duarte A., Sebastião A.M., Ribeiro

J.A. (2007) Influence of age on BDNF modulation of hippocampal synaptic

transmission: interplay with adenosine A2A receptors. Hippocampus. 17, 577-

85.

The following chapters of this thesis are in preparation to be submitted as

manuscripts to international journals:

Chapter 4.3 “Influence of age on the BDNF modulation of long-term potentiation:

interplay with adenosine A2A receptors.”

Chapter 4.4 “Influence of the activation of adenosine A2A receptors on the

protective BDNF action against apoptosis induced by amyloid beta peptide.”

Other papers where the author of this thesis participate during her doctoral studies:

Pousinha P.A., Diógenes M.J., Ribeiro J.A., Sebastião A.M. (2006) Triggering of

BDNF facilitatory action on neuromuscular transmission by adenosine A2A

receptors. Neuroscience Letters 404, 143–147

Fontinha B.M., Diógenes M.J., Ribeiro J.A., Sebastião A.M. (2007) Enhancement

of long-term potentiation by BDNF requires adenosine A2A receptor activation by

endogenous adenosine. Neuropharmacology 54,924-933.

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1. INTRODUCTION 1.1 NEUROTROPHINS

In 1953, Rita Levi-Montalcini and Victor Hamburger discovered that a mouse sarcoma

tumor implanted close to the spinal cord of developing chicks in ovo secreted a

soluble factor that induced the hypertrophy and fiber outgrowth of sympathetic

neurons. Later, this factor was isolated and named as nerve growth factor (NGF)

(Levi-Montalcini and Cohen, 1956). The discovery of this molecule opened a new field

in neurobiology research. After NGF identification, Barde and collaborators (1982)

isolated a different neurotrophin (NT), from a pig brain, designated as brain-derived

neurotrophic factor (BDNF). Since then other members of the family of NTs have

been identified in mammals namely, NT-3 and NT-4, and in fish NT-6 e NT-7 (Gotz et

al., 1994; Lai et al., 1998).

1.1.1 Neurotrophin synthesis All NTs are generated first as precursor proteins, pre-pro-neurotrophins

(approximately 240-260 amino acids long, Figure 1.1.1), which then are cleaved

intracellularly to mature proteins of 118-120 amino acids. The pre-mRNA sequence

directs the synthesis of the nascent protein in the endoplasmic reticulum (ER)

attached ribosomes, leading to the sequestration of the newly formed polypeptide

chain into the ER (see Lessmann et al., 2003). The signal peptide is cleaved off

immediately after sequestration in the ER. The resulting pro-neurotrophins can

20

spontaneously form non-covalently-linked homodimers in the ER. The pro-

neurotrophins in the ER then transit the Golgi apparatus most likely via intermediate

non-clathrin-coated transport vesicles and finally accumulate in the membrane stacks

of the trans-golgi network (TGN). There are three fates for intracellular pro-

neurotrophins: 1) intracellular cleavage followed by secretion, 2) secretion followed by

extracellular cleavage, or 3) secretion without subsequent cleavage. All of these

products serve as signalling molecules (see Lee et al., 2001).

Two different types of secretory vesicles may be generated (Figure 1.1.2),

filled with one or a combination of different neuropeptides. Thus, BDNF release can

occur through two different pathways: 1) a regulated pathway, where neurotrophins

are secreted in response to a stimuli or 2) a constitutive pathway, where these

molecules are spontaneously secreted. The secretory granules of the constitutive

pathway are smaller (50-100 nm in diameter) and independent of intracellular Ca2+

concentration elevation. Vesicles can fuse with the plasma membrane to release their

content in the absence of any specific triggering mechanisms. In the regulated

pathway, vesicles are large (100-300 nm in diameter) and their release depends on

intracellular Ca2+ concentration elevation. Secretion of neurotrophins can be regulated

by neuronal activity, potassium, and glutamate; this release is also dependent on the

stimulus-frequency (Lim et al., 2003). Interestingly, constitutive and regulated

secretion of BDNF can coexist in the same cell (see Lessman et al., 2003)

1.1.2 Neurotrophin receptors

Neurotrophins function by activating two distinct classes of transmembrane receptors:

the p75 neurotrophin receptor (p75NTR) and the Trk family of receptor tyrosine

kinases that includes TrkA, TrkB and TrkC. Unlike the non-selective p75NTR, which

21

has a similar affinity for all neurotrophins, each Trk receptor selectively binds a

different neurotrophin.

Figure 1.1.1- Structure of the rat BDNF gene and protein. (A) Each of the four 5’-exons (I–IV) includes its own promotor, and is combined with the 3’exon (V) to yield an mRNA (B) coding for pre-pro-BDNF. The short white stretch in exon V represents an alternative splice site, giving rise to two differentially spliced mRNA variants for each of the four transcripts. Each of these mRNAs is expressed in a tissue-specific and developmentally regulated manner. The gray portion in exon V codes for the pre-pro-BDNF protein. (C) Primary structure and sequence of the BDNF protein. The pre-sequence (18 aa, black) is cleaved off immediately after sequestration of the nascent protein into the ER. The mature BDNF protein (black) is excised from the pro-BDNF precursor by virtue of specific protein convertases residing either in the TGN or in immature secretory granules. (Adapted from Lessmann et al., 2003.)

A

B

C

22

Figure 1.1.2- Diagram illustrating the pathways of BDNF synthesis and secretion. ER represents the endoplasmic reticulum and TGN is the trans-golgi network. (Adapted from Lessmann et al., 2003.)

1.1.2.1 p75 receptor

p75NTR is a member of the tumor necrosis receptor family with an extracellular

domain that includes four cysteine-rich motifs, a single transmembrane domain and a

cytoplasmic domain, that includes a ‘death’ domain as represented in Figure 1.1.3

(Liepinsh et al., 1997; He and Garcia 2004).

All neurotrophins, including the pro-neurotrophins, can bind to p75NTR

(Figure1.1.3) (see Lu et al., 2005). This receptor can interact with several proteins

and forms multimeric receptor complexes that transmit important signals for regulating

neuronal survival and differentiation as well as synaptic plasticity (see e.g. Reichardt,

2006). P75NTR also regulates the responsiveness of Trk receptors to neurotrophins.

The presence of p75NTR enhances the specificity of TrkA and TrkB for their primary

ligands, NGF and BDNF, respectively (see Reichardt, 2006).

23

1.1.2.2 Trk receptors

The name TrK, from tropomyosin-related kinase, derives from the oncogene that

resulted in its discovery (Barbacid, 1991). The oncogene was discovered in a

carcinoma and it was found to consist in the first seven of eight exons of nonmuscle

tropomyosin fused to the transmembrane and cytoplasmic domains of a novel

tyrosine kinase. Consequently, the protooncogene was named tropomyosin-related

kinase (Trk), commonly referred as TrkA. The TrkB and TrkC genes were identified

because of their homology with TrkA. Seventeen years ago, it was demonstrated that

NGF binds to TrkA and that it activates the tyrosine kinase activity of the receptor

(Kaplan et al., 1991; Klein et al., 1990). Subsequently, TrkB was identified as the

receptor for BDNF and NT-4, and TrkC as the receptor for NT-3 (see for review

Huang and Reichardt, 2001; Bibel and Barde, 2000). Although NT-4 and BDNF bind

to the same receptor, several observations indicate that the biological effects

mediated by these neurotrophins are not identical, which may mean that neurons can

discriminate between these two molecules (see e.g. Lewin and Barde, 1996). The

extracellular domain of each Trk receptor consists of a cysteine-rich cluster, followed

by three leucine-rich repeats, another cysteine-rich cluster and two immunoglobulin-

like domains. Each receptor terminates with a cytoplasmic domain consisting of a

tyrosine kinase domain surrounded by several tyrosines that serve as

phosphorylation-dependent docking sites for cytoplasmic adaptors and enzymes.

The major site at which neurotrophins interact with these receptors is in the

membrane-proximal immunoglobulin-like domain (Figure 1.1.3).

Splicing generates isoforms of TrkB and TrkC that include comparatively

short cytoplasmic motifs without a tyrosine kinase domain; these receptors are

designated as truncated receptors. Different TrkB isoforms can be generated: T1, T2

and T-Shc. Expression of these non-kinase-containing isoforms has been shown to

inhibit productive dimerization of kinase-containing Trk receptors, thereby inhibiting

24

responses to neurotrophins (Eide et al. 1996). Moreover, recent data indicates that

some truncated isoforms help to regulate the expression of full-length TrkB receptors

(Haapasalo et al., 2002).

Figure 1.1.3- Representation of the domain structures of the Trk and p75 receptors. Extracellular domains of cysteine rich clusters (C), leucine-rich repeats (LRR) and immunoglobin-like domains (Ig) are illustrated. Trk and p75 have one transmembrane domain and one cytoplasmic tyrosine kinase domain. In both TrkA and TrkB, the Ig2 is the major ligand-binding interface. The p75 receptor is depicted on the far left. The extracellular domain of this receptor has four cysteine-rich motifs (CR) in which CR2 and CR3 are involved in the neurotrophin-binding interaction. (Adapted from Reichardt, 2006.)

25

1.1.2.3 Trk-mediated signalling pathways

The Trk receptors are activated specifically by the mature and not by the pro-forms of

the neurotrophin gene products (Lee et al., 2001). Binding of mature neurotrophins to

their specific Trk receptors induces dimerization and autophosphorylation at specific

tyrosine residues within intracellular domains. The generation of phosphotyrosine

residues in turn catalyses the formation of large signalling complexes. (Segal and

Greenberg, 1996). Phosphorylation at Y515 (in the case of TrkB receptors) creates

the binding site for Shc (Src homology 2/α-collagen-related protein), whereas

phosphorylation at Y816 (in case of TrkB receptors) forms the adaptor site for

Phospholipase C (PLC)γ as represented in Figure 1.1.4. The major signalling

pathways activated by the Trk receptors are; (1) phosphatydilinositol-3 kinase (PI3K);

(2) Ras-MAPK pathway and (3) PLCγ pathway, and their downstream effectors.

These include PI3K stimulation of Akt kinase, Ras stimulation of mitogen-activated

protein MAPK cascades and PLC-γ dependent generation of inositol 1,4,5-

triphosphate (IP3) and diacylglycerol (DAG), which results in mobilization of Ca2+

stores and activation of Ca2+ and DAG-regulated protein kinase (Segal and

Greenberg, 1996). These intracellular signalling pathways modulate gene expression

in a cell type-specific manner and are responsible for most of the effects of

neurotrophins related to neuronal growth, survival and differentiation, along with

synaptic transmission and plasticity.

Neurotrophins and activated Trk receptors are transported together in

endocytotic vesicles (see e.g. Lessman et al., 2003). Internalization and transport of

Trk receptors serve two functions: 1) to bring activated Trk receptors into proximity of

cell compartments, such a the nucleus, where signalling is required to enable specific

cellular responses, such as gene transcription and 2) to transport activated Trk

receptors to membrane compartments, where signalling effectors are concentrated

(see Reichardt, 2006). The classic retrograde route is destined to exert the long-

26

lasting effects of neurotrophins, which are, for example, mediated by new gene

transcription; but, there is also an additional anterograde targeting that occurs in the

axon towards synaptic terminals which is involved in the fast actions of neurotrophins

(see e.g. Lessmann et al., 2003). Mechanisms underlying the fast action of

neurotrophins include intracellular Ca2+ signalling, neuronal excitation, augmentation

of synaptic excitation by modulation of N-methyl-D-aspartate (NMDA) receptor activity

and control of synaptic inhibition through the regulation of the K+/Cl- cotransporter

KCC2. The fastest action of brain-derived neurotrophic factor and neurotrophin-4/5

occurs within milliseconds, and involves activation of TrkB and the opening of the Na+

channel Nav1.9. Through these rapid actions, neurotrophins orchestrate neuronal

activity, modulate synaptic transmission and produce instructive signals for the

induction of long-term changes in the efficacy of synaptic transmission (see

Kovalchuk et al., 2004).

27

Figure 1.1.4- TrK-mediated neurotrophin signalling. The activated signalling pathways mediate effects of neurotrophins on neuronal survival, differentiation, and gene expression as well as acute effects on synaptic transmission and plasticity, as indicated. Abbreviations represent the following: P, phosphorylation; Y, tyrosine residue; PI3K, phosphatidylinositol 3-kinase; AKT, AKT kinase; Ras, small GTP-binding protein Ras; PIP2, phosphatidylinositol 4,5-bisphosphate; DAG, diacylglycerol; IP3, inositol 1,4,5-trisphosphate. (Adapted from Blom and Konnerth, 2005).

P

P P

P

P P P

P

P

P

Shc PI3K AKT

MAPK Ras

1-PI3K pathway

2- Ras pathway

PLCγγγγ

3- PLC pathway

PIP2

DAG

IP3

Survival

Growth Differentiation

Synaptic transmission Plasticity

Ca2+

PKC

BDNF

Cytosol

Nucleus

Gene expression

+

Y515-Shc site

Y816-PLC site

28

1.1.3 Pathophysiological implications of BDNF actions

The first suggestion to use a neurotrophin for therapeutic purposes appeared in the

1980s, when NGF was the only known member of the NT family. This proposal was

based on the knowledge that in the CNS, NGF promotes the survival and function of

cholinergic neurons in the basal forebrain (Fisher et al., 1987). These neurons project

to the hippocampus and are believed to be important in the memory process, which is

specifically affected in patients with Alzheimer’s disease. Thus, in 1993, a group of

investigators from the Institute of Karolinska developed a way to administer NGF

directly to the brain of Alzheimer’s patients (Seiger et al., 1993). This method

consisted in a canule placed in the lateral ventricles connected to an infusion pump,

enabling administration of NGF to patients (Figure 1.1.5). However this route of

administration is troublesome since it requires neurosurgery.

Figure 1.1.5- NGF administration directly to the brain. NGF was delivered through a canule implanted in the lateral ventricle connected to an infunsion pump. (Adapted from Sieger et al., 1993.)

29

The discovery of other members of the neurotrophin family, in particular the

discovery of BDNF and the knowledge of its physiological functions, contributed to the

large number of studies correlating the pathogenesis of human neurodegenerative

disorders with alteration in BDNF actions. These changes can be a result of genetic

polymorphisms in the BDNF gene or in the genes for BDNF receptors (TrKB full

length, truncated, and P75 receptors), alterations in the levels of mRNA for BDNF,

modifications in levels of BDNF protein and changes in BDNF receptor densities.

Regarding modifications in the BDNF gene, mRNA for BDNF and protein

levels, a polymorphism in BDNF gene leading to a different BDNF protein (BDNFmet)

appears to alter susceptibility to neuropsychiatric disorders, such us Alzheimer’s

disease (AD) (Ventriglia et al., 2002), Parkinson’s disease (Momose et al., 2002),

depression (Sen, 2003), and bipolar disorders (Sklar et al., 2002; Neves-Pereira et

al., 2002). In 1991, Phillips and colleagues reported a selective reduction of BDNF

mRNA expression in the hippocampus of individuals with Alzheimer’s desease, which

was later, confirmed by others (Murray et al., 1994). Moreover, a decrease in BDNF

protein levels in the hippocampus has been associated with depression (Russo-

Neustadt et al., 2001; Chen et al., 2001a). Furthermore, a decrease in BDNF protein

levels has been reported in nigrostriatal dopamine regions in the brains of Parkinson’s

patients (Mogi et al., 1999, Parai et al., 1999). A decrease in the expression of the

BDNF protein in the hippocampus of people with dementia exhibiting lewy bodies

(Immamura et al., 2005) in patients with diabetic brain neuropathies (Nitta et al.,

2002) and in the striatum of individuals with Huntington’s disease (see Zuccato and

Cattaneo, 2007) was also reported. There is also evidence that brain BDNF content is

diminished in the cortex and the hippocampus of AD patients (Connor et al., 1997,

Ferrer et al., 1999).

Concerning modifications in TrkB receptors, a study demonstrated that a

mutation in the kinase domain of TrkB, that leads to impaired intracellular signalling is

30

associated with obesity and developmental delay (Yeo et al., 2004). On the other

hand, the expression of different TrkB splice variants is associated with cognitive

capacity, while over-expression of the kinase-containing form enhances memory and

learning in transgenic mice (Koponen et al., 2004); conversely, over-expression of the

TrkBT1 isoform in adult neurons impairs long-term memory (Saarelainen et al., 2000).

Interestingly, increased content of the TrkBT1 isoform and decreased TrkB

expression has been observed in AD patients (Ferrer et al., 1999).

Finally, genetic modification in the gene encoding P75NTR can be involved in

pathology. Polymorphisms in this gene can lead to an increased susceptibility to

depressive disorders (Kunugi et al., 2004).

The regulatory actions of neurotrophic factors in neuronal functions and in

response to neuronal injury, together with the evidence that changes in BDNF

signalling are associated with a wide variety of pathologies, led to the working

hypothesis that the delivery of BDNF to the brain could ameliorate those diseases. A

number of clinical trials involving the use of BDNF for neurodegenerative disorders

such as amyotrophic lateral sclerosis and diabetic neuropathy has been carried out in

the past decade. However, BDNF had minimal beneficial effects and produced side

effects such as pain and gastrointestinal symptoms (Thoenen and Sendtner, 2002).

The lack of promising results can be attributed to a short half-life for BDNF, a non-

controlled delivery of the neurotrophin, lack of controlled levels of BDNF at the site of

action and the presence of endogenous compensative processes that may regulate

BDNF levels. Therefore further basic studies are needed in order to better understand

BDNF actions in neurons, which may help in a better design of future clinical trials.

31

1.2 ADENOSINE

Adenosine (Figure 1.2.1) is a nucleoside formed when N9 of adenine (a purine) is

covalently bound to the C1’ position of ribose.

Adenosine is an ubiquitous homeostatic substance present in all cells, and it

is released from apparently all cells, including neurons and glia (see Ribeiro et al.,

2003). Adenosine plays a major role in the cardiovascular system, in the central

nervous system, in the gastrointestinal tract, in the immune system, in mast cell

degranulation, and asthma, as well as cell growth, proliferation and apoptosis (see

Schultle and Fredholm, 2003).

Figure 1.2.1 – Adenosine chemical structure. Adenosine contains adenine (in the blue square) and ribose (in the red square) attached by a C1’-N9 glycosideic bond.

1.2.1 Adenosine synthesis

Adenosine can be formed by the action of the enzyme endo-5’-nucleotidase on

adenosine monophosphate (AMP) (Phillis and Newsholme, 1979) and by the

hydrolysis of S-adenosylhomocysteine (SAH) (Nagata et al., 1984) catalysed by SAH

hydrolase. The intracellular concentration of adenosine at equilibrium is about 100 nM

(Meghji, 1992). This level is controlled by adenosine kinase (AK), which

32

phosphorylates adenosine to produce AMP, and by adenosine deaminase (ADA),

which catalyzes the formation of inosine (Figure 1.2.2). Adenosine is neither stored

nor released as a classical neurotransmitter since it does not accumulate in synaptic

vesicles, but is instead released from the cytoplasm into extracellular space through a

nucleoside transporter (see e.g. Ribeiro et al., 2003). Adenosine is released from the

presynaptic component, from the activated postsynaptic component or from non-

synaptic regions of neurons (see Cunha, 1997). The extracellular adenosine

concentration in all body fluids is rather constant under basal conditions (30-300 nM)

(see Schultle and Fredholm, 2003).

Figure 1.2.2- Pathways of adenosine production, metabolism and transport Abbreviations are as follows: ADA, adenosine deaminase; AK, adenosine kinase; es, equilibrative-sensitive nucleoside transporters; ei, equilibrative-insensitive nucleoside transporters; SAH, S-adenosyl homocysteine. (Adapted from Latini and Pedata, 2001.)

Equilibrative

nucleoside transporter ei es

ADENOSINE

Adenosine receptors activation

33

1.2.2 Adenosine receptors

Adenosine receptors have been intensively studied, and to the present date, four

different receptors have been cloned and designated as A1, A2A, A2B and A3 receptors.

These receptors are classified as seven transmembrane domain G-protein-coupled

receptors. The original receptor classification was based on the effect on adenosine

binding to the receptor affecting the cyclic AMP (cAMP) levels in different tissues, with

A1 decreasing and A2 increasing cAMP levels (van Calker et al., 1979; Londos et al.,

1980).

The adenosine A1 receptors are highly expressed in brain cortex, cerebellum,

hippocampus, and the dorsal horn of spinal cord (Figure 1.2.3) (see e.g. Fredholm et

al., 2001). These receptors are present at pre-, post- and non-synaptic sites. When

they are located pre-synaptically, they inhibit neurotransmitter release (Dunwiddie and

Hass, 1985) whereas post-synaptically and in neuronal cell bodies they inhibit calcium

influx through voltage-sensitive calcium channels, inhibit NMDA receptor and inhibit

potassium currents, leading to membrane hyperpolarization (reviewed in Fredholm et

al., 2005). A1 receptors are coupled to inhibitory G-proteins (Gi/Go) that lead to an

inhibition of the activity of the enzyme adenylate cyclase (Figure 1.2.4) (see Linden,

2001).

The A2A receptors are highly expressed postsynaptically in the striato-pallidal

GABAergic neurons and olfactory bulb (see e.g. Fredholm et al., 2001); they are also

expressed in hippocampus and cortex (Figure 1.2.3) (see e.g. Ribeiro et al., 2003),

where they have a predominant presynaptic localization (Rebola et al., 2005b).

