Petar - University of Toronto
152
Direct Effects of Glucocorticoids and Serotonin on Developing Hippocampal Cells Petar Erdeljan A thesis submitted with the requirernents for the degree of Master of Science Graduate Department of Physiology University of Toronto O Copyright by Petar Erdeljan 2000
Transcript of Petar - University of Toronto
Direct Effects of Glucocorticoids and Serotonin on Developing
Hippocampal Cells
Petar Erdeljan
A thesis submitted with the requirernents for the degree of
Master of Science
Graduate Department of Physiology
University of Toronto
O Copyright by Petar Erdeljan 2000
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Direct Effects of Glucocorticoids and Serotonin on Developing
Hippocam pal Cells
Petar Erdeljan
MSc., 2000
Department of Physiology, University of Toronto
We hypothesized that dexamethasone. corticosterone/cortisol and
serotonin exposure wouid modify GR and MR mRNA expression within fetal
rnouse and fetal guinea-pig hippocampal ce11 cultures. In mouse cultures. 4 day
exposure to dexamethasone or corticosterone (IOnM or 100nM) down-regulated
levels of GR mRNA within neurons and exposure to serotonin (100nM)
significantly up-regulated expression of GR rnRNA. MR mRNA levels were
unaffected by any of the treatments. Dexamethasone, corticosterone or serotonin
exposure did not alter expression of GR mRNA within glial cells. In guinea pig
cultures, 4 day exposure to dexamethasone or cortisol (1 00nM) down-regulated
levels of GR mRNA within neurons and exposure to serotonin (100nM)
significantly up-regulated expression of GR mRNA. Finally, multidrug resistance
(MDRla) gene expression excludes dexamethasone from the brain. We found
that the MDRla gene is expressed in the hippocampus. dentate gynis. specific
thalamic nuclei and cortex of the fetal brain. Patterns of MDRla changed dunng
developrnent.
ACKNOWLEDGEMENTS
I whish to thank the following individuals:
Dr. Stephen G. Matthews: For being my first mentor in science. Through Our
friendship I became aware of new dimensions in thinking and approach to
science. For his supervision. guidance, support dumg my studies. i whish to
thank him for his patience and for allowing me to express my sometimes "radicaln
creativity .
Dr. John F. MacDonald: For allowing me to work in his laboratory and for his
expert scientific input into rny project.
Dr. Denise Belsham: For being a member of my supetvisory committee and for
her suggestions and advice.
Elrzbieta Czewinska: For her
this study.
would have
Li Liu and
Without her expert
been impossible.
help in developing the culture system utilized in
advice, suggestions and patience this project
Lucy McCabe, who ensured cuntinuous support of experimental
animais used in this study.
Rania Lingas and Joanne Kotsopoulos who provided friendly and very
enjoyable atmosphere in the lab.
My parents for their endless support and encouragement.
CHAPTER 3
Regulation of GR and MR mRNA during Development: Mouse
Studies. ............................................................................................. 34
3.1 Introduction .................................................................................... 34
..................................................................................... 3.2 Objectives 35
3.3 Hypothesis ..................................................................................... 36
3.4 Methods ....................................................................................... -36
3.4.1 Mouse Hippocampal Culture ............................................................ 36
3.4.2 In situ Hybridization ....................................................................... 38
3.4.3 Oligonucleotide Probes ................................................................... 38
3.4.4 Probe Labeling ............................................................................. 38
.............................................................................. 3.4.5 Hybridization -39
3.4.6 Washing ...................................................................................... 40
3.4.7 Emulsions ................................................................................... 40
3.4.8 Developing and Staining Emulsions .................................................. 41 . *
3.4.9 Cell Viability ................................................................................. 41
3.5 Data analysis .................................................................................. 42
.......................................................................................... 3.6 Results 43
..................................................................................... 3.7 Discussion 45
CHAPTER 4
Reguiation of GR and MR mRNA During Development: Guinea Pig Studies.
Gestational Age 40 Days ............................................~....................... ..61
Introduction .................................................................................... 61
Objectives .................................................................................... -63 Hypothesis .................................................................................... -63
Methods ....................................................................................... -64
Development of Fetal Guinea-pig Culture ........................................... 64
Mouse Cerebellar Culture ............................................................... 65
Guinea-pig Hippocampal Culture, Gestational Day 40 .......................... 65
4.4.4 In situ Hybridization ....................................................................... 68
.................................................................. 4.4.5 Oligonucleotide Probes 68
............................................................................... 4.4.6 Hybridization -68
...................... 4.4.7 Washing, Emulsions. Developing and Staining Emulsions 68
............................................................................... 4.5 Data Analysis 69
......................................................................................... 4.6 ResuIts -70
.................................................................................... 4.7 Discussion -71
CHAPTER 5
Regulation of GR and MR mRNA During Development: Guinea Pig Studies.