Adenosine A2A receptors are mostly coupled to stimulatory G-proteins (Gs), which

consequently increase intracellular cAMP (Figure 1.2.4). In striatum, they are also

coupled to Golf (Corvol et al., 2001); and, in the hippocampus, there is evidence that

this receptor can be coupled to Gi/Go (Cunha et al., 1999), in addition to its usual

34

coupling to Gs proteins. Moreover, adenosine A2A receptors overexpressed in COS-7

cells were shown to couple to G15/16, but there is no evidence that this interaction

occurs in vivo (Offermanns and Simon, 1995). Investigating endogenously expressed

adenosine A2A receptors in human endothelial cells, Sexl and collaborators cautiously

proposed signalling via G12/13 proteins without providing direct experimental evidence

for this hypothesis (Sexl et al., 1997). Activation of the adenosine A2A receptors also

induces formation of inositol phosphates in COS-7 cells via pertuxis toxin-insensitive

Gα15 and Gα16 proteins (see Jacobson and Gao, 2006).

The A2B receptors display low levels of expression in the brain (Dixon et al.,

1996), however their expression is noted in astrocytes (van Calker et al., 1979). A2B

receptors are coupled to Gs proteins (Figure 1.2.4).

The A3 receptor is found frequently in peripheral tissues, in particular in mast

cells and testis (Schubert et al., 1994); and, it has apparent intermediate levels of

expression in the human cerebellum and hippocampus (Figure 1.2.3) and low levels

in the rest of the brain (see Fredholm et al., 2001). The affinity of this receptor for

adenosine (Ki=1000 nM) is considerably lower than the adenosine affinity of A1

receptors (Ki=10 nM) and A2A (Ki=30 nM). This receptor is usually coupled to

inhibitory G-proteins (Gi and Go) (Figure 1.2.4)(see Linden, 2001).

35

Figure 1.2.3 Distribution of adenosine receptores (A1, A2A and human A3) in brain regions. High levels of expression are indicated by larger font. (From Ribeiro et al., 2003.)

36

Figure 1.2.4- Adenosine receptor-signalling pathways. Activation of the A1 and A3 adenosine receptors inhibits adenylyl cyclase activity through activation of pertussis toxin-sensitive Gi proteins resulting in increased activity of phospholipase C (PLC). Activation of the A2A and A2B adenosine receptors increases adenylyl cyclase activity through activation of Gs proteins. Activation of the A2A receptor induces formation of inositol phosphates. A2B receptor-induced activation of PLC occurs via Gq proteins. All four subtypes of adenosine receptors can couple to mitogen-activated protein kinase (MAPK), giving them a role in cell growth, survival, death and differentiation. Abbreviations represent the folowing: CREB, cAMP response element binding protein; DAG, diacylglycerol; IP3, inositol 1,4,5-trisphosphate; PI3K, phosphatidylinositol 3-kinase; PIP2, phosphatidylinositol-4,5-bisphosphate; PK, protein kinase; PLD, phospholipase D; NF-kB, nuclear factor-kB. (Adapted from Jacobson and Gao, 2006.)

A1

A3

A2A

A2B

37

1.2.3 Pathophysiological implications of adenosine actions

Adenosine has the ability to modulate the release of neurotransmitters and

neuromodulators. The adenosine receptors have a crucial function in the regulation of

the activity of other receptors that affect several biological functions such as

differentiation, synaptic transmission, plasticity and apoptosis. Thus, adenosine

receptors regulate the function of neuropeptide receptors, nicotinic autofacilitatory

receptors, metabotropic glutamate receptors, NMDA receptors and neurotrophic

factor receptors (see e.g Ribeiro et al., 2003).

Adenosine is apparently involved in many functions that can play a role in the

pathology of the nervous system. Therefore, the modifications of extracellular

adenosine levels or the pharmacological or molecular manipulation of adenosine

receptors will interfere with the action of other important molecules that regulate brain

functions. This may prove relevant in the treatment of several diseases, where

activation or inhibition of adenosine receptors through modulation of certain pathways

may change the fate of the diseases.

The pharmacological manipulation of adenosine has been suggested for the

treatment of several health conditions (see Ribeiro et al., 2003). Adenosine functions

as a natural sleep-promoting agent mostly through activation of A1 receptors

(Benington et al., 1995; Porkka-Heiskanen et al., 1997). It was suggested that

adenosine participates in resetting of the circadian clock by manipulation of

behavioral stats (Antle et al., 2001). Thus, it emerges that there exists a potential role

for adenosine-related compounds and of A1 receptor agonists as sleep promoters and

adenosine receptor antagonists as arousal stimulators. In addition, adenosine A1

receptor agonists have anxiolytic activity suggesting that drugs that facilitate

adenosine A1 receptor-mediated actions may be effective for the treatment of anxiety

(Jain et al., 1995; Florio et al., 1998). Adenosine A1 receptor antagonists have also

38

been proposed for the treatment of memory disorders (see Ribeiro et al., 2003).

Caffeine, which is an antagonist of adenosine receptors, has cognitive effects mostly

due to its ability to antagonize adenosine A1 receptors in the hippocampus and cortex,

the brain areas actively involved in cognition. Positive actions of caffeine on

information processing and performance might also be attributed to improvement of

behavioral routines, arousal enhancement and sensorimotor gating (Fredholm et al.,

1999). This is futher supported by the observation that theophylline, an adenosine

receptor antagonist, enhances spatial memory performance only during the light

period, which is the time of sleepiness in rats (Hauber and Bareiß, 2001). However,

some cognitive actions of caffeine were also described in relation to A2A receptors

antagonism (Chen et al., 2007).

In vivo and in vitro studies have demonstrated the neuroprotective role of

adenosine A1 receptor activation. Evaluating the action of hypoxia on synaptic

transmission in hippocampal slices, it has been shown that substances that are

released during hypoxia, such as GABA, acetylcholine, and even glutamate through

metabotropic receptors, may have a neuroprotective role; however, their action is

evident only when activation of adenosine A1 receptors is impaired, leading to the

proposal that adenosine A1 receptors play a pivotal role in response to hypoxia

(Sebastião et al., 2001).

The pharmacological manipulation of adenosine receptors has also been

evaluated in the context of Alzheimer’s disease treatment. Caffeine intake has been

associated with a significantly lower risk for AD (Maia and de Mendonça, 2002). In

recent studies with a model of AD in mice, chronic caffeine protected against

cognitive impairment and resulted in reduced brain levels of amyloid beta protein

(Arendash et al., 2006). The most widely used drugs to treat AD patients increase the

availability of acetylcholine in central cholinergic pathways by inhibiting the enzyme

acetylcholinesterase (Doody et al., 2001). Another strategy to promote cholinergic

39

transmission might be to activate adenosine A2A receptors, which facilitate

acetylcholine release, or to block adenosine A1 receptors, which inhibit acetylcholine

release (see Sebastião and Ribeiro, 1996). Thus, either adenosine A2A receptor

agonists or adenosine A1 receptor antagonists (or the combination of both) might be

useful as cognitive enhancers. However, the adenosine A2A receptor antagonist has

also been studied to improve cognition of AD patients (see Chen et al., 2007).

Furthermore, the pharmacologic manipulation of A2B-adenosine receptor have been

proposed as possible therapeutic agents for Alzheimer’s disease patients (Rosi et al.,

2003). AD is associated with glial activation and increased levels of pro-inflammatory

cytokines. Epidemiological results suggest that anti-inflammatory therapies can slow

the onset of AD (see Rojo et al., 2008). Adenosine, acting at A2A receptors, is an

effective endogenous anti-inflammatory agent that can modulate inflammation both in

the periphery and the brain (Mayne et al., 2001).

Moreover, antagonists of adenosine A2A receptors emerged as potential anti-

parkinsonian agents, based, in part, on the CNS distribution of the A2A receptors. A

key finding in this process is the co-localization and reciprocal antagonistic

interactions between A2A and D2 receptors in the striatum, initially observed in rats

(Ferré et al., 1999), but also described in humans (Díaz-Cabiale et al., 2001). A2A

receptor antagonism not only diminishes Parkinsonian-like muscle rigidity in rats but

also potentiates the effect of L-DOPA (Wardas et al., 2001), which may allow the use

of lower doses of L-DOPA and hence minimize or retard side effects and

tachyphylaxis caused by L-DOPA treatment in Parkinson’s disease.

In Huntington’s disease (HD) both adenosine A1 receptor agonists and

adenosine A2A receptor antagonists appear to exert neuroprotective actions. The

adenosine amine congener (ADAC), an adenosine A1 receptor agonist, attenuates the

striatal lesion, as well as the dystonia, induced in a rat model of HD by the

administration of the mitochondrial toxin 3-nitropropionic acid (3-NP) (Blum et al.,

40

2002). Since A2A receptors are mainly localized on the neurons, which degenerate

early in HD, and given their ability to stimulate glutamate outflow and inflammatory

gliosis, it was hypothesized that A2A receptors could be involved in the pathogenesis

of HD, thus A2A receptors antagonists could be neuroprotective.

In addition other pathologies such as schizophrenia, epilepsy, drug addiction,

pain, and control of ventilation can also be improved through the use of adenosine

receptor agonists and antagonists (see Ribeiro et al., 2003).

41

1.3 HIPPOCAMPUS

Buried deep within the medial temporal lobe of the human brain lies a group of many

million of neurons organized into a network quite different from that found anywhere

else in the nervous system. The hippocampal formation is a brain area consisting of

the dentate gyrus, hippocampus, subiculum, presubiculum, parasubiculum, and

entorhinal cortex (Andersen et al., 2007).

The hippocampus has played a central role in brain investigations. There are

several reasons for the interest in this specific brain area. It has a relatively simple

organization of principal cell layers coupled with the highly-organized laminar

distribution of its inputs. There is also increasing information concerning the

involvement of the hippocampus in several physiological processes, such as, memory

and in pathological processes, such as, epilepsy or AD. In addition, the hippocampus

is a brain area extremely susceptible to aging (see e.g., Smith, 1996), which can

contribute to several age related changes observed in older subjects. Moreover, the

basic layout of cells and fiber pathways is much the same in all mammals (Figure

1.3.1), although the volume of the hipocampus is about 10 times larger in monkeys

than in rats and 100 times larger in humans than in rats. Since the basic hippocampal

architecture is common in mammalian species (Andersen et al., 2007), the study of

hippocampal functions in rat models allows correlation to human hippocampal

processes.

The anatomist Giulio Cesare Aranzi was the first to coin the name

“hippocampus”, undoubtedly because of its similarity to the tropical fish Hippocampus

leria (Figure 1.3.2) (Andersen et al., 2007).

After the advent of microscopy, the hipocampus was deemed even more

impressive, with its characteristic, neatly regimented cellular arrangement. The

entorhinal cortex can be considered the first step in the intrinsic hippocampal circuit

42

because much of the neocortical input reaching the hippocampal formation does so

through the entorhinal cortex. Cells in the superficial layers of entorhinal cortex give

rise to axons that project, among other destinations, to the dentate gyrus and CA3

area. These projections from the entorhinal cortex to the dentate gyrus and CA3

form part of the major hippocampal input pathway called the perforant path. These

pathways are not reciprocal since dentate gyrus and CA3 do not project back to

entorhinal cortex. Neurons of entorhinal cortex also project to the CA1 field and to

the subiculum via the perforant and alvear pathways. Both the CA1 and the

subiculum project back to the deep layers of the entorhinal cortex (see Amaral and

Lavenex, 2007 and Figure 1.3.3).

The granule cells, which are the principal cells of the dentate gyrus, give rise

to axons called mossy fibers that connect with pyramidal cells of the CA3 field of the

hippocampus. However, CA3 cells do not project back to the granule cells. The

pyramidal cells of the CA3 field are in turn the source of major input to the CA1

hippocampal field (the Schaffer collateral axons). Again, CA1 neurons do not project

back to the CA3 field. The CA1 field projects to the subiculum, providing its major

excitatory input. Subiculum does not project back to CA1 field of the hippocampus

(see Amaral and Lavenex, 2007 and Figure 1.3.3).

43

Figure 1.3.1- Hippocampus in rat and human. Although there are some differences in certain regions, the most striking feature is the general similarity of hippocampus sctruture across phylogeny. A- Brain and hippocampus of the rat (adapted from Squire and Kandel, 2000). B- Brain and hippocampus of humans (adapted from Amaral and Lavenex, 2007).

Figure 1.3.2 Human hippocampus dissected free (left) compared to a specimen of Hippocampus leria (right). (Adapted from Andersen et al., 2007.)

A B

44

Figure 1.3.3 The hippocampal formation anatomy. The hippocampus forms a principally uni-directional network with input from the entorhinal cortex that forms connections with the dentate gyrus and CA3 pyramidal neurons via the perforant path. CA3 neurons also receive input from the DG via the mossy fibers. The dendrites of pyramidal cells in the CA1 regions form a thick band (stratum radiatum), where they receive synapses from Shaffer collaterals, the axons of pyramidal cells in the CA3 region (see Lopes da Silva and Arnolds, 1978). CA1 neurons also receive input directly from the perforant path and project axons into the subiculum. These neurons, in turn, send the main hippocampal output back to the entorhinal cortex, forming a loop. Abbreviations: EC, entorhinal cortex; DG, dentate gyrus; Pre, presubiculum; Para, parasubiculum and Sub, subiculum. (Adapted from Amaral and Lavenex , 2007.)

45

2. AIMS

As briefly reviewed in the Introduction, adenosine is a modulator of modulators; the

activation of its specific receptors can modify the action of other molecules interfering

with several biologic functions. In particular, adenosine A2A receptor activation

induces phosphorylation of TrkB receptors (Lee and Chao, 2001), the specific

receptor for the neurotrophin BDNF, which, is lacking in several neurodegenerative

diseases (see introduction 1.1.3). However the clinical use of BDNF is difficult and it

remains necessary to clarify if it can be administrated peripherally. Thus, it is crucial

to identify small molecules that can potentiate BDNF actions in the brain. Adenosine,

through A2A receptor activation, was considered as a good candidate. Therefore, the

first aim of this work was to understand if activation of adenosine A2A receptors

modulates TrkB receptor mediated actions in the hippocampus and to characterize

the possible mechanisms that underlie this cross talk.

Aging is an inevitable life event and the hippocampus is a brain area

extremely susceptible to aging (see e.g. Smith, 1996). Besides functional evidence for

age-related dysfunctions in this area (Lynch, 1998; Barnes, 2003; Gooney et al.,

2004), cellular and molecular mechanisms are also modified. A decrease in the

expression of brain-derived neurotrophic factor (BDNF) has been implicated in

neuronal death occurring with aging, as well as in some neurological disorders (Murer

et al., 2001). It is also known that the expression of the high-affinity receptor for

BDNF, TrkB, as well as TrkB mRNA expression, is decreased in aged rats (Silhol et

al., 2005) and in old humans (Webster et al., 2006). The neuromodulatory action of

adenosine and the expression of its membrane receptors also change with age

(Sebastião et al., 2000, Lopes et al., 1999a; Rebola et al., 2003).

46

Considering that the therapeutic potential for BDNF-based strategies is

greater in aged subjects, that the hippocampus is particularly vulnerable to aging, and

that A2A receptor-mediated actions are more evident in older subjects, it was

considered of interest to investigate how the BDNF actions in the hippocampus

change with age, as well as whether these actions may depend on adenosine A2A

receptors activation.

During normal aging, the brain and in particular the hippocampus suffers

changes that might contribute to age-related memory deficits. On the other hand, the

use of BDNF has been attempted in treating patients with AD, where memory is

dramatically affected. Since, both adenosine and BDNF can modulate long-term

potentiation (LTP), and LTP is considered the neuropsychological correlate of

synaptic plasticity and memory it was considered important to evaluate the influence

of A2A/TrkB receptor cross talk in a model of synaptic plasticity during the aging

process.

Finally, apoptosis is a type of cellular death present in the brain of

Alzheimer’s patients as a result of abnormal amyloid-beta deposition. It is currently

accepted that BDNF, acting through TrkB, protects neurons from apoptosis induced

by numerous agents. Moreover, adenosine can also modulate apoptosis in several

tissues including the brain. Thus, it is of vital importance to understand if the

activation of adenosine A2A receptors modulates the anti-apoptotic neuroprotective

role of BDNF.

47

In summary, the work now reported aims to identify and to understand the

functional consequences of the cross talk between adenosine A2A and TrkB

receptors, having the following specific objectives:

1- To understand whether activation of adenosine A2A receptors can modulate

BDNF action on synaptic transmission.

2- To evaluate age-related changes in synaptic transmission in the density of

A2A and TrkB receptors and in the cross talk between A2A and TrkB receptors.

3- To evaluate the age-related changes that could result from changes in the

cross talk between A2A and TrkB receptors, using an in vitro synaptic plasticity model,

LTP.

4- To evaluate the effect of the activation of A2A receptors on the

neuroprotective action of BDNF against apoptosis.

49

3. METHODS

3.1 BIOLOGIC SAMPLE PREPARATIONS

3.1.1 Hippocampal slices

3.1.1.1 Hippocampus

McIlwain and collaborators were pioneers in developing methods for ex vivo CNS

preparations to perform biochemistry studies. In 1957, Li and McIlwain published the

first electrophysiological study performed in cortex slices. In spite of these early

reports, these biological preparations were believed to maintain their normal

physiological properties only after the studies by Yamamoto and McIlwain (1966) and

Richards and McIlwain (1967), showing that hippocampal slices, sectioned

perpendicularly to the long axis of the hippocampus, maintained synaptic activity and

that the evoked responses were similar to those recorded in vivo. Since then, slices of

CNS are commonly used as experimental models in pharmacological, biochemical

and neurophysiological studies. The hippocampus is used extensively in these

techniques because of the unique laminated organization of neuronal pathways in this

area (see e.g. Andersen and Colingridge, 1971 and previous Chapter 1.3). The

arrangement of neurons in this brain structure allows it to be sectioned such that most

of the relevant circuitry is left intact. In this preparation, the cell bodies of the

pyramidal neurons lie in a single packed layer that is easily visualized.

Thus, the regular sequential arrangement of hippocampal neurons that

facilitates electrophysiological studies, the prominence of adenosine and BDNF

receptors in the hippocampus and the impact of aging and neurodegenerative

50

diseases on the hippocampus prompted us to choose this structure to perform the

studies here reported.

3.1.1.2 Hippocampus isolation and slice preparation

Male Wistar rats were decapitated after halothane anesthesia. The skull was exposed

by cutting the skin at the top of the head. The brain was removed as illustrated in

Figure 3.1.1 A,B as already described by others (e.g. Palkovits et al., 1983), and

placed into ice-cold Krebs’ solution (124 mM NaCl, 3 mM KCl, 1.25 mM NaH2PO4, 26

mM NaHCO3, 1 mM MgSO4, 2 mM CaCl2, and 10 mM glucose) previously gassed

with 95% O2 and 5% CO2, pH 7.4. The cerebellum was cut off and discarded and the

cerebrum was bisected along the midline, separating the two hemispheres. The

hemispheres were placed with the medial surface facing up, as illustrated in Figure

3.1.1 C. The neocortex was peeled off toward the caudal surface, and the midbrain

was pulled away ventrally by pulling in opposite directions at the location marked by

the arrows, using small spatulas. At this point, the dentate surface of the

hippocampus was revealed. Care was taken not to touch the hippocampus with the

spatulas. The fornix was cut by pushing the point of the spatula into the brain at the

point indicated by the dotted line. Next, one spatula was inserted gently under the

fimbria and then further under the hippocampus (area of insertion indicated by the

large arrows). The hippocampus was flipped out of the brain by lifting and pushing on

the spatula, and then rotating the spatula tip around the long axis of the hippocampus.

Once the hippocampus was isolated (Figure 3.1.1 D), it was trimmed at the line

indicated by the two arrows, and the rest of the brain was pulled away from the

hippocampus. Slices (400 µm thick) were cut perpendicularly to the long axis of the

hippocampus (Figure 3.1.1 E,F) with a McIlwain tissue chopper and allowed to

51

recover for at least one hour in a resting chamber (Figure 3.1.2) in Krebs’ solution at

room temperature.

Figure 3.1.1- Hippocampal slices preparation. (A) Scissor introduction into the foramen magnum to remove the occipital bone. (B) Brain extraction. (C) Separation of the hemispheres and hippocampus isolation. (D) Isolated hippocampus. (E) Orientation used to prepare hippocampal slices for electrophysiological recordings. (F) Hippocampal slices. (Adapted from Palkovits and Brownstein, 1983).

A B C

F E D

52

Figure 3.1.2- Resting chamber for hippocampal slices. 1 is a support with a Teflon net to hold the slices; 2 is the inlet tub through which the gas mixture composed by 95% O2 and 5% CO2 is delivered and 3 represents the place where the support with a Teflon net (1) is located. (Adapted from Harvard apparatus catalog).

3.1.2 Hippocampal homogenates After isolation, the hippocampus was disrupted with a Teflon pestle in 0.32 M

sucrose-Tris pH 7.5 and supplemented with protease inhibitors (Complete; Roche

Applied Science, Mannheim, Germany).

3.1.3 Neuronal cell cultures Primary cultures of rat hippocampal or cortical neurons were prepared from 18- to 19-

day-old fetuses of Wistar rats as previously described (Brewer et al., 1993). Pregnant

rats were anesthetized with halothane and then decapitated. The fetuses were

collected in Hanks’ balanced salt solution (HBSS-1; Invitrogen, Grand Island, NY,

USA) and rapidly decapitated. After removal of meninges and white matter, the brain

1

2 3

1

2 3

53

cortex or hippocampus were collected in HBSS without Ca2+ and Mg2+ (HBSS-2;

Invitrogen, Grand Island, NY, USA). The cortex or hippocampus were then

mechanically fragmented, transferred to HBSS-2 solution containing 0.025% trypsin,

and incubated for 15 minutes at 37°C. Following trypsinization, the cells were washed

twice in HBSS-2 with 10% fetal bovine serum and resuspended in Neurobasal

medium (Invitrogen, Grand Island, NY, USA), supplemented with 0.5 mM L-glutamine,

25 µ M L-glutamic acid, and 2% B-27 supplement (Invitrogen, Grand Island, NY,

USA), and 12 mg/ml gentamicin. The cells were then plated on tissue culture plates

(2 x 106 cells/ml) precoated with poly-D-lysine and maintained at 37°C in a humidified

atmosphere of 5% CO2. On the third day, the medium was removed and replaced by

medium without B-27 supplement until the end of the experiment. All assays were

performed on cells cultured for 5 days. Cells were characterized by

immunofluorescence labelling, confirming that neuronal cultures contained < 5% of

glia.

3.1.3.1 Immunofluorescence characterization of cultured neuronal cells

The media was removed from the culture and cells were rinsed with phosphate

buffered saline (PBS) and the cells were then fixed for 30 minutes in

paraformaldehyde (4% in PBS). Cells were subsequently rinsed with PBS and

permeabilized with 0.2% Triton X-100 in PBS for 15 minutes. After washing with PBS,

blocking with 3% BSA in PBS for 30 minutes was performed. Cells were then

incubated overnight at 4°C with primary antibodies: mouse monoclonal antibody to

glial fibrillary acid protein (GFAP) (1:200; see table 3.3) and/or rabbit polyclonal

antibody to neurofilament (1:200; see table 3.3), with subsequent treatment with

secondary antibodies (anti rabbit IgG AlexaF488 and/or anti mouse IgG AlexaF568;

see table 3.3). Fluorescence was visualized with a 40 x (0.45 NA) using an inverted

54

fluorescence microscope Axiovert 135 TV (Zeiss) as previously described by others

(Rodrigues et al., 2002).

3.1.4 Cytosolic protein isolation Cells were homogenized in buffer A (composed by: 10 mM Tris pH 7.6, 5 mM MgCl,

1.5 mM KAc) supplemented with protease inhibitors and 2 mM 1,4-dithiothreitol

(DTT) as previously described with some modifications (Rodrigues et al., 2002).

Then, cells were centrifuged at 500 g for 10 minutes at 4°C. Supernatant was

recovered and the pellet was again resuspended in buffer A and centrifuged at 500 g

for 10 minutes at 4°C. The supernatants were combined and centrifuged at 3160 g for

10 minutes at 4°C. Pellets were discarded and supernatants containing cytosolic

proteins were rapidly frozen and stored at -20°C until used for experimentation.

55

3.2 DRUGS AND ANTIBODIES 3.2.1 Drugs

Table 3.2- Drugs used in the experimental work.

* Aliquots of stock solutions were kept frozen at –20º C until used. ŧ The concentration of DMSO added to the slices (0.001% v/v) was well bellow the concentration that influences glutamatergic synaptic transmission (0.02% v/v, Tsvyetlynska et al., 2005).

Abbreviation Designation Function Supplier Stock solution

ADA Adenosine deaminase (EC 3.5.4.4) Adenosine deamination promoting enzyme

Roche Diagnostics Corporation (Germany)

AP5 DL-2-amino-5-phosphonopentanoate NMDA receptor antagonist

Tocris (Bistol, UK)

Aβ 25-35 Amyloid beta fragment 25:35 Apoptosis inducer Bachem AG (Bubendorf, Switzerland)

100 mM in water

BDNF Brain-derived neurotrophic factor Neurotrophin Regeneron Pharmaceuticals (Tarrytown, NY)

Supplied in 1 mg/ml solution in 150 mM NaCl, 10 mM Na2PO4 buffer, 0.004% Tween 20*

CGS 21680 4-[2-[[6-Amino-9-(N-ethyl-β-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid hydrochloride

Adenosine A2A receptor agonist

Tocris (Bistol, UK)

5 mM stock solutions in DMSO*ŧ

DbcAMP 5’-cAMP Tris salt, N6, 2’-o-dibutyryladenosine-3’:5’-cAMP

cAMP analogue Sigma (ST Luis, USA)


DPCPX 1,3-Dipropyl-8- cyclopentylxanthine Adenosine A1A receptor antagonist

Tocris (Bistol, UK)

5 mM stock solution in DMSO* ŧ

H-89 N-(2-[p-bromocinnamylamino] ethyl)-5-isoquinolinesulfonamide hydrochloride

Protein kinase A inhibitor

Sigma (ST Luis, USA)


ITU 5-iodotubercidin Adenosine kinase inhibitor

Sigma (ST Luis, USA)

5mM stock solution in DMSO*ŧ

[3H]ZM 241385

3H-4-(2-[7-amino-2-(2-furyl)-[1,2,4]triazolo[2,3-a][1,3,5]triazinylamino]ethyl)phenol

Tritiated adenosine A2A receptor antagonist

American Radiolabed Chemicals, Inc. (ST Luis, USA)

Supplied as a 36.5µ M solution in ethanol

K-252a (8R*,9S*,11S*)-(-)-9-hydroxy-9-methoxycarbony-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b,11a-triazadibenzo(a,g)cycloocta(cde)trinden-1-one

Inhibitor of tyrosine protein kinase activity

Calbiochem (La Jolla, CA)

1 mM stock solution in DMSO*ŧ

TrkB-Fc Recombinant human TrkB/Fc chimera BDNF scavenger R&D Systems (Minneapolis, USA)

50 µg/ml stock solution in PBS with BSA 0.1% *

SCH 58261 7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1 ,2,4-triazolo[1 ,5-c]pyrimidine

Adenosine A2A receptor antagonist

Tocris (Bistol, UK)

5 mM stock solution in DMSO* ŧ

XAC 8-4-[(2-aminoethyl)amino]carbonylmethyloxy phenylxanthine

Adenosine receptor antagonist

RBI (Natick, MA, USA)

ZM 241385 4-(2-[7-Amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol

Adenosine A2A receptor antagonist

Tocris (Bistol, UK)

5 mM stock solutions in DMSO*

56

3.2.2 Antibodies

Table 3.3- Antibodies used in the experimental work.

Description Supplier Dilution Use

Primary antibodies Mouse monoclonal antiboy to Adenosine A2A receptor Upstate Biotechnology

(Lake Placid, NY, USA) 1:500 Western Blotting

Mouse monoclonal antibody to TrkB receptor BD Transduction Laboratories (Mississauga, ON Canada)

1:1000 Western Blotting

Rabbit polyclonal antibody to beta-actin Abcam (Cambridge, UK)


Mouse monoclonal antibody to GFAP Abcam (Cambridge, UK)

1:200 Immunofluorescence

Rabbit polyclonal antibody to neurofilament 200 kD Chemicon (Temecula, CA,USA)


Rabbit polyclonal antibody to caspase 3 Santa Cruz Biotechnology (Santa Cruz, CA, USA)


Secondary antibodies

Anti-mouse HRP Biorad laboratories (Hercules, CA, USA)


Anti-rabbit HRP Biorad laboratories (Hercules, CA, USA)


Anti-rabbit IgG AlexaF488 Invitrogen (Grand Island, NY, USA)


Anti-mouse IgG AlexaF568 Invitrogen (Grand Island, NY, USA)


57

3.3 TECHNIQUES 3.3.1 Microelectrophysiological recordings After 1 hour of functional and energetic recovery, slices were transferred to a

recording chamber for submerged slices and continuously superfused at 3 ml/min

with bathing solution gassed with 5% CO2 and 95% O2 at 31-32°C (Figure 3.3.1). The

drugs were added to this superfusion solution for experiments.

Recordings were obtained with an Axoclamp 2B amplifer and digitized (Axon

Instruments, Foster City, CA). Individual responses were monitored, and averages of

eight consecutive responses were continuously stored on a personal computer with

the LTP program (Anderson and Collingridge, 2001).

Figure 3.3.1- Setup for extra-cellular microelectrophysiology recordings. 1-reference electrode; 2-stimulation electrode; 3-recording electrode and 4-temperature sensor.

1

2

3

4

1

2

3

4

58

Field excitatory post-synaptic potentials (fEPSPs) were recorded (Figure

3.3.2C) through an extracellular microelectrode (4 M NaCl, 2–6 MΩ resistance)

placed in the stratum radiatum of the CA1 area (Figure 3.3.2A). Stimulation

(rectangular 0.1 ms pulses, once every 15 seconds) was delivered through a

concentric electrode placed on the Schaffer collateral-commissural fibers, in the

stratum radiatum near the CA3–CA1 border except as otherwise indicated. The

intensity of stimulus (80–200 µA) was initially adjusted to obtain a large fEPSP slope

with a minimum population spike contamination.

Alteration on synaptic transmission was evaluated as the % change in the

average slope of the fEPSP in relation to the average slope of the fEPSP measured

during the 10 minutes that preceded the addition of drugs as previously described

(Diógenes et al., 2004).

3.3.1.1 LTP induction and quantification

In LTP experiments, stimulation (rectangular 0.1 ms pulses, once every 10 seconds)

was delivered alternatively to two independent pathways through two bipolar

concentric electrodes placed on the Shaffer collateral/commissural fibers in the

stratum radiatum (Figure 3.3.2B). LTP was induced by θ-Burst with two or three trains

of 100 HZ, 3 stimuli, separated by 200 ms (Figure 3.3.2D).

The intensity of the stimulus was never changed during these induction

protocols. LTP was quantified as the % change in the average slope of the fEPSP

taken from 46 to 60 minutes after LTP induction in relation to the average slope of the

fEPSP measured during the 14 minutes that preceded the induction of LTP. In each

individual experiment the same LTP-inducing paradigm was delivered to each

pathway. One hour after LTP induction in one of the pathways, BDNF was added to

59

the superfusion solution and LTP was induced in the second pathway, no less than 30

minutes after BDNF perfusion and only after stability of fEPSP slope values was

observed for at least 10 minutes. The effect of BDNF upon LTP was evaluated by

comparing the magnitude of LTP in the first pathway in the absence of BDNF (control

pathway), with the magnitude of LTP in the second pathway in the presence of BDNF

(test pathway); each pathway was used as control or test on alternate days. To test

the modification of the effect of BDNF upon LTP, the modulatory drugs (agonist and

antagonist of adenosine A2A receptors, or the inhibitor of Trk receptors) were added to

the superperfusing bath at least 30 minutes before induction of LTP in the first

pathway and remained in the bath up to the end of the experiment. BDNF was added,

as usual, 60 minutes after induction of LTP in the first pathway. Thus, modulatory

drugs were present during both LTP-inducing periods, whereas BDNF was only

present during the second induction of LTP. This protocol allows the comparison

between the effect of BDNF upon LTP under different experimental conditions,

keeping the magnitude of LTP under the same drug condition, without BDNF in the

same slice as an internal control. When testing the effect of a drug upon LTP (rather

than the modulation of the BDNF effect on LTP), the drug was added to the bath 30

minutes before induction of LTP in the second pathway and the magnitude of the

resulting LTP was compared with that previously obtained (first pathway) in the

absence of the drug as previously described (Fontinha et al., 2008).

60

Figure 3.3.2- Extracellular recordings in hippocampal slices. Representation of the stimulation of one pathway (A) or two pathways (B) in a hippocampal slice to record field excitatory postsynaptic potentials (fEPSP) (C). Traces obtained after stimulation are composed of the stimulus artifact (1), followed by the presynaptic volley (2) and the fEPSP (3). A schematic representation of the stimulation paradigms used in plasticity experiments is represented in D.

3.3.2 Western blotting

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to

evaluate the level of the receptor TrKB immunolabeling as previously described

(Diógenes et al., 2007). Hippocampi were disrupted in sucrose-tris solution with a

S1

DG

CA3

CA1

S1

S2

DG

CA3

CA1

A- Stimulation of one pathawy

B- Stimulation of two pathawy

1 mV

5 ms

C- Traces recorded

D- θ- burst stimulation paradigms

1

2

3

I I I I I I I I I

I I I I I I

200 ms

3x3

2x3

100 Hz

S1

DG

CA3

CA1

S1

S2

DG

CA3

CA1

S1

S2

DG

CA3

CA1

A- Stimulation of one pathawy

B- Stimulation of two pathawy

1 mV

5 ms

C- Traces recorded

D- θ- burst stimulation paradigms

1

2

3

I I I I I I I I I

I I I I I I

200 ms

3x3

2x3

100 Hz

61

Teflon pestle. Hippocampal proteins (20 µg) were separated on 8% SDS-

polyacrylamide electrophoresis gels, and than transferred onto nitrocellulose

membranes. Immunoblots were incubated with 15% H2O2 for 15 min at room

temperature. After blocking with 5% milk solution, the blots were than incubated

overnight at 4°C with primary mouse monoclonal antibodies recognizing TrkB

receptors, adenosine A2A receptors, caspase-3 or β-actin; and, finally the membranes

were incubated with secondary antibodies conjugated with horseradish peroxidase for

3 hours at room temperature. The proteins of interest were detected using Super

SignalTM substrate (Pierce, Rockford, IL, USA). β-actin was used as a loading control.

Protein concentrations were quantified using the Bio-Rad protein assay according to

Bradford (Bradford, 1976).

3.3.3 Binding assays

[3H]ZM 241385 binding studies were performed as previously described (Palmer et

al., 1995), with some modifications. The experiments were performed with

hippocampal homogenates (90 µg protein per assay). After incubation with 2 U/mL

adenosine deaminase (ADA) for 30 minutes at 37°C, [3H]ZM 241385 binding was

carried out in an incubation solution containing 50 mM Tris-HCl buffer and 10 mM

MgCl2 (pH 7.4) for 60 minutes at room temperature, in a final volume of 200 µL.

Specific binding was calculated by subtraction of the nonspecific binding, defined in

the presence of 2 µM XAC. The reaction was stopped by addition of cold incubation

buffer and vacuum filtration through glass fiber filters (FilterMAT for receptor binding,

Skatron Instruments, Lier, Norway) using a semi-automatic cell harvester from

Skatron Instruments. The samples were transferred to scintillation vials and the

radioactivity of each sample measured using a liquid scintillation analyser (Tri Carb

62

2900TR, Perkin Elmer, Illinois, USA). Membrane protein content was quantified with

the Bio-Rad protein assay according to Bradford (Bradford, 1976).

3.3.4 Apoptosis detection

To induce apoptosis, rat neurons cultured for 4 days, were incubated with amyloid-β

peptide (Aβ) 25-35 (25 µ M) one hour after the administration of all drugs. 24 hours

after the induction of apoptosis by Aβ 25-35, cells were analyzed with the following

assays: hoescht staining for morphologic analysis, caspase activity assay and

caspase-3 cleavage detection as described previously by others (Rodrigues et al.,

2002).

3.3.4.1 Morphologic Analysis

Analysis of nuclear morphology was performed as described previously (Rodrigues et

al., 2002). Cell culture medium was gently removed from the cultured plates at the

end of the incubation period. Cells were fixed with 4% formaldehyde in PBS (pH 7.4)

for 10 minutes at room temperature, incubated with Hoechst dye 33258 (Sigma, St.

Luis, USA) at 5 µ g/mL in PBS for 5 minutes, washed with PBS, and mounted using

an aqueous mounting medium (Sigma, St. Luis, USA). Fluorescence was visualized

at 40x magnification (0.45NA) with an inverted fluorescence microscope Axiovert 135

TV (Zeiss). Fluorescent nuclei were independently scored by different observers and

categorized according to the condensation and staining characteristics of chromatin.

Normal nuclei showed noncondensed chromatin dispersed over the entire nucleus.

Apoptotic nuclei were identified by condensed chromatin, contiguous to the nuclear

membrane, as well as nuclear fragmentation of condensed chromatin. Five random

63

microscopic fields per sample were counted and mean values expressed as the

percentage of apoptotic nuclei in the counted cell population.

3.3.4.2 Pro-caspase-3 cleavage

To determine caspase-3 processing, cytosolic proteins were separated by 15% SDS-

PAGE. Blots were probed with primary rabbit polyclonal antibodies to caspase-3 at a

dilution of 1:1000, and subsequently incubated with horseradish peroxidase–

conjugated secondary anti-rabbit antibody. Finally, membranes were processed for

caspase-3 cleavage detection (32 KDa and 20 KDa) (Rodrigues et al., 2002).

3.3.4.3 Caspase activity assay

Caspase activation was evaluated in cytosolic protein extract as decribed previously

(Solá et al., 2003). Cells were harvested and homogenized in buffer A (previously

described in 3.1.4). General caspase-3-like activity was evaluated by enzymatic

cleavage of the chromophore p-nitroanilide (pNA) from the substrate N-acetyl-Asp-

Glu-Val-Asp (DEVD)-pNA. The proteolytic reaction was preceded in isolation buffer

containing 50 µ g of cytosolic protein and 50 µ M DEVD-pNA. The reaction mixtures

were incubated at 37° C for 1 hour, and the liberation of pNA determined by

measuring absorbance at 405 nm using a 96-well plate reader.

64

3.4 STATISTICS

The values presented are mean ± SEM of n experiments. To test the significance of

the effect of a drug versus control, a paired Student´s t-test was used. When making

comparisons from different set of experiments with control, a one-way variance

analysis (ANOVA) was used, followed by a Dunnett’s test. One-way variance analysis

(ANOVA) followed by a Bonferroni test was used when different sets of experiments

were compared between them. Values of p<0.05 were considered to be statistically

significant.

65

4. RESULTS

4.1 INTERPLAY BETWEEN TrkB AND ADENOSINE A2A RECEPTORS IN INFANT

RATS

4.1.1 Rationale Adenosine and adenosine agonists can induce TrkB phosphorylation through a

mechanism involving the adenosine A2A receptor (Lee and Chao, 2001). These data

together with the finding that adenosine A2A receptors and neurotrophin receptors

have a considerable overlap in their distribution (Lewin and Barde, 1996; Fredholm et

al., 2001), prompted the author to evaluate how endogenous activation of adenosine

A2A receptor could influence the action of BDNF on hippocampal synaptic

transmission. Because the excitatory action of BDNF on synaptic transmission at the

developing neuromuscular junction is facilitated by depolarisation (Boulanger and

Poo, 1999a) and depolarising conditions are able to induce adenosine release

(Pazzagli et al., 1993), it was also investigated how depolarising conditions influence

BDNF actions and how this could be related to adenosine receptor activation. Since

the actions of neurotrophins are expected to be more relevant at young ages, 3-week-

old animals were used.

4.1.2 Pre-depolarisation induced by high K+ facilitates BDNF excitatory action

on hippocampal synaptic transmission through TrkB receptors

As illustrated in Figure 4.1.1A, BDNF (20 ng/ml) when applied alone to the

hippocampal slices did not significantly influence the slope of fEPSP (n=7). Even at a

higher concentration (100 ng/ml), BDNF could not enhance synaptic transmission

66

(n=2) (Figure 4.1.1B). However, when applied to slices that had been shortly

depolarised (Figure 4.1.2A) with a pulse of high-K+ (10 mM) for 2 minutes, 46 minutes

before application of the neurotrophin, BDNF (20 ng/ml) caused a significant increase

in fEPSP slope (41 ± 9.8% increase; n=9; p<0.05). The depolarising pulse of high-K+

(10 mM) for 2 minutes, caused a transient enhancement of fEPSP slope, which

returned to the basal level, within 35 minutes after returning to the normal K+

concentration in the bath; in slices where BDNF was not applied (n=3), the fEPSP

slope remained within those basal levels for at least 60 minutes. To evaluate the type

of receptor involved in the excitatory action of BDNF on synaptic transmission, we

studied the effect of this neurotrophin in the presence of K252a, an inhibitor of Trk

receptors phosphorylation (Berg et al., 1992). This inhibitor was added to the

perfusion solution 30 minutes before the pulse of high-K+ and remained in the bath up

to the end of the experiment. The slices were therefore perfused with K252a (200 nM)

for 76 minutes before BDNF application. K252a (200 nM) was virtually devoid of effect

on synaptic transmission; and, as shown in Figure 4.1.2B, it abolished the excitatory

effect of BDNF (n =3; p<0.05).

67

Figure 4.1.1- The averaged time course of changes in fEPSP slope induced by application of BDNF alone 20 ng/ml (A) or 100 ng/ml (B). Values obtained in individual experiments at time 0 and 60 minutes are shown as a scatter representation in (A). The horizontal bars represent the application of BDNF. All values are mean ± SEM; 100% (averaged fEPSP slopes obtained during 10 minutes immediately before BDNF application): -0.64 ± 0.05 mV/ms, n=7 (A), -0.70±0.03 mV/ms, n=2 (B).