Gestational Age 50 Dayç: .........................~......~.......~...m...~..~.........~.... **a3
.................................................................................... Introduction 83
.................................................................................... Objectives -83
.................................................................................... Hypothesis -84
........................................................................................ Methods 85
Development of Fetal Guinea-pig Culture system ................................. 85
Mouse cerebellar culture on inserts ....................... ,... ................... 85
Guinea-pig hippocampal culture. gestational day 50 ......................... ... . 86 ...................................................................... In situ hybridization -86
.................................................................................. 5.5 Data analysis 86
.......................................................................................... 5.6 Results 87
..................................................................................... 5.7 Discussion 88
CHAPTER 6: Multi drug resistance studies ...................................m.......m 95
.................................................................................... 6.1 Introduction 95
.................................................................................... 6.2 Objectives -96
..................................................................................... 6.3 Hypothesis 96
....................................................................................... 6.4 Methods -97
............................................ ......... ............... 6.4.1 Animal Work ,. ,,. 97
6.4.2 Tissue Preparation and Sectioning ................... .... .......................... 97
6.4.3 ln situ Hybridizaüon ...................................................................... 98
........................................................... 6.5 Autoradiography and Analysis -98
.......................................................................................... 6.6 Results 99
................................................................................... 6.7 Discussion 701
CHAPTER 7: Summaw and Conclusions ........m..............m.m.....m......~...... 114
APPENDlX 1: Culture solutions ..............m........m.........................m........ 118
APPENDIX 2: Derivation of treatments for mouse hippocampal cultures.120
.......................................... APPENDIX 3: Molecular biology solutions A22
REFERENCESmemm*eeem..... ............................................................. ..l28
LIST OF FIGURES
Figure 3.la: Mouse hippocampal neurons in vitro and . . ................................................... autoradiographic images.. .51
Figure 3.1 b: Silver emulsion grains over mouse hippocampal neurons.. ......... 53
Figure 3.2a: Analysis of GR mRNA in the mouse neurons following
................................................. dexamethasone treatment.. .55
Figure 3.2b: Analysis of MR mRNA in the mouse neurons following
dexamethasone treatment.. ................................................. .55 Figure 3.3a: Analysis of GR mRNA in the mouse neurons following
corticosterone treatment, .................................................... .57
Figure 3.3b: Analysis of MR mRNA in the mouse neurons following
corticosterone treatment. ..................................................... .57
Figure 3.4a: Analysis of GR mRNA in the mouse neurons following
serotonin treatrnent,. ......................................................... .59 Figure 3.4b: Analysis of MR mRNA in the mouse neurons following
.......................................................... Serotonin treatment.. .59
Figure 4.la: Analysis of GR mRNA in the guinea-pig neurons following
corticosterone treatment, .................................................... .80
Figure 4.1 b: Analysis of MR mRNA in the guinea-pig neurons following
corticosterone treatment.. ................................................... .80
Figure 4.2: Analysis of GR mRNA in the guinea-pig neurons following
dexamethasone treatmenf.. ............................,.................... .82
Figure 4.3: Analysis of GR mRNA in the guinea pig neurons following
serotonin treatment.. .......................................................... .82
Figure Sala: Analysis of GR mRNA in the guinea-pig neurons (gd50) following
dexamethasone exposure.. ................................................. -92 Figure 5.l b: Analysis of MR mRNA in the guinea-pig neurons (gd50) following
dexamethasone exposure.. ................................................ -92 Figure 5.2a: Analysis of GR mRNA in the guinea-pig neurons (gd50) foliowing
serotonin exposure.. .......................... .. ......................... ..94
Figure 5m2b: Analysis of MR mRNA in the guinea-pig neurons (gd50) following
Figure 6.1:
Figure 6.2:
Figure 6.3:
Figure 6.4:
Figure 6.5:
Figure 6.6:
Figure 6.7:
Figure 6.8:
Figure 6.9:
Table 1 :
serotonin exposure.. ........................................................... 94
Color-enhanced image of MDRI a in coronal section of a fetal
............................................................ guinea-pig brain.. -1 05
Images of coronal sections that underwent silver emulsion
............................................................. autoradiography. .IO5
MDRla mRNA levels within the CAIICA.2 region of the
................................................................ hippocampus.. -1 07
MDRla mRNA levels within the CA3 region of the
Hippocampus.. ............................................................... -1 07
MDRl a mRNA levels within the CA4 region of the
................................................................ hippocampus.. .l 09
MDRl a mRNA levels within the dentate gyms.. ....................... 109
MDRl a mRNA levels within the cortex.. ............................... .l Il M DR? a mRNA levels within the thalamic nuclei ...................... . I l 1
MDRla mRNA levels in the limbic regions of fetal guinea-pig
following dexamethasone exposure.. .................................. .Il 3
Effects of dexarnethasone, corticosterone and serotonin on GR
mRNA expression in non-neuronal cells.. .............................. .60
CHAPTER 1 : Introduction
1 .el Overview
Fetal glucocorticoid exposure or prenatal stress can permanently modify
the activity of the h ypothaiamic-pituitary-adrenai (HPA) axis. This modification or
"programmingn is at least in part mediated by an alteration in expression of
corticosteroid receptor genes in the brain reg ions involved in g l ucocorticoid
feedback (Matthews, 2000). Evidence from animal models indicated that the
hippocampus, a key locus for glucocorticoid feedback control, is central to the
process of programming (Jacobson and Sapolsky, 1991).