A

B

68

Figure 4.1.2- Pre-depolarisation induced by high K+ facilitates BDNF excitatory action on hippocampal synaptic transmission through TrkB receptors. (Aa) illustrates the averaged time course of changes in fEPSP slope under pre-depolarisation (n=9). Slices were superfused with BDNF (20 ng/ml, corresponding to approximately 0.8 nM BDNF) 46 minutes after treatment with high-K+ (10 mM) for 2 minutes. In (Ab) are shown traces obtained in a representative experiment in (a); each trace is the average of eight consecutive responses obtained immediately before (1) and during (2) BDNF application, and is composed of the stimulus artifact, followed by the presynaptic volley and the fEPSP. In (B) is shown the averaged time course of the effect of BDNF (20 ng/ml) 46 minutes after treatment with high-K+ (10 mM) for 2 minutes in the presence of an inhibitor of TrkB receptors, K252a (200 nM), which was added to the slices 30 minutes before the pulse of high-K+ (n=3). Values obtained in individual experiments at time 0 and 60 minutes are shown as a scatter representation in (Aa). The arrows represent the beginning of the 2 minutes high-K+ application and the horizontal bars represent the application of the different drugs. All values are mean ± SEM; 100% (averaged fEPSP slopes obtained during 10 minutes immediately before BDNF application): -0.57 ± 0.03 mV/ms (A), and -0.50 ± 0.10 mV/ms (B). Note that BDNF increased fEPSP slope only in pre-depolarised slices, an action prevented by the treatment with K252a.

Aa

Ab

B

69

4.1.3 Theta Burst stimulation paired with BDNF can elicit synaptic potentiation

The K+ depolarising stimulus could still be effective if applied after BDNF

administration. Indeed, when BDNF (20 ng/ml) was added to the slice at least

30 minutes before the K+ depolarising pulse (10 mM, 2 minutes) and remained in the

bath up to the end of the experiment, an increase (81 ± 2.7%; n= 2) in fEPSP slope

was observed (Figure 4.1.3), which is clearly different from what occurred when the

BDNF (20 ng/ml) was applied without the K+ pulse, or when the K+ pulse was applied

without BDNF (see above). Because the K+ pulse causes a broad depolarisation in all

cells, including glial cells and interneurons, we evaluated whether a focal

depolarisation delivered through the stimulation electrode placed at the synaptic

afferents (Schaffer collaterals) was able to elicit the BDNF effect. Weak theta Burst

stimulation (three Bursts of three pulses each at 100 Hz, delivered 100 ms apart) was

applied to slices that had been previously perfused with BDNF (20 ng/ml) for at least

10 minutes, and fEPSPs were increased by 91 ± 9.5% at 60 minutes after stimulus

(n=3, p<0.05 as compared with values before stimulation) (Figure. 4.1.3B); as

expected (de Mendonça and Ribeiro, 2000), the same stimulation applied in the

absence of BDNF caused a smaller and non-sustained increase in fEPSP slope (20 ±

16.1% increase 60 minutes after stimulation; n=3; p< 0.05).

4.1.4 Adenosine A2A receptors activation facilitates BDNF excitatory action on

synaptic transmission

To evaluate if adenosine A2A receptors activation could influence the action of BDNF

on synaptic transmission, the effect of a selective agonist of the adenosine A2A

receptor CGS 21680 (Jarvis et al., 1989) upon BDNF action was tested. CGS 21680

(10 nM) was added to the slices at least 30 minutes before BDNF application and was

70

virtually devoid of effect on fEPSP slope (Figure 4.1.4C). In the presence of CGS

21680 (10 nM), BDNF increased (Figure 4.1.4D) the slope of fEPSPs by 43 ± 6.7%

(n=6; p<0.05) in slices that had not been predepolarised by the high-K+ pulse.

Blockade of adenosine A2A receptors with the selective antagonist (Poucher et al.,

1995), ZM 241385 (50 nM), which was added 30 min before CGS 21680 (10 nM),

prevented this excitatory effect of BDNF (n =3; p<0.05 as compared with absence of

ZM 241385) (Figure 4.1.4D). Indeed, in the presence of ZM 241385 (50 nM), BDNF

(20 ng/ml) even decreased (26 ± 6.5%), rather than increased, fEPSP slope (Figure

4.1.4B). When added alone, ZM 241385 (50 nM) was virtually devoid of effect on

fEPSP slope (Figure 4.1.4A). To know whether the adenosine A2A receptor could play

a role in the excitatory action of BDNF observed after K+ depolarisation, the influence

of adenosine A2A receptor blockade upon the effect of BDNF was evaluated. In the

presence of the adenosine A2A receptor antagonist, ZM 241385 (50 nM), which was

applied for at least 30 minutes before the K+ pulse, the excitatory action of BDNF (20

ng/ml) was completely prevented (n=3; p<0.05) (Figure 4.1.4B). Under these

conditions BDNF (20 ng/ml) even decreased (17 ± 8.0%) fEPSP slope as it occurred

in the presence of CGS 21680 (10 nM) plus ZM 241385 (50 nM).

71

Figure 4.1.3-Theta-Burst stimulation paired with BDNF can elicit potentiation effect. (A) Shows the averaged time course of changes in fEPSP slope induced by BDNF when the high-K+ pulse was applied in the presence of the neurotrophin. In (Ba) is illustrated the averaged time course of changes in fEPSP slope induced by BDNF (20 ng/ml) after a theta-Burst stimulus (3 Bursts at 5 Hz, each composed of three pulses at 100 Hz) (n=3) () and the changes of fEPSP slope in the same conditions of stimulation but without administration of the neurotrophin (n=3) (). Slices were superfused with BDNF (20 ng/ml) at least 10 min before electrical stimulation. In (Bb) are shown traces obtained in representative experiments in (Ba); each trace is the average of eight consecutive responses obtained immediately before (1) and after (2) theta-Burst stimulation in the absence (n=3) () or in the presence (n=3) () of BDNF, and is composed of the stimulus artifact, followed by the presynaptic volley and the fEPSP. The arrow represents the beginning of stimulation and the horizontal bars represent the application of the BDNF. All values are mean ± SEM; 100% (averaged fEPSP slopes obtained during 10 minutes immediately before stimulation): -0.61 ± 0.07 mV/ms, n=2 (A), -0.70± 0.02 mV/ms, n=3 (Ba,), -0.66 ± 0.01 mV/ms, n=3 (Ba,).

A

Ba

Bb

72

Figure 4.1.4- Adenosine A2A receptors activation facilitates BDNF excitatory action on synaptic transmission. (A) ilustrates the averaged time course of changes in fEPSP slope induced by application of 50 nM ZM 241385 alone (n=3). (B) represents the averaged time course of the effect of BDNF after a depolarising stimulus of potassium in the presence of an A2A adenosine receptor antagonist (ZM 241385). Slices were superfused with BDNF (20ng/ml) 46 min after treatment with high-K+ (10 mM) for 2 min and in the presence of ZM 241385 (50 nM) which was added for at least 30 min before the potassium pulse. In (C) averaged time course of changes in fEPSP slope induced by application of 10 nM CGS 21860 alone (n=3) is ilustrated. In (Da), the averaged time course of the effect of BDNF (20 ng/ml) in the presence of the A2A receptor agonist, CGS 21680 (10 nM) () or in the presence of both CGS 21680 (10 nM) and the A2A receptor antagonist, ZM 241385 (50 nM) () are shown. CGS 21680 was applied at least 30 minutes before BDNF application ( and ) and ZM 241385 was applied 30 minutes before CGS 21680 application (). In (Db) are shown traces obtained in a representative experiment in (Da,), each trace is the average of eight consecutive responses obtained immediately before (1) and during (2) BDNF application, and is composed of the stimulus artifact, followed by the presynaptic volley and the fEPSP. The arrow represents the beginning of the 2 minutes high-K+ application and the horizontal bars represent drug application. All values are mean ± SEM; 100% (averaged fEPSP slopes obtained during 10 min immediately before ZM 241385 application in (A), before BDNF application in (B and Da), or before CGS 21860 application in (C): -0.54 ± 0.090 mV/ms (A), -0.58 ± 0.06 mV/ms (B), -0.60 ± 0.09 mV/ms (C), -0.61 ± 0.01 mV/ms (Da,) and -0.53 ± 0.14 mV/ms (Db,). Note that activation of adenosine A2A receptors facilitates the excitatory action of BDNF on synaptic transmission, an action prevented by A2A receptor blockade.

73

4.1.5 The excitatory action of BDNF is facilitated by the selective adenosine

kinase inhibitor 5-iodotubercidin

The findings that a brief depolarisation by a pulse of high-K+ (10 mM) and subsequent

application of BDNF resulted in a significant increase in fEPSP slope, together with

previous findings that treatment of slices with high-K+ Krebs’ solution results in an

increase of adenosine release (Pazzagli et al., 1993), prompted the next question:

does an enhancement of the extracellular adenosine levels mimic the action of the

pulse of high-K+? A selective adenosine kinase inhibitor, 5-iodotubercidin (ITU), which

enhances the concentration of endogenous extracellular adenosine in hippocampal

slices (Pak et al., 1994) was used. The application of ITU (100 nM) caused a

decrease in synaptic transmission, which may be attributed to activation of adenosine

A1 receptors (the predominant adenosine receptors in the hippocampus) because it

was prevented by the DPCPX (50 nM), a specific adenosine A1 receptor antagonist

(Lohse et al., 1987), (Figure 4.1.5A).

In the experiments where the action of BDNF in the presence of ITU was

tested, the neurotrophin was applied when the full effect of ITU was achieved, and the

slope values of the fEPSPs recorded under these conditions were taken as 100%. As

shown in Figure 4.1.5B, perfusion of BDNF (20 ng/ml) under these conditions resulted

in a significant increase in fEPSP slope (44 ± 8.7% increase; n =3; p<0.05); this did

not occur when A2A receptors were blocked with ZM 241385 (50 nM) before ITU

application (n=3) (Figure 4.1.5B). When ZM 241385 (50 nM) was added only

50 minutes after ITU, BDNF (20 ng/ml) caused the expected enhancement (51%) of

synaptic transmission (one experiment). This may suggest that A2A receptors are

required to trigger the action of BDNF, but they do not need to remain activated

during the BDNF action to enhance synaptic transmission.

74

4.1.6 The activation of the cAMP–PKA transducing system is a critical step in

the excitatory action of BDNF

Because ITU and the pulse of high K+ mimicked the action of the adenosine A2A

receptor agonist, CGS 21680, in respect to its ability to trigger BDNF actions, and

because the activation of A2A receptors stimulates the formation of cAMP–PKA

transducing system (Fredholm et al., 2001), the next series of experiments were

designed to evaluate if a selective PKA inhibitor H-89 (Chijiwa et al., 1990) could

modify the action of BDNF. H-89 (1 µ M) was added for at least 30 minutes before K+

pulse application (Figure 4.1.6A), CGS 21680 perfusion (Figure 4.1.6B), or ITU

administration (Figure 4.1.6C) and remained in the bath up to the end of the

experiments. In all cases, H-89 abolished (p<0.05) the excitatory action of BDNF on

fEPSPs (n=3 for each experimental condition), but by itself H-89 was virtually devoid

of effect on fEPSPs (Figure 4.1.6D). To further evaluate the involvement of cAMP-

dependent PKA activity on the synaptic action of BDNF, experiments were designed

to test if a membrane-permeable cAMP analog, dibutyryl cAMP (dbcAMP) (Henion et

al., 1967), influences BDNF action. By itself dbcAMP (0.5 mM) was virtually devoid of

effect on fEPSP slope (Figure 4.1.7A), and it was added to the slices at least 30

minutes before BDNF application. In the presence of dbcAMP (0.5 mM), BDNF

caused a significant increase (49 ± 13.6%; n=4; p<0.05) in fEPSP slope (Figure

4.1.7B), an effect fully blocked by H-89 (1 µ M; n=2; p<0.05) (Figure 4.1.7B). In slices

predepolarised by a pulse of high-K+ (10 mM), dbcAMP caused only a small increase

in the fEPSP slope (17 ± 1.4%; n=2; p<0.05 as compared with dbcAMP alone),

suggesting that cAMP-mediated actions are slightly influenced by predepolarisation.

75

Figure 4.1.5- The excitatory action of BDNF is facilitated by a selective adenosine kinase inhibitor, 5-iodotubercidin. (A) Represents the averaged time courses of changes of fEPSP slope in the presence of 5-iodotubercidin (ITU, 100 nM) () and in the presence of both ITU (100 nM) plus DPCPX (50 nM) (), an A1 adenosine receptor agonist. Note that the application of DPCPX inhibited the decrease of synaptic transmission induced by ITU (n=3). (Ba,) shows the averaged time courses of changes in fEPSP slope induced by BDNF (20 ng/ml) in the presence of ITU (100 nM), (n=3) and (Ba,) in the presence of both ITU (100 nM) plus ZM 241385 (50 nM), an A2A adenosine receptor antagonist. The horizontal bars represent drug application. In (Bb) are shown traces obtained in a representative experiment in (Ba,). Each trace is the average of eight consecutive responses obtained immediately before (1) and during (2) BDNF application, and is composed of the stimulus artifact, followed by the presynaptic volley and the fEPSP. All values are mean ± SEM; 100% (averaged fEPSP slopes obtained during 10 minutes immediately before the ITU application in (A) or before BDNF application in (Ba)): -0.65 ± 0.06 mV/ms, n=3 (A,), -0.57 ± 0.03 mV/ms, n=3 (A,), -0.52 ± 0.02 mV/ms, n=3 (Ba,) and -0.50 ± 0.10 mV/ms, n=3 (Ba,).

76

Figure 4.1.6- The activation of the cAMP–PKA transducing system is a critical step for the excitatory action of BDNF. Averaged time course of changes in fEPSP slope induced by BDNF in the absence () and in the presence () of the PKA inhibitor H-89 (1 µM) are represented under the following experimental conditions: (A) after a pre-depolarising pulse of high-K+ (10 mM, 2 min); (B) in the presence of CGS 21680 (10 nM); (C) in the presence of 5-iodotubercidin (ITU; 100 nM). H-89 (1 µM) was added to the slices 30 minutes before the application of the K+ pulse (A), CGS 21680 (B), or ITU (C) and alone was virtually devoid of effect on fEPSP slope, as shown in D. In all panels the horizontal bars represent drug application, and the arrow in A represents the beginning of the 2 minutes high-K+ application. All values are mean ± SEM; 100% (averaged fEPSP slopes obtained at times -10–0): -0.57 ± 0.03 mV/ms, n=9 (A,), -0.48 ± 0.23 mV/ms, n=3 (A,), -0.61 ± 0.01 mV/ms, n=6 (B,), -0.47 ± 0.06 mV/ms, n=3 (B,), -0.50 ± 0.10 mV/ms, n=3 (C,), -0.43 ± 0.17 mV/ms, n=3 (C,), -0.59 ± 0.08 mV/ms, n=3 (D). Note that the PKA inhibition prevented the action of BDNF in all experimental conditions.

A

B

C

D

77

Figure 4.1.7- Presynaptic cAMP mimics the effect of high-K+, ITU, or CGS 21680. A shows the averaged time course of changes in fEPSP slope induced by application of dbcAMP (0.5 mM), and Ba shows the averaged time course of changes in fEPSP slope induced by BDNF (20 ng/ml) in the presence of dbcAMP (0.5 mM), either in the absence () and or in the presence () of the PKA inhibitor H-89 (1 µM). Slices were superfused with dbcAMP 30 minutes before BDNF application. Bb shows traces obtained in a representative experiment in Ba; each trace is the average of eight consecutive responses obtained immediately before (1) and during (2) BDNF application, and is composed of the stimulus artifact, followed by the presynaptic volley and the fEPSP. In all panels the horizontal bar represents the application of the different drugs. All values are mean ± SEM; 100% in A and B (averaged fEPSP slopes obtained at times -10–0: -0.70 ± 0.2, n=2 (A), -0.56 ± 1.60 mV/ms, n=4 (Ba,), and -0.64 ± 0.04 mV/ms, n=2 (Ba,).

78

4.1.7 Discussion

The present results show that the excitatory action of BDNF on hippocampal synaptic

transmission is facilitated by predepolarisation and that this effect is dependent on

adenosine A2A receptor activation. These actions were revealed by using a BDNF

concentration that on its own did not significantly modify the slope of fEPSPs and was

mediated by a receptor of the tyrosine kinase receptor family, because pretreatment

with the Trk receptor inhibitor K252a (Berg et al., 1992), prevented the excitatory

effect of this neurotrophin on fEPSPs. BDNF possesses a greater affinity for TrkB

receptors than for TrkA or TrkC receptors (Lewin and Barde, 1996) and, therefore, the

observed actions of BDNF are probably caused by activation of TrkB receptors, which

are present in the hippocampus (Klein et al., 1990; Lewin and Barde, 1996).

The findings that after the brief K+ depolarising pulse, BDNF could facilitate

synaptic transmission are in accordance with the results of Boulanger and Poo

(1999a) at developing neuromuscular synapses showing that presynaptic

depolarisation is a critical factor in causing the synaptic action of neurotrophins. This

could result from the following mechanisms: (1) the prepulse of K+ triggers secretion

of endogenous neurotrophin that could act synergistically with applied BDNF to

potentiate synapses; (2) potassium treatment could induce the expression of more

TrkB receptors, and (3) treatment with high-K+ induces release of adenosine

(Pazzagli et al., 1993), which could gate the excitatory action of BDNF on synaptic

transmission. The results obtained with the adenosine A2A selective agonist, CGS

21680 and with ITU, a selective inhibitor of adenosine kinase that enhances the

extracellular amount of adenosine (Pak et al., 1994), support this last hypothesis.

Thus, in the presence of CGS 21680, BDNF increased the slope of fEPSP, even in

slices that had not been predepolarised by the high-K+ pulse. Moreover the adenosine

79

A2A receptor antagonist ZM 241385 prevented the excitatory effects of BDNF in

conditions of predepolarisation, as well as in the presence of CGS 21680. Finally, ITU

was also able to unmask the BDNF excitatory action, as did CGS 21680 and the brief

K+ pulse, an action also prevented by the A2A atagonist ZM 241385.

There are two sources of extracellular adenosine: release of adenosine by

facilitated diffusion, through membrane adenosine transporters that are equilibrative

and bidirectional (Gu et al., 1995), and extracellular conversion of released adenine

nucleotides into adenosine through a series of ectoenzymes, the last one in the

cascade and the rate-limiting step for adenosine formation being ecto-5’-nucleotidase

(Zimmermann and Braun, 1999). ATP is co-stored with most neurotransmitters, and

therefore neuronal activity causes the release of ATP and extracellular formation of

adenosine in most brain areas, including the hippocampus (Wieraszko et al., 1989;

Cunha et al., 1996). Depolarisation by K+ predominantly induces the release of

adenosine as such (see Latini and Pedata, 2001). The intracellular adenosine levels

are kept low mainly because of the activity of adenosine kinase (see Arch and

Newsholme, 1978) and when this enzyme is inhibited (e.g., by ITU) there is a marked

increase in the release of adenosine (Pak et al., 1994). One may question why K+-

induced adenosine release did not cause an A1-receptor mediated decrease in

synaptic transmission, as ITU did. This most probably results from the time course of

A1-receptor mediated actions (Sebastião et al., 1990; Lupica et al., 1992): any fast

inhibitory actions, because of adenosine released during the 2 minutes application of

K+, should be masked below the transient excitation induced by depolarisation. In

contrast, activation of A2A receptors for a similar period of time might lead to long

lasting enhancement in intracellular cAMP caused by adenylate cyclase activation

(Fredholm et al., 2001), which could gate BDNF actions (Boulanger and Poo, 1999b).

In line with this possibility are the observations that the PKA inhibitor, H-89, prevented

the excitatory action of BDNF in the presence of K+ pulse, CGS 21680, or ITU.

80

Moreover in the presence of the cAMP analog dbcAMP, BDNF enhanced synaptic

transmission, an effect blocked by H-89.

Figure 4.1.8- Mechanism of the facilitatory action of adenosine A2A receptor activation on the BDNF hippocampal synaptic transmission effects on infant rats. The activation of adenosine A2A receptors by the selective agonist, CGS 21680 or through the increase of endogenous adenosine by the addition of the inhibitor of adenosine kinase, ITU or by the pulse of K+, facilitates the excitatory action of BDNF on hippocampal synaptic transmission. This mechanism is dependent on the transducing system cAMP/ PKA since its inhibition by H-89 blocks the BDNF excitatory action. The addition of an analogue of cAMP, dbcAMP, also facilitates the BDNF excitatory action. K252a, the inhibitor of Trk phosphorylation totally prevents BDNF effect on synaptic transmission.

It is known that cAMP can induce a variety of cellular processes in different

systems, including expression of mRNA for Trk receptors and neurotrophins in

primary astroglial cultures (Condorelli et al., 1994) and recruitment of TrkB receptors

to the plasma membrane of CNS neurons (Meyer-Franke et al., 1998). Cytosolic

cAMP can be positively modulated by depolarisation and synaptic activity (Ferrendelli

et al., 1980), and activation of cAMP signalling has been shown to potentiate

TrKB

Gs

AC

A2A

?

cAMP/PKA

BDNF

ITU

K+

ADO

CGS 21680

ZM 241385

(-)

(-)

(+)

(+)

(+)

(+)

H-89

K252a

(-)

dbcAMP(+)


TrKB

Gs

AC

A2A

?

cAMP/PKA

BDNF

ITU

K+

ADO

CGS 21680

ZM 241385

(-)

(-)

(+)

(+)

(+)

(+)

H-89

K252a

(-)

dbcAMP(+)


81

facilitatory actions of BDNF at the developing neuromuscular junction (Boulanger and

Poo, 1999b). A cAMP—PKA—CREB-dependent pathway is also involved in the

ability of adenosine A2A receptor agonists to potentiate nerve growth factor-induced

neurite outgrowth in cultured PC12 cells (Chen et al., 2002). The results now

described show that the interactions between neurotrophins and adenosine A2A

receptors can occur in CNS neurons that are not in culture, therefore not at

developing stage. The finding that blockade of adenosine A2A receptors prevents the

action of BDNF after a K+ predepolarising pulse, suggests that the predepolarisation

pulse per se is not an essential requisite to unmask BDNF excitatory effects,

eventually by a K+-induced increase in cAMP levels (Ferrendelli et al., 1980). In

contrast, activation of adenosine A2A receptors either by released adenosine or by a

selective agonist (CGS 21680) appears to be an essential step to trigger BDNF

excitatory action on synaptic transmission.

Clear excitatory actions of BDNF on hippocampal synaptic transmission have

been observed by some authors (Kang and Schuman, 1995, 1996), whereas others

reported minimal or no effects of this neurotrophin in the same brain area (Figurov et

al., 1996; Gottschalk et al., 1998). Whether this discrepancy results from different

levels of endogenous activation of adenosine A2A receptors, awaits further

investigation.

Neurotrophins have been suggested to have an important role in protecting

mature neurons from neuronal atrophy in the degenerating human brain. A decrease

in BDNF levels might be involved in neurodegenerative disorders such as Alzheimer’s

disease, Parkinson’s disease, Huntington’s disease (Connor and Dragunow, 1998), or

diabetic neuropathies (Nitta et al., 2002), making the use of the naturally occurring

neurotrophic factors very promising for treatment of these disorders. However, until

now the pharmacological administration of in vivo BDNF has not been easy. One of

the reasons is that these molecules are unable to cross the blood–brain barrier,

82

making invasive application strategies like intracerebroventricular infusion necessary.

Intravenous administration of BDNF has been attempted, but it involves a complex

molecular reformulation of the neurotrophin (Wu and Pardridge, 1999). The results

described in this chapter open a new prospective to potentiate BDNF actions on CNS

neurons, i.e., by co-activation of a specific type of adenosine receptors, the A2A

receptors. This possibility expands the pathophysiological implications of adenosine

receptor functioning in the brain (Ribeiro et al., 2003) and points toward new

strategies to interfere with neurotrophic factors in the therapeutics of some

neurodegenerative disease.

In summary, the results described in this chapter clearly indicate a way

to enhance the excitatory action of BDNF on synaptic transmission via

activation of adenosine A2A receptors, which can be achieved either by

enhancing adenosine release or by pharmacological manipulation of the A2A

receptors.

83

4.2 INFLUENCE OF AGE ON BDNF MODULATION OF HIPPOCAMPAL SYNAPTIC

TRANSMISSION: INTERPLAY WITH ADENOSINE A2A RECEPTORS

4.2.1 Rationale

As reported in chapter 4.1 (see also Diógenes et al., 2004), adenosine, through A2A

receptor activation, potentiates synaptic actions of brain-derived neurotrophic factor

(BDNF) in the hippocampus of infant (3-4 weeks) rats. Since A2A receptor mediated

actions are more relevant in older than in young rats and since the therapeutic

potential for BDNF-based strategies is greater in old subjects, the next step was

focused upon 1) the synaptic actions of BDNF, 2) the levels of TrkB receptors, and 3)

the levels of adenosine A2A receptors, in the hippocampus of three groups of adult

rats: young adult rats (10-16 weeks), an age where reproductive behavior is fully

established (Havenaar et al., 1993), old adult rats (36-38 weeks), the time where rats

get the third part of life expectancy (Havenaar et al., 1993), and aged rats (70-80

weeks old); a group of infant (3-4 weeks) rats was also used again for comparison.

4.2.2 BDNF facilitates synaptic transmission in young adult rats in a “LTP-like”

process

As previously observed (see chapter 4.1), BDNF (20 ng/ml) did not significantly

influence the slope of fEPSP when applied alone to hippocampal slices from infant

rats (3-4 weeks old) (n=7, Figure 4.2.1A and 4.2.2). However, in hippocampal slices

from young adult (10-16 week-old) rats, BDNF (20 ng/ml) significantly increased

fEPSP slope by 32 ± 5.3% (n=8, P<0.05, Figure 4.2.1B and 4.2.2). This excitatory

action of BDNF (20 ng/ml) was lost (n=9) in old adults (36-38 weeks, Figure 4.2.1C

84

and 4.2.2), though a higher BDNF concentration (100 ng/ml, Figure 4.2.3) increased

synaptic transmission by 18 ± 2.5%, n=5. Finally, in the aged group of rats (70-80

weeks), hippocampal synaptic transmission was enhanced by approximately 18 ±

4.4% (n=5; P<0.05, Figure 4.2.1D and 4.2.2) by BDNF (20 ng/ml).

The excitatory action of BDNF (20ng/ml) on synaptic transmission of young

adult rats (10-16 week-old) can be attributed to Trk receptor activation since it was

prevented (n=3; p<0.05, Figure 4.2.1B) by K252a (200 nM), an inhibitor of Trk

receptors phosphorylation (Berg et al., 1992). K252a (200 nM) was added to the

perfusion solution 30 minutes before BDNF administration and K252a alone was

devoid of effect on synaptic transmission (n=3).

Neurotrophins, and in particular BDNF, modulate long-term potentiation (LTP)

in the CA1 area of the hippocampus by presynaptically enhancing synaptic responses

to tetanic stimulation (Figurov et al., 1996; Gottschalk et al., 1998). N-methyl-D-

aspartate (NMDA) glutamate receptors play a key role in LTP induction but are not

required for LTP maintenance (Bliss and Collingridge, 1993). To evaluate if the

increase of synaptic transmission induced by BDNF in young adult rats was

dependent on NMDA receptor activation we studied the effect of BDNF in the

presence of AP5, an antagonist of NMDA receptors, at a concentration (50 µM)

known to prevent NMDA responses in the hippocampus (e.g. Sebastião et al., 2001).

As shown on Figure 4.2.4A, when AP5 (50 µM) was applied to hippocampal slices 30

minutes before BDNF (20 ng/ml), the neurotrophin failed to increase fEPSP slope

(n=4, p<0.05 as compared with absence of AP5). When AP5 (50 µ M) was added 60

minutes after BDNF (20 ng/ml) perfusion, therefore, after the full effect of BDNF on

synaptic transmission, it did not revert the action of the neurotrophin (n=3, Figure

4.2.4B). Therefore, as it occurs with LTP (Collingridge et al., 1983), NMDA receptors

are required for the induction of the excitatory action of BDNF on synaptic

85

Ba

C

Da

Bb Db

A

transmission but they do not need to remain activated during BDNF action. The AP5

(50 µ M) by it self, did not significantly change synaptic transmission (n=4).

Figure 4.2.1- The action of BDNF on excitatory synaptic transmission varies in rats from different age groups. In A-D are shown the averaged time courses of changes in fEPSP slope induced by application of 20 ng/ml (corresponding to ≈ 0.8 nM) BDNF to hippocampal slices taken from 3-4 week-old rats (A), 10-16 week-old rats (B), 36-38 week-old rats (C) and 70-80 week-old rats (D) in the absence (•) or in the presence () of the inhibitor of Trk phosphorylation, K252a (200 nM), which was applied at least 30 minutes before BDNF superfusion. The horizontal bars represent the application of the BDNF. Averaged fEPSPs (Bb and Db) obtained in a representative experiment in B and D are also shown below the respective panel; each trace is the average of eight consecutive responses obtained immediately before (1) and during (2) BDNF application, and is composed of the stimulus artifact, followed by the presynaptic volley and the fEPSP. In all panels the values are mean ± SEM; 100% represents the averaged fEPSP slopes recorded for 10 minutes immediately before BDNF, and were: -0.64 ± 0.05 mV/ms, n=7 (A), -0.73 ± 0.06 mV/ms, n=8 (B,•), -0.62 ± 0.04 mV/ms, n=3 (B,), -0.62 ± 0.04 mV/ms, n=9 (C) and -0.52 ± 0.05 mV/ms, n=5 (D).

86

Figure 4.2.2- Comparison between the averaged effects of BDNF in rats from different age groups. The ordinates shows the % change of fEPSP slope induced by BDNF (20 ng/ml) 50-60 minutes after its application to hippocampal slices taken from infant (3-4 weeks), young adult (10-16 weeks), old adult (36-38 weeks) and aged (70-80 weeks) rats, as indicated below each bar *p<0.05, **p<0.001 versus infants (one way ANOVA with the Dunnett’s correction).

Infants Young adult Old adult Aged

50

100

150

* **

fEP

SP

slo

pe

(% o

f ch

ang

e)

87

A

B

Figure 4.2.3- A high concentration (100 ng/ml) of BDNF increases synaptic transmission in slices taken from old adult rats. In A the averaged time courses of changes in fEPSP slope induced by application of 100 ng/ml BDNF to hippocampal slices taken from 36-38 week-old rats are shown. Averaged fEPSPs obtained in a representative experiment in A are shown in B; each trace is the average of eight consecutive responses obtained immediately before (1) and during (2) BDNF application, and is composed of the stimulus artifact, followed by the presynaptic volley and the fEPSP. The values in A are mean ± SEM; 100% represents the averaged fEPSP slopes recorded for 10 minutes immediately before BDNF, and were: -0.52 ± 0.05 mV/ms, n=5 (A).

88

A

B

Figure 4.2.4- BDNF facilitates synaptic transmission in young adult rats in a “LTP-like” process. A and B show the averaged time course of changes in fEPSP slope induced by BDNF (20 ng/ml) in hippocampal slices taken from young adult (10-16 week-old) rats; in the experiments shown in (A), the antagonist of NMDA receptors, AP5 (50 µM), was applied at least 30 minutes before BDNF application; whereas in the experiments shown in (B), AP5 was applied 60 minutes after BDNF, as indicated by the horizontal bars. All values are mean ± SEM; 100% (averaged fEPSP slopes recorded for 10 minutes immediately before BDNF: -0.55 ± 0.01 mV/ms, n=4 (A) and -0.52 ± 0.03 mV/ms, n=3 (B). Note that AP5 inhibited the induction but not the maintenance of the effect of BDNF.

89

4.2.3 Adenosine A2A receptors are involved in the excitatory action of BDNF in

the hippocampus of young adult rats and aged rats.

As shown previously (Chapter 4.1), to trigger an excitatory action of BDNF in infant

rats (3-4 weeks) it is necessary to co-activate adenosine A2A receptors. It was

therefore evaluated if the excitatory action of BDNF observed in young rats could also

be under the influence of adenosine A2A receptors. As illustrated in Figure 4.2.5A and

Figure 4.2.6, the A2A receptor selective antagonist, ZM 241385 (50 nM, added 30

minutes before BDNF) prevented the excitatory effect of BDNF (20ng/ml) in

hippocampal slices taken from young adult (10-16 weeks old) rats (n=5; p<0.05 as

compared with the absence of ZM 241385), suggesting that in this group of animals

the action of BDNF upon hippocampal synaptic transmission requires active

adenosine A2A receptors.

In the oldest group of rats (70-80 weeks) the small increase of the fEPSP

induced by application of the neurotrophin alone was also prevented by ZM 241385

(50 nM, n=3, Figure 4.2.5B and Figure 4.2.6, p<0.05 as compared with absence of

ZM 241385), which was applied 30 minutes before BDNF.

Considering that a pre-depolarisation pulse, by inducing adenosine release

and A2A receptor activation, can trigger a facilitatory action on synaptic transmission in

infant (3-4 weeks old) rats (Chapter 4.1), and that pre-depolarisation also triggers

BDNF actions in other preparations (Boulanger and Poo, 1999a; Pousinha et al.,

2006), it was now evaluated whether a similar facilitation of BDNF actions could occur

in the old adult (36-38 weeks) rats. As before, the slices were shortly depolarised by a

pulse of high-K+ (10 mM) for 2 minutes and BDNF was applied 46 minutes after the

pulse. As shown in Figure 4.2.7A, the depolarising pulse of high-K+ caused a transient

enhancement of fEPSP slope, which returned to the basal level, within 35 minutes

after returning to the normal K+ concentration in the bath. When BDNF was applied to

90

the pre-depolarised slices, it enhanced fEPSP slope by 18 ± 2.4% (n=4) (Figure

4.2.7A). This facilitatory action of BDNF upon synaptic transmission in slices from old

adult (36-38 weeks) rats was dependent on A2A receptor co-activation since it was

prevented by the adenosine A2A receptor selective antagonist, ZM 241385 (50 nM,

Figure 4.2.7B) (n= 3; p< 0.05).

In all age groups, ZM 241385 (50 nM), on its own, was virtually devoid of

effect on the EPSPs (n=3-5).

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Figure 4.2.5- Involvement of adenosine A2A receptors in the excitatory action of BDNF in synaptic transmission. In (A,B) are shown the averaged time course of changes in synaptic transmission induced by application of BDNF (20 ng/ml) to hippocampal slices taken from young adult (10-16 weeks) and aged rats (70-80 weeks) in the absence (•), or the presence of the selective A2A receptor antagonist, ZM 241385 (50 nM) (), which was applied at least 30 minutes before BDNF. The horizontal bars represent time of BDNF application; 100% (averaged fEPSP slopes recorded for 10 minutes immediately before BDNF): -0.73 ± 0.06 mV/ms, n=8 (A,•), -0.51 ± 0.04 mV/ms, n=5 (A,), -0.52 ± 0.05 mV/ms, n=5 (B,•), -0.62 ± 0.08 mV/ms, n=3 (B,). The data in (A,•) is the same as shown in Figure 4.2.1B(•) and the data in (B,•) is the same as in Figure 4.2.1D(•).

A

B

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Figure 4.2.6- Involvement of adenosine A2A receptors in the excitatory action of BDNF in synaptic transmission. The percentage of change of fEPSP slope (ordinates) induced by BDNF (20 ng/ml) 50-60 minutes after its application to the young adult and aged rats in the absence (filled bars) and presence (open bars) of ZM 241385 (50 nM) is shown. *p<0.05 versus the same age group in the absence of the A2A receptor antagonist.

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Figure 4.2.7- Pre-depolarisation induced by high K+ facilitates BDNF excitatory action on hippocampal synaptic transmission in slices taken from old adult (36-38 weeks old) rats. In A are shown the averaged time courses of changes in fEPSP slope induced by application of BDNF (20 ng/ml), which was perfused 46 minutes after treatment with high-K+ (10 mM) for 2 minutes. Averaged fEPSPs (Ab) obtained in a representative experiment in A are shown; each trace is the average of eight consecutive responses obtained immediately before (1) and during (2) BDNF application, and is composed of the stimulus artifact, followed by the presynaptic volley and the fEPSP. B shows the averaged time course of the effect of BDNF (20 ng/ml) applied 46 minutes after treatment with high-K+(10 mM) for 2 minutes in the presence of the selective antagonist of adenosine A2A receptor, ZM 241385 (50 nM), which was added to the slices 30 minutes before the pulse of high-K+. In all panels, the arrows represent the beginning of the 2 minutes high-K+ application, and the horizontal bars represent the application of the different drugs. All values are mean ± SEM; 100% (averaged fEPSP slopes recorded for 10 minutes immediately before BDNF): -0.57 ± 0.04 mV/ms, n=4 (A), -0.55 ± 0.02 mV/ms, n=3 (B).

Aa

Ab

B

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4.2.4 The density of TrkB full length receptors and adenosine A2A receptors is

altered in older rats

To evaluate if the changes in the BDNF excitatory effect on hippocampal slices taken

from animals with different ages could be related to changes in the number of TrkB

full-length receptors, the levels of this receptor in hippocampal homogenates taken

from the same animals used in electrophysiology were assessed by western blot. As

illustrated in Figure 4.2.8A and B, there was a small, though non-significant, increase

in TrkB receptor density in the hippocampus of young adult (10-16 weeks old) rats as

compared with TrkB receptor density in infant (3-4 weeks old, which was taken as

100% reference level) rats. TrkB receptor levels in the hippocampus of old adult (36-

38 weeks old) rats were significantly (p<0.05) reduced, and the decrease was even

more pronounced in the hippocampus from aged (70-80 weeks old) rats.

Since the excitatory action of BDNF on synaptic transmission is dependent on

adenosine A2A receptors activation, the density of A2A adenosine receptors in the

same animals was also evaluated. The existence of commercially available tritiated

ligands for A2A receptors allowed receptor binding assays, which are frequently

preferred for quantitative analysis of receptors. The A2A receptor antagonist [3H]ZM

241385 was used as ligand for A2A receptors (Palmer et al., 1995). Figure 4.2.9

shows the saturation isotherms for the binding of [3H]ZM 241385 to hippocampal

homogenates taken from the same hippocampi as those used in Western blot

analysis of TrkB receptors. It is evident that the specific [3H]ZM41385 binding is

higher in aged (70–80 weeks old) than in young adult (10–16 weeks old) rats, the

difference being more pronounced for higher ligand concentrations. The Bmax values

obtained by nonlinear regression analysis were 28 ± 7.7 fmol mg/protein (n = 5) for

the 10–16-week-old rats, and 67 ± 16 fmol mg/protein (n = 4) for the 70–80-week-old

rats (p<0.05). No significant differences in the Kd values were found (young adult: 1.7

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± 1.1 nM, n = 5; aged: 2.4 ± 1.2 nM, n = 4, p>0.05) and a ZM 241385 concentration

near the Kd (2 nM) was therefore used in subsequent experiments. Binding of 2 nM

[3H]ZM 241385 was significantly higher in aged (70–80 weeks old, p<0.01) and old

adult (36–38 weeks, p<0.05) rats than in infant (3–4 weeks old) rats (Figure 4.2.9B).

DISCUSSION

Figure 4.2.8- Changes in the density of TrkB full length receptors in different age groups. In (A) are shown the Western blots of TrkB full length (FL) (145 KDa) receptors and for control purposes, β-actin protein (43 KDa), in homogenates of rat hippocampus taken from infant (3 weeks), young adult (10-16 weeks), old adult (36-38 weeks) and aged (70-80 weeks) rats, as indicated above each lane. In (B) are shown the averaged of TrkB FL receptor density (n=5-6). All values are mean ± SEM. 100% was taken as the density of TrkB (B) receptors in infant rats. *p<0.05 and **p<0.01 versus infants (one way ANOVA with Bonferroni correction). The hippocampus homogenates were taken from the same animals that were used in electrophysiology experiments.

A

B

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Figure 4.2.9- Specific binding to adenosine A2A receptor receptors in the hippocampus of rats from different age groups. In (A) are shown saturation isotherms for the binding of the selective A2A receptor antagonist [3H]ZM 241385 to the hippocampal homogenates of young adult (,n=5 ) and aged (•,n=4) rats. In (B) is shown the comparison between the specific binding of 2 nM [3H]ZM 241385 to the hippocampal homogenates of infant (3-4 weeks), young adult (10-16 weeks), old adults (36-38 weeks) and aged (70-80 weeks) rats (n=3-5). In both panels the ordinates represent the specific binding of [3H]ZM 241385 obtained upon subtraction of the non-specific binding (determined in the presence of 2 µM XAC) from total binding. The specific binding of 2 nM [3H]ZM 241385 corresponded to 18 ± 1.9% of total binding for all age groups; no appreciable differences in the proportion of specific binding over total binding occurred among the different groups. All values are mean ± SEM. *p<0.05 versus infant rats; **p<0.01 versus infant rats; §p<0.01 versus young adults (one way ANOVA with Bonferroni correction). The hippocampus homogenates were taken from the same animals used in electrophysiology experiments, and from the same hippocampi as those used in western blot analysis.

A

B

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4.2.5 Discussion

The present results show that the effect of BDNF on rat hippocampal synaptic

transmission changes according to the age of the animals and that this could be

related to changes induced by age in the density of TrkB and of adenosine A2A

receptors.

BDNF when applied alone to hippocampal slices from infant rats (3-4 weeks)

does not affect synaptic transmission, but it is able to do so when adenosine A2A

receptors are exogenously activated or when extracellular adenosine levels are

increased (see Chapter 4.1). This ability of A2A receptors to trigger a BDNF action in

infant rats requires cAMP formation and protein kinase A (PKA) activity (Chapter 4.1).

Moreover, in A2A receptor knockout mice BDNF is unable to facilitate hippocampal

synaptic transmission, reinforcing the idea that BDNF-induced modulation of synaptic

transmission requires functional adenosine A2A receptors (Tebano et al., 2008). These

new results show that when BDNF is applied to hippocampal slices taken from young

adult rats (10-16 weeks), it induces a significant increase in fEPSPs. This effect is

mediated by a tyrosine kinase receptor, since it was prevented by K252a, and it also

requires co-activation of adenosine A2A receptors, since it was abolished in the

presence of the selective A2A receptor antagonist, ZM 241385. Therefore, in spite of

not being necessary to exogenously activate A2A receptors to trigger a BDNF action in

young adult rats, A2A receptors are still required for the effect of BDNF on synaptic

transmission.

BDNF enhances glutamate release (Canas et al., 2004) and increases

activation (Levine et al., 1998) and phosphorylation of glutamate NMDA receptors

(Suen et al., 1997) in the hippocampus. By operating pre- and postsynaptic

mechanisms, BDNF also facilitates LTP induction (Figurov et al., 1996; Gottschalk et

al., 1998; Kang and Schuman, 1995; Xu et al., 2000). In the present work it was

98

observed that induction (but not the maintenance) of the excitatory action of BDNF

upon synaptic transmission is abolished by the NMDA receptor antagonist, AP5. It

thus appears that BDNF can by itself induce a long-lasting enhancement of synaptic

transmission that does not require high-frequency stimulation but involves, as LTP

does, NMDA receptor activation. This BDNF-induced “LTP-like” phenomenon is

clearly observed in young adult rats (10-16 weeks old) but disappears in old adult

rats. At this age (36-38 weeks old) animals have clearly lower density of TrkB

receptors and this may account for the loss of effect of BDNF on synaptic

transmission. Interestingly, after a pre-depolarising potassium pulse, a condition

known to increase extracellular adenosine levels (Pazzagli et al., 1993), the

neurotrophin increased synaptic transmission in an adenosine A2A receptor-

dependent manner, suggesting that in this age group an increase in the A2A receptor-

mediated adenosinergic tonus is able to trigger a BDNF action upon hippocampal

synaptic transmission.

A decrease in the amount of TrkB receptor with aging fits to previously

reported data (Silhol et al., 2005). The decrease was evident in old adult animals but

was much more pronounced in aged animals. Surprisingly, BDNF could enhance

synaptic transmission in aged (70-80 weeks old) animals. In this age group we could

observe: 1) a marked increase in Bmax value for A2A receptor binding, indicating

higher density in A2A receptors and 2) that the effect of BDNF on synaptic

transmission requires A2A receptors since it was prevented in the presence of the

selective antagonist of these receptors, ZM 241385. Taken together, these data

indicate that the ability of BDNF to enhance synaptic transmission in aged rats is

probably due to an increase in A2A receptor levels that partially compensate for the

marked loss of TrkB receptors. It is possible that critical levels of A2A and TrkB

receptors need to be present together to allow the facilitatory effect of BDNF on

synaptic transmission. However, concomitant changes in the transducing pathways

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operated by A2A (Rebola et al., 2003) or TrkB receptors (Gooney et al., 2004), or in

the levels of endogenous extracellular adenosine (Cunha et al., 2001) or BDNF

(Narisawa-Saito and Nawa, 1996; Katoh-Semba et al., 1997; Croll et al., 1998) may

also account for a change in BDNF effects throughout age. Indeed, the changes in

TrkB and A2A receptors density in the hippocampus from infant (3-4 weeks old)

toyoung adult (10-16 weeks old) animals are very mild and probably do not account

entirely for the quantitative changes of the BDNF effect in the two age groups.

Unfortunately, the complexity of the transducing pathways operated by TrkB receptors

(Huang and Reihardt, 2003), or even A2A receptors (e.g. Sebastião and Ribeiro, 2000)

makes it difficult to directly address this issue.

The tonic action of adenosine results from a balance between inhibitory A1

and facilitatory A2A receptor-mediated actions. It is known that in the limbic cortex

there is a lower density of A1 receptors and a higher density of A2A receptors (Cunha

et al., 1995). These changes result in a decreased ability of A1 receptor agonists to

inhibit (Sebastião et al., 2000) and an enhanced efficiency of A2A receptors to

facilitate (Rebola et al., 2003) hippocampal synaptic transmission. Furthermore, the

A1/A2A cross talk also changes with age (Lopes et al., 1999b). How this A1/A2A

adenosine receptors interplay contributes to the observed age related changes in the

facilitatory effect of BDNF on synaptic transmission deserves further investigation.

After the pioneer publication by Kang and Schuman (1995) showing that

BDNF enhances synaptic transmission in the rat hippocampus, several papers

reported the absence of an effect of the neurotrophin on evoked EPSPs induced by

low frequency stimulation (e.g. Figurov et al., 1996; Gottschalk et al., 1998; Tanaka et

al., 1997). Methodological differences (e.g. flow rate, temperature, type of chamber)

may not be the sole reason for this discrepancy. Thus, Kang and Schumman (1995)

used young adult (≅ 50 days old) rats whereas the other studies used hippocampus

from developing (12-18 postnatal days old) rats. From the data now presented, where

100

the effect of BDNF was compared in the same experimental conditions in animals

from different ages, it is clear that at least two physiological variables that are not fully

independent may contribute to the variability of the effect of BDNF on fast excitatory

transmission in the hippocampus: age and adenosine through A2A receptor activation.

In summary, the present results show, for the first time, a relationship

between age-related changes in the density of TrkB receptors and adenosine

A2A receptors BDNF-induced enhancement of synaptic transmission in the

hippocampus. This interplay should be taken into consideration whenever

evaluating BDNF actions in nerve cells, and may prove relevant in the design of

BDNF-based therapeutic strategies.

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4.3 INFLUENCE OF AGE ON THE BDNF MODULATION OF LONG TERM

POTENTIATION: INTERPLAY WITH ADENOSINE A2A RECEPTORS

4.3.1 Rationale

During normal aging and in some pathologies such as Alzheirmer’s disease, the

whole brain, and in particular the hippocampus, suffers changes that might contribute

to memory deficits. It is commonly accepted that the neurophysiological basis for

learning and memory involve modifications in the efficiency of synapses. Using

specific patterns of stimulation, it is possible to experimentally induce long-term

modifications in synaptic strength, for instance long-term potentiation (LTP) (Bliss and

Collingridge, 1993). Neurotrophins have a key role in LTP, in particular through

activation of TrkB receptors (Poo, 2001).

Besides functional evidence of dysfunctions in the hippocampus (Lynch,

1998; Barnes, 2003; Gooney et al., 2004), cellular and molecular mechanisms are

also modified during aging. A decrease in the expression of BDNF high-affinity

receptor, TrkB, in rats (Silhol et al., 2005, Diógenes et al., 2007, chapter 4.2) and also

in humans (Webster et al., 2006) has been reported. Furthermore, the

neuromodulatory action of adenosine and the expression of its receptors also change

with age. Thus, the inhibitory adenosine A1 receptor is less efficient in aged animals

(Sebastião et al., 2000), whereas the functioning and number of the adenosine A2A

receptors in the hippocampus is higher in aged rats (Lopes et al., 1999a; Rebola et

al., 2003, Diógenes et al., 2007, chapter 4.2 of this thesis).

Considering the relevance of the action of BDNF upon synaptic plasticity

phenomena and considering the change of TrkB and A2A receptors density upon

aging, the next step was developed to study the influence of BDNF upon LTP during

aging and the influence of adenosine A2A receptors on the effect of BDNF.

102

4.3.2 BDNF effect on LTP induced by θ-Burst stimulation (3 Bursts, 100Hz, 3

stimuli, separated by 200 ms).

As illustrated in Figure 4.3.1, when a θ-Burst (3 Burst of 3 stimuli each, 3x3) was

applied to hippocampal slices taken from 4 week-old rats, a very small LTP was

observed (12 ± 1.0% increasing in fEPSP slope, n=4). In contrast, when BDNF (20

ng/ml) was present, the same θ-Burst stimuli elicited a robust LTP (50 ± 5.1 %

increase in fEPSP slope, n=4, p<0.05). This facilitation of LTP induced by BDNF was

totally abolished when the adenosine A2A receptors were antagonized by the selective

antagonist, SCH 58261 (100 nM) (n=3, p<0.05, Figure 4.3.2). In these experiments

SCH 58261 was added to the superperfusing bath at least 30 minutes before

induction of LTP in the first pathway and remained in the bath up to the end of the

experiment, whereas BDNF was added 60 minutes after induction of LTP in the first

pathway and at least 30 minutes before induction of LTP in the second pathway. No

significant differences in the magnitude of the first LTP were found in the presence or

in the absence of SCH 58261 (100 nM, compare first and third columns of the Figure

4.3.2).

103

Aa Ab

Figure 4.3.1- Effect of the BDNF (20 ng/ml) on θ-Burst (3x3) induced LTP in slices taken from 4 week-old rats. Aa shows averaged time courses changes of fEPSP slope induced by a θ-Burst (3x3) stimulation in the absence () or in the presence () of BDNF (20 ng/mL). The neurotrophin () was applied 60 minutes after induction of LTP in the first pathway (). The ordinates represents normalized fEPSP slopes where 0% corresponds to the averaged slopes recorded for 14 minutes before θ-Burst stimulation (-0.61 ± 0.06 mV/ms, n= 4 () and -0.52 ± 0.06 mV/ms, n= 4 ()) and the abscissa represents the time of every recordings. The recordings obtained in representative experiments are shown in Ab; each recording is the average of eight consecutive responses obtained before (1) and 46-60 minutes after (2) LTP induction in the presence (upper panel) or in the absence of BDNF (lower panel), and is composed of the stimulus artifact, followed by the presynaptic volley and the fEPSP. All values are mean ± SEM.

104

A

B

Figure 4.3.2- Effect of the BDNF (20 ng/ml) on θ-Burst (3x3) induced LTP in slices taken from 4 week-old rats is dependent on adenosine A2A receptors. A shows averaged time course changes of fEPSP slope induced by the θ-Burst (3x3) stimulation in the absence () or in the presence () of BDNF (20 ng/mL). The adenosine A2A receptor antagonist, SCH 58261 (100 nM), was applied 30 minutes before the LTP induction in the first pathway () and remained in the bath up to the end of the experiment. BDNF (20 ng/ml) was applied 60 minutes () after the induction of LTP in the first pathway (). The ordinates represents normalized fEPSP slopes where 0% corresponds to the averaged slopes recorded for 14 minutes before θ-Burst stimulation (-0.52 ± 0.03 mV/ms, n=3 (), -0.55 ± 0.08 mV/ms, n=3 () and the abscissa represents the time of every recordings. All values are mean ± SEM. Panel B depicts the magnitude of LTP (change in fEPSP slope at 46-60 minutes) induced by θ-Burst (3x3) stimulation in relation to pre-θ-Burst values (0%) in the absence of any drugs (control), in the presence of BDNF (20 ng/mL) alone, in the presence of SCH 58261 (100 nM) alone or BDNF (20 ng/mL) together with SCH 58261 (100 nM, n=3) as indicated below each column. *p<0.05 (one-way ANOVA with the Bonferroni´s correction).

105

A similar result was observed in slices taken from 10-16 week-old rats (Figure

4.3.3). Thus, the same θ-Burst stimulus (3x3) only induced a moderate LTP, (13 ±

1.8% increase in fEPSP slope, n=5) which in the presence of the BDNF (20 ng/ml)

was significantly higher (33 ± 3.2 % increase, n=5, p<0.05). In the presence of SCH

58261 (100 nM) the facilitation of LTP induced by the neurotrophin was prevented

(n=5 p<0.05, Figure 4.3.4). As before, SCH 58261 was added to the superperfusing

bath at least 30 minutes before induction of LTP in the first pathway and remained in

the bath up to the end of the experiment. No significant differences in the magnitude

of the first LTP were found in the presence or in the absence of SCH 58261 (100 nM,

compare first and third columns of the Figure 4.3.4).

Figure 4.3.3- Effect of BDNF (20 ng/ml) on θ-Burst (3x3) induced LTP in slices taken from 10-16 week-old rats. Averaged time course changes of fEPSP slope induced by a θ-Burst (3x3) stimulation in the absence () or in the presence () of BDNF (20 ng/mL). The neurotrophin () was applied 60 minutes after induction of LTP in the first pathway (). The ordinates represents normalized fEPSP slopes where 0% corresponds to the averaged slopes recorded for 14 minutes before θ-Burst stimulation (-0.58 ± 0.06 mV/ms, n= 4 () and -0.57 ± 0.06 mV/ms, n= 4 ()) and the abscissa represents the time of every recordings. All values are mean ± SEM.

106

A

B

Figure 4.3.4- Effect of the BDNF (20 ng/ml) on θ-Burst (3x3) induced LTP in slices taken from 10-16 week-old rats is dependent on adenosine A2A receptors. A shows averaged time course changes of fEPSP slope induced by the θ-Burst (3x3) stimulation in the absence () or in the presence () of BDNF (20 ng/mL). The adenosine A2A receptor antagonist, SCH 58261 (100 nM), was applied 30 minutes before the LTP induction in the first pathway () and remained in the bath up to the end of the experiment. BDNF (20 ng/ml) was applied 60 minutes () after the induction of LTP in the first pathway (). The ordinates represents normalized fEPSP slopes where 0% corresponds to the averaged slopes recorded for 14 minutes before θ-Burst stimulation (-0.54 ± 0.03 mV/ms, n=5 (), -0.63 ± 0.06 mV/ms, n=3 ()) and the abscissa represents the time of every recordings. All values are mean ± SEM. Panel B depicts the magnitude of LTP (change in fEPSP slope at 46-60 minutes) induced θ-Burst (3x3) stimulation in relation to pre-θ-Burst values (0%) in the absence of any drugs (control), in the presence of BDNF (20 ng/mL) alone, in the presence of SCH 58261 (100 nM) alone or BDNF (20 ng/mL) together with SCH 58261 (100 nM, n=5) as indicated below each column. *p<0.05 (one-way ANOVA with the Bonferroni´s correction).

107

As illustrated in Figure 4.3.5A, in slices taken from 36-38 week-old rats, the θ-Burst

stimulation (3x3) was able to induce LTP (37 ± 7.4 % increase in fEPSP slope, n=4).

In parallel, in slices from the same animals, BDNF (20 ng/ml) did not cause a further

significant enhancement of LTP (42 ± 7.0 %, n=4, p>0.05 as compared with the

absence of BDNF). Since the synaptic effects of BDNF are potentiated by adenosine

A2A receptors activation (Chapters 4.1 and 4.2), it was next studied if the agonist of

this receptor also potentiates the effect of BDNF on LTP. The presence of a selective

agonist of the adenosine A2A receptor, CGS 21680 (10 nM), did not trigger an effect of

the neurotrophin (20 ng/ml) upon LTP (n=3, Figure 4.3.5B). CGS 21680 was added

to the superperfusing bath at least 30 minutes before induction of LTP in the first

pathway and remained in the bath up to the end of the experiment.

Finally, in the oldest group of rats (70-80 wek old rats), θ-Burst stimulation

was able to induce LTP (35± 3.5 % increase in fEPSP slope, n=4, Figure 4.3.6), but

BDNF (20 ng/ml) did not induce any further significant increase in the magnitude of

LTP (37 ± 1.4% increase of fEPSP slpe, n=4 p>0.05, when compared with the

absence of BDNF Figure 4.3.6).

In Figure 4.3.7 are sumarized the results from the experiments with all age

groups, where it is clear that BDNF gradually looses the ability to facilitate LTP but it

also emerge that LTP is progressively larger with aging.

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Figure 4.3.5- Absence of effect of the BDNF (20 ng/ml) on θ-Burst (3x3) induced LTP in slices taken from 36-38 week-old rats. A and B show the averaged time courses changes of fEPSP slope induced by a θ-Burst (3x3) stimulation in the absence () or in the presence () of BDNF (20 ng/mL). In panel B, the adenosine A2A receptor agonist, CGS 21680 (10 nM), was applied 30 minutes before the LTP induction in the first pathway () and remain in the bath up to the end of the experiment. BDNF (20 ng/ml) was applied 60 minutes () after the induction of LTP in the first pathway (). The ordinates represent normalized fEPSP slopes where 0% corresponds to the averaged slopes recorded for 14 minutes before θ-Burst stimulation (-0.69 ± 0.07 mV/ms, n=4 (A,), -0.68 ± 0.05 mV/ms, n=4 (A,), -0.59 ± 0.07 mV/ms, n=3 (B,) and -0.59 ± 0.05 mV/ms, n=3 (B,)) and the abscissa represent the time of every recordings . All values are mean ± SEM.

A

B

109

Figure 4.3.6- Absence of effect of the BDNF (20 ng/ml) on θ-Burst (3x3) induced LTP in slices taken from 70-80 week-old rats. Averaged time course changes of fEPSP slope induced by a θ-Burst (3x3) stimulation in the absence () or in the presence () of BDNF (20 ng/mL). The neurotrophin () was applied 60 minutes after induction of LTP in the first pathway (). The ordinates represents normalized fEPSP slopes where 0% corresponds to the averaged slopes recorded for 14 minutes before θ-Burst stimulation (-0.69 ± 0.07 mV/ms, n= 4 () and -0.68 ± 0.07 mV/ms, n= 4 ()) and the abscissa represent the time of every recordings. All values are mean ± SEM.

110

Figure 4.3.7- Age-related modulation of long-term potentiation (LTP) by BDNF. The bars represent the % change of fEPSP slope 46-60 minutes after LTP induction in the presence (black bars) and the absence (white bars) of BDNF (20 ng/ml). BDNF (20 ng/ml) significantly increases LTP magnitude in hippocampi taken from 4 (n=4) and 10-16 (n=5) week old rats (first and second black bars respectively) when compared to controls (first and second white bars). In slices taken from 36-38 (n=4) and 70-80 (n=4) week old, BDNF was not able to increase LTP (fourth and fifth black bars) magnitude compared to the control LTPs (fourth and fifth white bars). *P<0.05 (paired Student´s t test) as compared with absence of BDNF in the same experiments (adjacent white bar to the left).

0

10

20

30

40

50

60

Without BDNF

With BDNF (20 ng/ml)

10-16 36-384 70-80 Age (weeks)

*

*

fEP

SP

slo

pe (

% o

f ch

an

ge)

111

4.3.3 BDNF effect on LTP induced by a weak θ-Burst stimulation (2 Bursts,

100Hz, 3 stimuli, separated by 200 ms).

The observed reduction in the effect of BDNF upon LTP in aged animals could be due

to the age-related increase in the magnitude of LTP in the absence of BDNF, which

would therefore contribute to masking the effect of BDNF. Thus, the effect of BDNF in

LTP induced by a weaker θ-Burst stimulation (2 Bursts, 100Hz, 3 stimuli, separated

by 200 ms - 2x3) was tested. In these conditions, the magnitude of LTP (without

BDNF) attained in hippocampal slices taken from both 36-38 week-old and 70-80

week-old rats was lower, (14 ± 3.5 % and 31 ± 2.7 %, increase in fEPSP respectively,

Figure 4.3.8) as compared with LTP observed in the animals of the same age but

after the stronger θ-Burst (3x3) stimulation protocol (see 4.3.2).

BDNF was not able to increase LTP induced by the weak θ-Burst (2x3) in

slices taken from 36-38 week-old rats (Figure 4.3.8A) even when adenosine A2A

receptors were pharmacologically activated by CGS 21680 (10 nM) (n=5, P>0.05,

Figure 4.3.8.B). In slices taken from 70-80 week-old rats, BDNF was also unable to

increase the magnitude of LTP (31 ± 7.1% increase, n=3, Figure 4.3.8C).

112

Figure 4.3.8- Absence of effect of the BDNF (20 ng/ml) on θ-Burst (2x3) induced LTP in slices taken from 36-38 week-old rats and 70-80 week-old rats. Averaged time course changes of fEPSP slope induced by a θ-Burst (2x3) stimulation in the absence () or in the presence () of BDNF (20 ng/mL) in hippocampal slices taken from 36-38 week-old rats (A and B) or taken from 70-80 week-old rats (C). The neurotrophin () was applied 60 minutes after induction of LTP in the first pathway () in the absence (A and C) or in the presence (B) of CGS 21680 (10 nM). CGS 21680 was added to slice 30 minutes before induction of LTP in the first pathway and remained in the bath up to the end of the experiment. The ordinates represents normalized fEPSP slopes where 0% corresponds to the averaged slopes recorded for 14 minutes before θ-Burst stimulation: -0.51 ± 0.03 mV/ms, n=3 (A,), –0.50 ± 0.18 mV/ms, n=3 (A,), -0.58 ± 0.03 mV/ms, n=5 (B,), -0.54 ± 0.02 mV/ms, n=5 (B,), -0.66 ± 0.14 mV/ms, n=3 (C,), –0.56 ± 0.07 mV/ms, n=3 (C,) and the abscissa represent the time of every recordings. All values are mean ± SEM.

A B

C

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4.3.4 Effects of a BDNF scavenger on LTP in hippocampal slices

In slices taken from 10-16 week-old rats, the magnitude of LTP after a θ-Burst (3x3) in

the presence of BDNF was similar to that observed on hippocampal slices taken from

36-38 week-old rats in the absence of the exogenous BDNF administration (Figure

4.3.7). This could suggest that the higher LTP in control conditions in older rats was

due to higher endogenous levels of BDNF. To test this hypotesis, hippocampal slices

from 36-38 week-old rats were treated with a BDNF scavenger, TrkB-Fc. Slices were

incubated as described by Patterson and collaborators (2001), submerged in either

gassed Krebs’ solution alone or in gassed Krebs’ solution containing 2 µ g/ml TrkB-Fc

at room temperature for 1–1.5 hours prior to use for electrophysiological experiments.

Slices were then moved to the interface-recording chamber and were stimulated

using the θ-Burst stimuli. The TrkB-Fc fusion protein (the ligand binding domain of

the native TrkB receptor coupled to the Fc fragment of human immunoglobulin)

scavenges unbound TrkB ligands (Shelton et al., 1995), such as the BDNF.

If extracellular BDNF levels in 36-38 week-old rats were higher and this was

the cause for a higher LTP, a lower LTP magnitude in the slices pre-treated with

TrkB-Fc would be expected. As shown in Figure 4.3.9, LTP magnitude in the

presence of Trk-Fc (2 µg/ml) was similar (n=3, p>0.05) to that observed without the

scavenger, suggesting that the age related increase of LTP magnitude may not be a

result of an increase of endogenous BDNF levels acting via TrkB receptors. However,

the slices were, as mentioned before, pretreated with TrkB-Fc for 1–1.5 hours prior to

use for electrophysiological experiments allowing the scavenging of the BDNF

available in that time. The slices were placed in the recording chamber continuously

perfused with Krebs’ solution, which probably removed the complex (BDNF)-(TrkB-

Fc) from the hippocampal slice. On the other hand, during the θ-Burst stimulation a

higher amount of BDNF can be released (Aicardi et al., 2004); since more TrkB-Fc

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was not administerd, the activation of TrkB receptors could not be prevented because

of execess of BDNF. Thus, it was next tested the effect of the K252a, the inhibitor of

Trk phosphorylation, on LTP magnitude of hippocampal slices taken from 36-38

week-old rats.

Figure 4.3.9- Effect of Trk-Fc (2 µµµµg/ml) on θ-Burst (3x3) induced LTP in slices taken from 38-38 week-old rats. Averaged time course changes of fEPSP slope induced by a θ-Burst (3x3) stimulation. In the pathway represented as (), slices were pre-incubated for 1.5 hr with Trk-Fc (2 µg/ml). In the pathway represented by (), no drug was added to slices. The arrow represents θ-Burst stimulation. All values are mean ± SEM; 0% (averaged fEPSP slopes recorded for 14 minutes immediately before θ-Burst stimulation: -0.58 ± 0.06 mV/ms, n=5 () and –0.53 ± 0.02 mV/ms, n=3 ()).

115

A B

K252a was applied to slices 60 minutes after the induction of the LTP in the

first pathway and 30 minutes before LTP induction in second pathway. As illustrated

in Figure 4.3.10, in slices taken fom 36-38 week-old rats, K252a (200 nM) reduced

significantly the magnitude of the LTP (22 ± 2.8 %, n=3, p<0.05). K252a did not

influence the basal synaptic transmission as well as does not influence LTP in infant

rats (Fontinha et al., 2008). This suggests that the increase of LTP observed in older

rats comparing to the younger can be in part attributed to the activation of Trk

receptors as a result of the increase in BDNF levels during the θ-Burst stimulation.

Further experiments using animals for the different age groups are required for further

clarification of the ages related increase in LTP due to an age related enhancement of

BDNF release.

Figure 4.3.10- Effect of the K252a (200nM) on θ-Burst (3x3) induced LTP in slices taken from 38-38 week-old rats. A shows averaged time course changes of fEPSP slope induced by the θ-Burst (3x3) stimulation in the absence () or in the presence () of K252a (200 nM). K252a was applied 60 minutes () after the induction of LTP in the first pathway (). 0 % in the ordinates: -0.60 ± 0.03 mV/ms, n=3 (), -0.82 ± 0.02 mV/ms, n=3 (). All values are mean ± SEM. Panel B depicts the magnitude of LTP (change in fEPSP slope at 46-60 minutes) induced θ-Burst (3x3) stimulation in relation to pre-θ-Burst values (0%) in the absence or in the presence of K252a (200 nM). *p<0.05 (paired Student´s t-test).

116

4.3.5 Discussion The aim of the work described in this chapter was to study the effect of BDNF on LTP

induced by θ-Burst stimulus during aging and the influence of adenosine A2A receptor

activation on the BDNF effects.

In previous Chapters (4.1 an 4.2) it was observed that activation of adenosine

A2A receptors is required for the excitatory action of BDNF on hippocampal synaptic

transmission of young and aged rats; but the reduced ability of BDNF to increase

synaptic transmission in aged rats even when adenosine receptors are activated,

could be related to reduced TrkB receptor levels (Diógenes et al., 2007). These

observations lead to the question that was addressed in this chapter: Does the effect

of BDNF on LTP change upon aging and what is the involvement of adenosine A2A

receptors in this process?

In experiments performed using hippocampal slices from the two youngest

groups of rats (4 week-old and 10-16 week-old rats), a marked increase on LTP

induced by BDNF was observed. This effect was totally prevented when adenosine

A2A receptors were blocked. These results reinforce the conclusions from previous

chapters that the activation of adenosine A2A receptors is required for BDNF synaptic

excitatory actions, extending it to synaptic plasticity phenomena.

In slices taken from 36-38 and 70-80 weeks old rats, BDNF no longer

increased the magnitude of LTP even when adenosine A2A receptors were

pharmacologically activated. On the other hand, a marked increase was observed in

LTP magnitude in slices from 36-38 and 70-80 weeks old rats when compared with

LTP magnitude obtained in slices from young rats (4 or 10-16 week-old). To

understand if the effect of BDNF was masked by the increase in LTP magnitude, the

effect of BDNF upon LTP induced by a weaker θ-Burst stimulation was evaluated.

However, under these weak LTP induction conditions, BDNF was still devoid of effect.

117

In slices taken from 10-16 week-old rats, the magnitude of LTP induced by θ-

Burst in the presence of BDNF was similar to that observed in hippocampal slices

taken from 36-38 week-old rats in the absence of exogenous BDNF. This could

suggest that the higher LTP in control conditions in older rats was due to higher

endogenous levels of BDNF and a consequent increase in TrkB activation. The BDNF

scavenger, Trk-Fc, did not reduce LTP observed in older animals, suggesting that the

high LTP in aged animals is not due to an increase of extracellular BDNF levels.

However, it is important to be aware of some technical aspects of these experiments.

When Trk-Fc was tested, the slices were pre-incubated with this scavenger during 1.5

hours immediately before electrophysiological recordings. Thus, Trk-Fc scavenges

the available endogenous BDNF during that time and any molecules of Trk-Fc that

became loosely bound to the tissue would be expected to scavenge BDNF released

afterwards. The slices were then continuously perfused for electrophysiological

recordings. For economical reasons and in accordance with a previously reported

protocol (Patterson et al., 2001), no Trk-Fc was added to the perfusion solution.

However, it is known that θ-Burst induces BDNF release (Aicardi et al., 2004), which

facilitates LTP (Figurov and et al., 1996). If the amount of BDNF released during θ-

Burst stimulus is higher in aged then in young animals, the remaining Trk-Fc

molecules may not be sufficiently able to scavenge it. Thus, experiments were

performed using the inhibitor of Trk phosphorylation, K252a. In the presence of this

inhibitor, the LTP magnitude was reduced in aged animals, which may suggest that

the increase of LTP magnitude observed in aged rats may indeed be a consequense

of higher BDNF levels in these animals.

Figurov and collaborators (1996), studied θ-Burst induced LTP in the

hippocampus from Sprague-Dawley rats with ages between one to 2.5 week-old

postnatal days and a group of adult rats. They also found that exogenous BDNF

enhanced LTP in the neonatal hippocampus where endogenous BDNF level is low;

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but, in the adult hippocampus, where endogenous BDNF is higher, the Trk-IgG

decreased LTP, suggesting that endogenous levels of this neurotrophin strongly

influenced LTP.

It is commonly accepted that in normal aging the hippocampus suffers

changes, which might contribute to memory deficits. Since LTP is related to learning

and memory (Lynch, 2004), the effect of aging on LTP is of great interest to

understand the age-related decline in cognitive function. However, the results relating

LTP and aging are controversial. The first reports indicated intact hippocampal LTP

induction at the Schaffer collateral-CA1 synapses in old rats (Landfield and Lynch,

1977; Landfield et al., 1978). On the other hand, further studies showed that age-

related deficits in cognitive function are accompanied by an impaired ability to sustain

LTP (see Barnes, 2003). An increase in LTP in slices from aged rats has ben

previously reported (Costenla et al., 1999).

What contributes to the changes here described in LTP, is a fundamental

question. It is known that 1) there is no loss of hippocampal CA1 pyramidal cells in old

rats (Rapp and Gallangher, 1996; Rasmussen et al., 1996), 2) most biophysical

properties of old pyramidal cells do not differ from those in young cells, and 3) there is

no change in spontaneous firing rates of single cells in the hippocampus of freely

behaving old rats comparing with young rats (see Barnes, 2003). However, 4) a deficit

in calcium regulation upon aging has been reported (Landfield and Lynch, 1977).

There are also results suggesting that an increase in microglia activation could be the

key factor for plasticity changes observed during aging (Griffin et al., 2006).

Additionally, changes in the density of NMDA subtype of glutamate receptors have

been discussed. With aging, declines in the expresion of mRNA for diferent NMDA

receptors subunits in rats (Magnusson, 2000) and in konkeys (Bai et al., 2004) were

already observed, as weel as, a decrease in the expression of several NMDA

subunits (Sonntag et al., 2000; Clayton et al., 2002). Yet, these changes do not

119

explain the now observed increase in LTP upon aging. But there are other points

which can, in fact, contribute to this increase in LTP: 1) a lower GABAergic inibition

observed in aged rat hippocampus (Potier et al., 1992; Billard et al., 1995); 2) a

possible increase in BDNF release or the release of other facilitatory substances upon

θ-Burst stimulation in aged animals; and 3) the increase of the levels of adenosine

A2A excitatory receptors in aged animals (Chapter 4.2). According to the results

observed with K252a, the second possibility seems to hold true, but this does not

exclude any of the others.

During the present work it was also observed that BDNF loses the ability to

increase LTP in old rats. The facilittatory effects of BDNF on plasticity have been

related to the activation of TrkB receptors (Figurov et al., 1996), and, TrkB receptor

density decreases with age (Chapter 4.2). Thus, it was postulated that BDNF would

be unable to increase LTP in aged rats. Since BDNF can activate both TrkB and p75

receptors and p75 receptor activation frequently has opposite effects to TrkB receptor

activation (Lu et al., 2005), further investigation needs to be done to understand the

role of p75 receptors in the age-related changes in LTP.

In summary, the work described here shows that BDNF increases θθθθ-

Burst induced-LTP in young and adult rats, that this facilitatory effect is

dependent on adenosine A2A receptor activation, that LTP magnitude increases

with the age of these animals and that this could be due to an increase of the

release of BDNF upon LTP induction, during aging and consequent increase in

TrkB receptor activation.

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4.4 INFLUENCE OF ADENOSINE A2A RECEPTORS ON THE PROTECTIVE

ACTION OF BDNF AGAINST APOPTOSIS INDUCED BY THE AMYLOID BETA

PEPTIDE

4.4.1 Rational

Neurotrophins have been suggested to have an important role in protecting neurons

from neuronal atrophy in the degenerating human brain. A decrease in BDNF levels

might be involved in neurodegenerative disorders such as Alzheimer’s disease (AD)

(Connor and Dragunow, 1998), making the use of this molecule very promising for

treatment of these disorders. However, as discussed in Chapter 1.1.3, the

pharmacological administration of in vivo BDNF has not been easy since these

molecules are unable to cross the blood–brain barrier, making invasive application

strategies like intracerebroventricular infusion necessary. Intravenous administration

of BDNF has been attempted, but it involves a complex molecular reformulation of the

neurotrophin (Wu and Pardridge, 1999).

As showed in previous chapters, a way to potentiate BDNF actions on

hippocampal neurons is the co-activation of a specific type of adenosine receptors,

the A2A receptors. This could open a new strategy for the treatment of

neurodegenerative diseases by the use of drugs that can induce activation of A2A

receptors and consequently potentiate the neuroprotective effect of the endogenous

BDNF. However, this type of adenosine receptors can also interfere with apoptosis.

Thus, it was observed that the blockade rather than the activation of A2A receptors is

involved in neuroprotection (Dall’lgna et al., 2007). However, adenosine A2A receptor

stimulation also has been associated with decreased apoptosis in the amygdala

(Boucher et al., 2006) and it is known that endogenous adenosine via the A2A

121

receptor subtype may be important in mediating protection of sympathetic neurons

from apoptosis (Ramirez et al., 2004).

Taking into account that adenosine A2A receptors activation facilitates BDNF

actions on synaptic transmission (Chapter 4.1), that the contribution of this adenosine

receptor in apoptosis is controversial and that apoptosis can be a key factor for some

neuodegenerative diseases where BDNF may play a role, it was decided to evaluate:

1) the effect of the adenosine A2A receptors on apoptosis in cultured neurons, and 2)

the function of the A2A receptor activation on the neuroprotective action of BDNF. The

amyloid beta peptide (Aβ) was used as an apoptotic stimulus since Aβ accumulates

in the brain of AD patients (Finder and Glockshuber, 2007), where it seems to induce

apoptosis (Loo et al, 1993), which has been implicated as a potential cause for the

neuronal loss in AD (Shimohama, 2000).

4.4.2 Apoptosis induced by amyloid beta in primary cultures of rat neurons is

prevented by BDNF and this protection is potentiated by adenosine A2A

receptors activation.

Since all the work described in previous chapters of this thesis was carried out in

hippocampus, the initial apoptosis studies were performed separately in hippocampal

and cortical cultured neurons. The results obtained were similar in the two brain

regions and therefore all remaining experiments were performed in cortical neuron

cultures to increase the amount of available biological material and thus decrease the

number of animals required.

Neuronal cells were incubated in the presence of BDNF (20 ng/ml), of the

adenosine A2A receptor agonist CGS 21680 (10 nM), or both CGS 21680 (10 nM) and

the neurotrophin (20 ng/ml) for 24 hours. Whenever both drugs were added, CGS

122

21680 was applied 30 minutes before BDNF. The characteristic morphologic nuclear

changes associated with apoptosis were observed using Hoechst staining (Figure

4.4.1). As expected, few apoptotic cells were detected in control cultures as well as

when BDNF, CGS 21680 or both were present. In contrast, almost 31 ± 4.4% of

neurons exhibited condensed chromatin and nuclear fragmentation with formation of

apoptotic bodies after treatment with 25 µM of amyloid beta (Aβ) 23-35 for 24 hours

(p<0.05 as compared with control, Figure 4.4.2). To evaluate if increased cell death

could be prevented by BDNF and/or CGS 21680, neurons were pre-exposed to these

drugs for 1 hour before Aβ application (Figure 4.4.3). BDNF, added prior to Aβ 25-35,

reduced the percentage of apoptotic cells to 19 ± 1.6% (n=5). CGS 21680 reduced

apoptosis to 24 ± 2.1% (n=7, p>0.05, as compared with Aβ 25-35 alone). When

added together, BDNF and CGS 21680 fully prevented Aβ 25-35-induced apoptosis

reducing apoptotic cells to a value of 11 ± 1.2% (n=7, P<0.05 as compared with Aβ

25-35 alone), similar to that obtained in the absence of Aβ 25-35 or any other drug.

The effect of the selective antagonist of A2A receptors, SCH 58261 (100 nM)

was tested in two cultures. SCH 58261 (100 nM) applied 1 hour before Aβ 25-35 (25

µM) did not appreciably change the percentage of apoptotic cells when compared

with Aβ 25-35 alone (n=3, p>0.05, Figure 4.4.4). This antagonist of adenosine A2A

receptors, added 30 minutes before BDNF completely prevented the neuroprotective

effect of the neurotrophin (Figure 4.4.4A). As expected when applied 30 minutes

before CGS 21680 (10 nM), SCH 58261 (100 nM) also reverted the slight decrease of

apoptosis induced by the A2A receptor agonist (Figure 4.4.4B). Moreover, in

experiments where BDNF and CGS 21680 were added together, SCH 58268 fully

reverted the marked neuroprotection already observed (Figure 4.4.4C).

123

Figure 4.4.1- Morphologic analysis of cells. Cortical neurons were isolated and cultured for 4 days, subsequently they were incubated with no drugs (control), BDNF (20 ng/ml), CGS 21680 (10 nM) or both BDNF and CGS 21680 as indicated. In co-incubation experiments CGS 21680 was added 30 minutes before BDNF application. 24 hours after drugs administration, cells were fixed and then assessed for nuclear morphologic alterations characterized by condensed chromatin, fragmentation, and formation of apoptotic bodies using fluorescence microscopy with Hoechst staining as shown in A. In B the mean ± SEM values obtained in five to eight experiments are ilustrated. Dotted line the value obtained without any drug.

Figure 4.4.2 Amyloid-beta (Aβ)-induced apoptosis in primary rat neurons. Cortical neurons were isolated and cultured for 4 days. When Aβ 25-35 (25 µM) was added alone it remain in the cell cultures for 24h, afterward cells were fixed and then assessed for nuclear morphologic alterations characterized by condensed chromatin, fragmentation, and formation of apoptotic bodies using fluorescence microscopy with Hoechst staining as shown in A. In B the mean ± SEM values obtained in eight experiments are illustrated. *P<0.05 (paired Student´s t-test).

Aβ

25

-35

(2

5 µ

M)

A B

A B

124

Figure 4.4.3 Apoptosis induced by amyloid-beta (Aβ) in primary rat neurons is prevented by BDNF and CGS 21680. Cortical neurons were isolated and cultured for 4 days; subsequently, they were incubated with BDNF (20 ng/ml), CGS 21680 (10 nM) or both BDNF and CGS 21680 as indicated. In co-incubation experiments CGS 21680 was added 30 minutes before BDNF application. Aβ 25-35 (25 µM) was added 1 hour after BDNF and/or CGS 21680 and it remained in the cell cultures for 24 hours. Cells were then fixed and assessed for nuclear morphologic alterations characterized by condensed chromatin, fragmentation, and formation of apoptotic bodies using fluorescence microscopy with Hoechst staining as shown in A. Arrows indicate apoptotic cells. In B, the mean ± SEM values obtained in four to eight experiments are illustrated. *P<0.05 (one-way ANOVA with the Dunnett correction). Dotted line the value obtained without any drug.

A B

125

Figure 4.4.4- SCH 58261 reverts the neuroprotection induced by BDNF. In panel A, black bars illustrate the neuroprotective effect of BDNF upon Aβ 25-35-induced apoptosis (already showed in Figure 4.4.3), grey bars show the effect of SCH 58261, an antagonist of adenosine A2A receptors, in the presence of Aβ 25-35 (first grey bar, n=3) and on the effect of BDNF with Aβ 25-35 treatment (second gray bar, n=2). In panel B, black bars illustrate the effect of CGS 21680 upon Aβ 25-35-induced apoptosis (already showed in Figure 4.4.3), grey bars show the effect of SCH 58261, in the presence of Aβ 25-35 (first grey bar, n=3) and on the effect of CGS 21680 (second gray bar, n=2). In panel C, black bars illustrate the neuroprotective effect of BDNF in the presence of CGS 21680 upon Aβ 25-35-induced apoptosis (already showed in Figure 4.4.3), grey bars show the effect of SCH 58261 in the presence of Aβ 25-35 (first grey bar, n=3) and on the effect of BDNF plus CGS 21680 (second gray bar, n=2). *p<0.05 (one-way ANOVA with the Dunnett correction). Dottted lines represent the control values (without drugs).

A B

C

126

4.4.3 Amyloid beta induced fragmentation of pro-caspase-3 is prevented by BDNF in the presence of adenosine A2A receptor activation Mitochondria are central life and death regulator, which release cytochrome c, leading

to the activation of caspase-3, which subsequently cleaves downstream substrates.

Processed caspase-3 in the cytosol of neuronal cells, assessed by immunoblot

analysis, was therefore used as measure of the degree of apoptosis in cultured cells.

Proteolytically activated caspase-3 (20 KDa) was low in cultured neurons after

incubation without drugs (control), BDNF (20 ng/ml) and or CGS 21680 (10 nM)

(Figure 4.4.5). However, when Aβ 25-35 (25 µM) was added, a significant increase

(more than 2-fold) in caspase-3 formation (p<0.05 when compared with control cells)

was observed (Figure 4.4.6). The pre-incubation with BDNF (20 ng/ml) reduced the

formation of caspase-3 induced by Aβ 25-35, however the decrease did not reveal

statistical significance. A similar trend occurred when cells were incubated with the

A2A receptor agonist (CGS 21680, 10 nM, Figure 4.4.7). However, when cells were

incubated with BDNF (20 ng/ml) in the presence of CGS 21680 (10 nM), the caspase-

3 formation from pro-caspase-3 was significantly reduced to levels similar to the

control (without drugs) (P>0.05 when compared to control cells, Figure 4.4.7).

127

Figure 4.4.5 Processing of caspase-3 in primary rat neurons in the absence of amyloid-beta. Cortical neurons were isolated and cultured for 4 days before incubation as described in Methods. Cells were incubated either with vehicle (control, no aded drugs), BDNF (20 ng/ml), CGS 21680 (10 nM) or both BDNF and CGS 21680. In coincubation experiments CGS 21680 was added 30 minutes before BDNF application. Procaspase-3 (32 KDa) and active caspase-3 (20 KDa) expression were analysed using western blotting. In A representative Western blots of protein expression are shown for cytosolic protein fractions. The histograme B show mean ± SEM values of the ratio caspase 3/ pro-caspase- 3 for 3-5 independent experiments. Dotted line represents the control values (without drugs).

B

A

Co

ntr

ol

BD

NF

CG

S 2

16

80

BD

NF

+ C

GS

21

68

0

Pro-caspase 3

Caspase 3

32 KDa

20 KDa

128

Figure 4.4.6 Amyloid-beta induced processing of caspase-3 in primary rat neurons. Cortical neurons were isolated and cultured for 4 days before incubation as described in Methods. Cells were incubated either with vehicle (control) or with Aβ 25-35 (25 µM) for 24 hours. Procaspase-3 (32 KDa) and active caspase-3 (20 KDa), expression were analysed using Western blotting. Representative western blots of protein expression are shown for cytosolic protein fractions in A. The histograme B show mean ± SEM values of the ratio caspase 3/ pro-caspase- 3 for 3 independent experiments. *p<0.05 (paired Student´s t-test). Dotted line represents the control values (without drugs).

Co

ntr

ol

Aβ

25

-35

32 KDa

20 KDa

Pro-caspase 3

Caspase 3

B

A

129

Figure 4.4.7-Amyloid-beta induced processing of caspase-3 in primary rat neurons, which is reduced by BDNF in the presence of adenosine A2A receptors activation. Cortical neurons were isolated and cultured for 4 days before incubation as described in Methods. Cells were incubated with vehicle (values represented as the dotted line) or with Aβ 25-35 (25 µM) alone or together with BDNF (20 ng/ml), with CGS 21680 (10 nM) or with both BDNF and CGS 21680. In coincubation experiments CGS 21680 was added 30 minutes before BDNF application. Aβ 25-35 (25µM) was added 1 hour after BDNF and/or CGS 21689 and it remained in the cell cultures for 24 hours. Procaspase-3 (32 KDa) and active caspase-3 (20 KDa) expression was mesured using western blotting. Representative Western blots of protein expression are shown for cytosolic protein fractions in A. The histograme B shows mean ± SEM values of the ratio caspase 3/ pro-caspase- 3 for 3 independent experiments. *p<0.05 (one-way ANOVA with the Dunnett correction).

BD

NF

A

CG

S2

16

80

32 KDa

20 KDa Pro-caspase 3

Caspase 3

+ Aβ 25-35

BD

NF

+

C

GS

21

68

0

B

130

A B

4.4.4 Amyloid beta induced increase in caspase-3 activity is prevented by BDNF

in the presence of adenosine A2A receptors activation

Caspase-3-like activity was evaluated by enzymatic cleavage of the chromophore

p-nitroanilide (pNA) from the substrate N-acetyl-Asp-Glu-Val-Asp (DEVD)-pNA and

the formation of pNA was measured at 405 nm, as mentioned in Methods (Chapter

3.4).

Neither BDNF nor CGS 21680 alone or together influenced significantly the

activity of caspase 3 (Figure 4.4.8). Aβ 25-35 (25 µM) significantly increased

caspase-3 activity (p<0.05, n=4, Figure 4.4.8B). When either BDNF (20 ng/ml) or

CGS 21680 (10 nM) were added alone, a slight redution in the increase in caspase-3

activity induced by Aβ 25-35 (25µM) was noted (Figure 4.4.9). The increase in

caspase-3 activity caused by Aβ 25-35 (25 µM) was fully prevented when cells were

incubated with BDNF (20 ng/ml) together with CGS 21680 (10 nM) (Figure 4.4.8C).

Figure 4.4.8- Activity of caspase-3 in primary rat neurons. Cortical neurons were isolated and cultured for 4 days before incubation as described in Methods. Cells were incubated with either vehicle (control), BDNF (20 ng/ml), CGS 21680 (10 nM) or both BDNF and CGS 21680 as indicated. In coincubation experiments CGS 21680 was added 30 minutes before BDNF application (A). When Aβ 25-35 (25 µM) was added alone it remained in the cell cultures for 24 hours (B). Caspase activity was measured in cytosolic protein fractions as described in Methods. The histogrames shows mean ± SEM values of caspase-3 activity for 3-4 experiments are represented. *p<0.05 (paired Student´s t-test). Dotted line represents the control values (without drugs).

131

Figure 4.4.9- Aβ-induced enhancement of caspase-3 activity in primary rat neurons is reduced by BDNF in the presence of adenosine A2A receptor activation. Cortical neurons were isolated and cultured for 4 days before incubation as described in Methods. Cells were incubated with either vehicle (represented as the dotted line), or with Aβ 25-35 (25µM) alone or together with BDNF (20 ng/ml), CGS 21680 (10 nM) or both BDNF and CGS 21680. In coincubation experiments CGS 21680 was added 30 minutes before BDNF application. Aβ 25-35 (25µM) was added 1 hour after BDNF and/or CGS 21680 and it remained in the cell cultures for 24 hours. Caspase activity was measured in cytosolic protein fractions as described in Methods. These results are the mean ± SEM values of caspase-3 activity for 3 independent experiments. *p<0.05 (one-way ANOVA with the Dunnett correction).

132

4.4.5 Discussion

Treatment of rat cortical neurons with Aβ 25-35 resulted in high levels of apoptosis

showing the consequent elevated levels of pro-caspase-3 cleavage into caspase-3

and also an increase in its proteolytic activity. The main finding in the work described

in this chapter was that the molecular modifications resulting from an increase in

neuronal death by apoptosis were totally prevented when cells were incubated with

BDNF in the presence of a selective agonist of adenosine A2A receptors, CGS 21680.

In addition, when the antagonist of adenosine A2A receptors, SCH 58261, was added

to cells prior to the other drugs, it completetly reverted the protective affects of BDNF,

CGS 21680 or both BDNF and CGS 21680 added together. Therefore, A2A adenosine

receptors need to be activated to allow BDNF neuroprotection against apoptosis

induced by Aβ.

The accumulation of Aβ in the brain has been implicated as a potential cause

for the neuronal loss that occurs in AD (Shimohama, 2000). Cell death from Aβ-

induced toxicity is a complex process that is thought to involve a number of different

pathways, including oxidative stress, perturbation of calcium homeostasis,

mitochondrial dysfunction, and activation of caspases (Selkoe, 2001). It is generally

believed that Aβ peptides contribute significantly to the pathogenesis of AD, although

the mechanisms for this pathogenesis are not yet fully understood. Accordingly, it was

reported that central administration of Aβ 25-35 caused a memory impairment in

mice, assessed by a decrease in performance in both inhibitory avoidance and

spontaneous alteration tasks (Maurice et al., 1996; Dall’Igna et al., 2007).

Neurotrophins, after binding to their Trk receptors, activate two main signal

transduction pathways that regulate the cell death machinery (Segal and Greenberg,

1996, Kaplan and Miler, 2000). The primary pathway is the phosphatidylinositol 3-

kinase (PI3K)–Akt pathway, which suppresses cell death by inhibiting the apoptotic

133

activities of forkhead (Brunet et al., 1999) and BCL2 (B-cell leukaemia/ lymphoma 2)-

associated death protein (BAD)(del Pesos et al., 1997; Datta et al., 1997). The other

pathway is the mitogen-activated protein kinase (MAPK)–MEK (MAPK/ERK

extracellular signal regulated kinase) signalling cascade, which stimulates the activity

and/or expression of antiapoptotic proteins, including BCL2 and the transcription

factor cyclic AMP response element binding protein (CREB) (Riccio et al., 1999).

Adenosine is an ubiquitous homeostatic substance present in all cells, which

modulates several functions in the brain, including neuronal survival. However, the

actions of adenosine can be diverse depending on the cell type, the aggressiveness

of stimuli and the age of animals or cells. In neurons, Lee and Chao (2001) observed

by measuring lactate dehydrogenase (LDH) release, that activation of adenosine A2A

receptors rescues neurons from death induced by the absence of BDNF, in a TRK

receptor dependent way. In PC12 cell cultures, adenosine A2A receptor activation

antagonizes apoptosis due to serum deprivation (Huang et al., 2001) and also

rescues the impairement of neurite outgrowth when the NGF-evoked MAPK cascade

is suppressed (Chen et al., 2002). Recently it was shown that the adenosine A2A

receptor contributes to motoneuron survival by transactivating the TrkB receptors

(Wiese et al., 2007). In primary glial cultures from cerebral cortices, the activation of

adenosine A2A receptors increases NGF expression (Heese et al., 1997), which in

turn can exert its neuroprotective effects. Adenosine also has important roles in

peripheral nervous system where it has neuroprotective effects on neurotrophin-

deprived sympathetic neurons through A2A receptor activation, via a pathway that

requires ERK signalling (Ramirez et al., 2004).

During hypoxia or ischemic conditions, adenosine is released in large

amounts and can mediate cellular protection. This neuroprotection against ischemia

or hypoxia is mainly due to A1 receptor activation (Ribeiro et al., 2003). However, in

liver, administration of A2A receptor agonists before induction of ischemia can

134

attenuate post-ischemic apoptotic hepatic injury and thereby minimize liver injury

(Ben-Ari et al., 2005). On the other hand, A2A antagonists have been reported to

reduce hypoxic-ischemic neuronal injury (von Lubitz et al., 1995; Phillis, 1995) as well

as to block the neurotoxicity induced by Aβ in primary cultures of cerebellar granule

cells prepared from 8-day-old Wistar rats (Dall’Igna et al., 2003). In fact, the ability of

A2A agonists to enhance glutamate release (see Sebastião and Ribeiro, 1996)

together with the frequent finding that these receptors operate in an opposite way to

the A1 receptors, which are widely accepted as neuroprotective receptors, leads to a

frequent assumption that A2A receptors enhance cell death. As shown in the work now

reported, and in line with data mentioned above, A2A receptors enhance the

neuroprotective action of neurotophins and, even more importantly, the

neuroprotective actions of neurotrophins are fully lost when A2A receptors are

blocked. This clearly point towards a putative neuroprotective role of A2A receptors

whenever neurotrophic actions are required. Therefore, studies are required on the

consequences of therapy using specific A2A receptor antagonists in

neurodegenerative diseases, where neurotrophic factors play a beneficial role (see

e.g. Muller and Ferré, 2007).

In summary, as shown in the work presented in this chapter, activation

of adenosine A2A receptors did not intensify the apoptosis induced by Aβ. On

the contrary, adenosine A2A receptor activation enhanced the ability of BDNF to

decrease cell death, strengthening the idea that adenosine A2A receptor

agonists may be usefull in potentiating the BDNF therapeutic actions on AD.

135

5. CONCLUSIONS

The goal of the present study was to investigate functional aspects in the cross talk

between adenosine A2A receptors and the brain-derived neurotrophic factor (BDNF).

This study was started using hippocampal slices taken from infant rats (3-4

week-old rats) to understand if the activation of adenosine A2A receptors modulates

BDNF action actions in basal synaptic transmission and the possible mechanisms

that underlie this cross talk. We found that adenosine A2A receptor activation

facilitates the excitatory effect of BDNF on synaptic transmission and that this

facilitatory action of adenosine A2A receptor activation is due to the activation of the

cAMP/PKA transducing system (Chapter 4.1).

Since BDNF has been pointed out as a promising drug to treat

neurodegenerative diseases, such as Alzheimer’s disease, a pathology more frequent

in old people, it was imperative to study the influence of adenosine receptors

activation upon the effect of BDNF on synaptic transmission during aging. With the

present work it became clear that BDNF has distinct actions throughout the lifetime of

animals, that the effects of BDNF are also dependent on adenosine A2A receptors

activation and that these changes can be in part due to a decrease of TrkB receptor

levels and an increase of A2A receptor densities (Chapter 4.2).

In addition, it was considered essential to evaluate the influence of adenosine

A2A receptors upon the BDNF effect on LTP, a commonly accepted way to study the

neurophysiological basis of learning and memory, and to understand if A2A receptors

would influence the neuroprotection induced by BDNF. The main reason for this

approach was that in neurodegenerative pathologies, such as Alzheimer’s disease,

there is both a memory deficit and neuronal loss probably due to enhanced apoptosis

as a consequence of amyloid beta peptide deposition. It was observed that BDNF

136

increases LTP in young and adult rats, and that this facilitatory effect is dependent on

adenosine A2A receptor activation. It was also observed that LTP magnitude

increases with age and this may be due, in part, to an increase in BDNF release upon

stimulation. Finally, BDNF loses the capacity to increase LTP in aged animals, which

can be attributed to a decrease of TrkB receptors density.

As stated earlier, BDNF has been proposed to be used in the treatment of

Alzheimer’s disease patients, a pathology characterized by the deposition of amyloid

plaques containing the amyloid beta peptide, the formation of neurofibrillary tangles

with hyperphosphorylated tau protein and cell death. Thus we studied the

neuroprotective effect of BDNF against cellular death induced by amyloid beta

peptide and the involvement of adenosine A2A receptors in this neuroprotective action.

This study showed that activation of adenosine A2A receptors enhances the ability of

BDNF to prevent apoptosis cellular death in cultured neurons.

The chronic actions of neurotrophins, such as differentiation, growth and

maturation of CNS cells, are already well established. A new area of investigation was

recently opened with the finding that these molecules also have important acute

actions upon synaptic transmission. The work presented in this thesis contributes to a

more elucidated understanding of these acute neuronal actions of BDNF and

describes a way to potentiate these actions through the activation of adenosine A2A

receptors.

137

6. FUTURE PERSPECTIVES

The importance of administering neurotrophic factors, in particularly BDNF, to

Alzheimer’s patients has been pointed out several times throughout this thesis. The

discovery of a molecule that crosses the blood-brain barrier and that could potentiate

BDNF actions would be a great therapeutic breakthrough. The first hint of the

potential benefits of adenosine or adenosine A2A receptor agonists as drugs to

potentiate BDNF action obtained in 2001 by Lee and Chao (2001), who found that a

selective adenosine A2A agonist induced phosphorylation of the BDNF receptors,

TrkB. But it remained to be known if this molecular interplay would be relevant to the

actions of BDNF. Answering this question was the global objective of this work.

As reported here, A2A receptor activation is an obligatory step for the synaptic

and neuroprotective function of BDNF, and this A2A facilitatory action occurs in a

cAMP-PKA transducion signal-dependent way. In addition to the possibilities already

discussed about the manner in which the cAMP-PKA transducing system can

modulate TrkB actions, one could envision that this cross talk could also occur

between the signalling transdution pathways initiated by adenosine receptors and Trk

receptors. Thus, the work here reported opens new perspectives for the investigation

of common players involved in this interplay between adenosine and BDNF receptors.

A schematic representation of the relationship between pathways initiated by

activation of both A2A and TrkB receptors is presented in Figure 5.1. As mentioned in

detail in the introduction of this thesis, adenosine A2A receptors generally couple to Gs

proteins. In addition, in the striatum they are also coupled to Golf proteins (Corvol et

al., 2001) and, in the hippocampus, they can be coupled to Gi/Go proteins (Cunha et

al., 1999). Furthermore, adenosine A2A receptors, when overexpressed in COS-7

cells, were shown to couple to G15/16 proteins (Offermanns and Simon, 1995). In

human endothelial cells, the signalling via G12/13 proteins has been hypothezied (Sexl

138

et al., 1997). There is also evidence that adenosine A2A receptors can induce

formation of inositol phosphates via pertussis toxin-insensitive Gα15 and Gα16 (see

Jacobson and Gao, 2006). Therefore, the adenosine A2A receptor couple, via G

proteins, to an intricate network of signalling pathways, which enables adenosine to

communicate with cells in a complex manner. Initially, signalling pathways were

thought to be linear, starting with activation of receptors in the cellular membrane and

a subsequent activation of different steps including small signalling molecules, called

second messengers, protein kinases and transcription factors that regulate gene

expression. In the particular case of A2A receptors, this involves the cAMP and cAMP-

dependent protein kinase (PKA) pathway (in blue in the Figure 5.1). However,

adenosine A2A receptors can also be involved in the MAPK signalling and as weel as

in PI3K signalling (Figure 5.1 blue dotted line).

Regarding to Trk receptors, as described in the introduction, there are three

major pathways involved in the signalling mediated by these receptors: 1)

phosphotydilinositol-3 kinase (PI3K); 2) Ras-MAPK pathway and 3) PLCγ pathway,

and their downstream effectors (Figure 5.1 brown dotted line).

Therfore, signalling cascades resulting from the activation of A2A and TrkB

receptors have common steps (Figure 5.1 in green), the Ras-MAPK and PI3K

pathways. These are involved in survival, growth and differentiation as well as in

synaptic transmission and in plasticity (Blom and Konnerth, 2005). Since adenosine

A2A receptors activation potentiates BDNF actions on synaptic transmission, plasticity

and neuroprotection, I advance the hypothesis that one or both of these pathways are

involved in the cross talk between A2A and TrkB receptors.

139

Figure 5.1 Cross talk between signalling pathways of adenosine A2A receptors and TrkB receptors. Possible interaction between the signalling pathways of both receptors A2A and TrKB.

In conclusion, the key finding in this thesis is that adenosine A2A receptors activation

potentiates BDNF actions. Furthermore, as clearly shown throughout this work, the

actions of this neurotrophin upon both synaptic trasmission and neuronal death are

fully lost when A2A receptors are blocked. Therefore, agonists of adenosine A2A

receptors may prove to be of high therapeutic interest where there is the need to

increase BDNF actions in neurodegenerative diseases. In other words, in the early

stages of neurodegenerative diseases, where an enhancement of neurotrophic

factors is highly desirable, A2A receptor agonists can be an important therapeutic

option and antagonists should be avoided in order to allow beneficial neurotrophic

influences. On the other hand, in the late stages of neurodegenerative diseases, A2A

receptor antagonists can be advantageous to inhibit excitatory processes.

AC

cAMP

CREB

SrcRas

Raf

MEK

Erk

SOS

PI3K

AKT

PLCγ

DAG IP3

PKC Ca2+

Adenosine A2A receptors Trk receptors

Gαs

G12/13

PKA

Rac1/Cdc42

p38

G15/16

AC

cAMP

CREB

SrcRas

Raf

MEK

Erk

SOSSOS

PI3K

AKT

PLCγPLCγ

DAG IP3

PKC Ca2+

Adenosine A2A receptors Trk receptors

Gαs

G12/13

PKA

Rac1/Cdc42

p38

G15/16

140

In the future, investigation of how to promote the expression of TrkB

receptors in aged subjects and how to avoid eventual deleterious consequences of

the upregulation of neurotrophic actions (e.g. tumor promotion) deserves to be

investigated.

141

7.ACKNOWLEDGEMENTS

Em primeiro lugar gostaria de expressar o meu profundo agradecimento aos meus

orientadores, Professor Joaquim Alexandre Ribeiro e Professora Ana Sebastião. Ao

professor Alexandre Ribeiro, por quem tenho um enorme respeito e admiração, por

me ter recebido no seu laboratório, pelo incentivo e pelos ensinamentos muito

experientes que me deu. À Professora Ana Sebastião, por quem tenho uma grande

admiração e amizade, por todos ensinamentos, palavras de apoio, positivismo e

discussão científica. Gostaria de agradecer ao Professor Alexandre de Mendonça,

que sempre esteve disponível para qualquer esclarecimento e discussão científica

principalmente em temas relacionados com plasticidade sináptica.

Uma palavra de agradecimento vai para todos os colegas que ao longo destes anos

se tornaram amigos. Gostaria de dirigir um agradecimento especial aos que

partilharam discussão científica, experiências e artigos são eles a Catarina

Fernandes, o António, a Ana Rita, a Natalia, a Paula, a Luisa e o Bruno Fontinha. Ao

Bernardo, estudante que orientei no decorrer do último ano, pelo entusiasmo e

colaboração nas experiências de apoptose. Não posso deixar de expressar o meu

reconhecimento a todos os técnicos em particular à Alexandra, ao Sr. João e á

Elvira.

À Professora Cecília Rodrigues da Faculdade de Farmácia da Universidade de

Lisboa por me ter recebido no seu laboratório o que possibilitou a realização de parte

das experiências de apoptose bem como pela sua orientação na realização das

mesmas. Um agradecimento muito especial à Susana Solá e todos os estudantes do

grupo pela constante disponibilidade, simpatia e paciência.

142

Ao Professor Rui Silva, à Adelaide e à Sofia da Faculdade de Farmácia da

Universidade de Lisboa por toda a ajuda prestada aquando de implementação das

culturas primárias de neurónios.

À Professora Dora Brites por me ter dado a oportunidade de entrar no mundo da

ciência e de ter acompanhado os meus primeiros passos ainda como estudante de

Ciências Farmacêuticas na Faculdade de Farmácia da Universidade de Lisboa.

Desde esses primeiros passos já decorreram 12 anos.

Ao Professor Silva Carvalho e à Professora Isabel Rocha do Instituto de Fisiologia

pelo constante e indispensável apoio na cedência de espaço do biotério bem como

da colaboração preciosa de todas as pessoas que nele trabalham.

I also would like to thank Lori, for the English review of this thesis, suggestions, good

humor and friendship.

E finalmente, à minha família: aos meus pais por me terem incentivado a continuar e

a apostar na procura do conhecimento sabendo não ser muitas vezes o caminho

mais fácil; à minha irmã e ao Henrique por toda a paciência, boa disposição e apoio

incondicional.

À Lúcia, a minha filha, a quem dedico esta tese pelo seu sorriso e olhar que tantas

vezes foi o meu consolo e força para continuar em dias menos bons.

Um agradecimento às instituições que financiaram o trabalho desenvolvido. À

Fundação para a Ciência e a Tecnologia (FCT) da qual recebi uma bolsa de

doutoramento (SFRH/BD/12167/2003) e que financiou o trabalho de investigação

143

(POCTI/43634/99) bem como a Fundação Caloust Gulbenkian e à Regeneron que

gentilmente cedeu o BDNF.

145

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