Permanent modification of the HPA axis is associated with alterations in
basal and stress-induced glucocorticoid secretion. Studies in rats demonstrated
that elevation of circulating glucocorücoid is linked to a decrease in hippocarnpal
glucocorticoid receptor levels, following prenatal dexamethasone (a synthetic
glucocorticoid) administration (Levitt et al., 1996). lncreases in the basal and
stress-induced levels of g lucocorticoid were also reported in the rhesus mon key
following prenatal synthetic glucocorticoid administration (Uno et al. 1994). Such
treatment is known to result in degeneration and depletion of pyramidal neuron
cells and granular neurons in the hippocampus, as well as in an overall reduction
in the hippocampal size and segmental volume (Uno et al. 1990). Unfortunately,
studies done in rhesus monkeys did not address potential changes in
corticosteroid receptor levels within the hippocarnpus.
Chronic increases in circulating glucocorticoid have been associated with
increased susceptibility to pathologies such as depression, hypertension,
immunosupression and diabetes (Baxter et al., 1987; Munck et al., 1984;
Sapolsky, 1994). Development of hypertension is strongly associated with
prenatal alteration of HPA function, at least in the rat model. In the rat, prenatal
dexamethasone treatment results in the adult offspring having elevated blood
pressure (BenediMsson et al., 1993). At present, the precise mechanisms
underlying the development of hypertension are unknown, as is the link between
hypertension, elevated glucocorticoids and reduction in the hippocampal
glucocorticoid receptor levels.
At present, 10% of al1 women, who are at nsk of pre-terni delivery are
treated with synthetic glucocorticoids. Synthetic glucocorticoid therapy is
administered in order to promote organ maturation and thus avoid complications
associated wit h pre-term delivery, such as respiratory distress syndrome,
intraventrîcular hemorrhage and necrotizing colitis (NIH consensus development
conference, 1995). Synthetic glucocorticoid therapy is used extensively and
often in a repeated fashion (French, et al. 1997). However, long-terrn effects of
synthetic glucocorticoid therapy on brain development (in the human) are not
known. To date, there is little information on the possible direct effects of
glucocorticoid exposure on expression of corticosteroid receptors in fetal
hippocampal neurons. The primary purpose of this study was to examine this
issue.
1.2 Stress and the Hypothalarno-Pituitary-Adrenal (HPA) axis
The brain can interpret a wide range of stress-related inputs into a general
class of neuroendocrine and autonomic responses designed to allow the
organisrn to cope with stress. The hypothalamic-pituitary-adrenal (HPA) systern
represents a principal component of the stress response. whose primary role is
to allow and to control secretion of glucocorticoids (GCs) by the adrenal cortex.
Glucocorticoid actions are diverse and wiil be considered in detail later.
The primary role of the stress response (particulariy glucocorticoids) is to
facilitate adaptation to stress and to restore homeostasis. Neurons in the
paraventrîcular nucleus (PVN) of the anterior hypothalamus are essential to the
stress response. Neurons within the PVN synthesize and secrete corticotrophin
release hormone (CRH), its CO-secretagogue vasopressin (VP) as well as other
neuropeptides that drive the activity of the sympatho-medullary systems. The
sympatho-medullary and HPA system act in a CO-ordinated fashion and exert
reciprocal control over the other activity (De Kloet, et al., 1998).
Upon stimulation from higher or lower brain centers, specialized
neurosecretory cells in the PVN release CRH, VP and oxytocin (Whitnall, 1993;
Plotsky, 1989). These hypophysiotrophic factors are released from nerve
teminals in the zona extema of the median eminence into the primary plexus of
the hypophyseal portal system (Antoni, 1986; Plotsky, 1987). Primary plexus
capillaries merge into the long portal vessels that lead to the infundibular stalk of
the pituitary gland (Ambrach et al., 1976). The released hypophysiotrophic
factors stimulate the synthesis and release of adrenocorticotrophin releasing
hormone (ACTH) from corücotroph cells in the anterior pituitary (Plotsky, 1987).
ACTH is foned from proopiomelanocortin (POMC) which is processed into
ACTHi-39, pendorphin, p-lipotrophin within corticotroph cells. In intermediate
pituitary melanotroph cells, a-MSH, CLIP and acetylated P-endorphin are primary
products of POMC processing (Eipper and Mains, 1980). ACTH, once released,
enters the systemic circulation and acts on adrenocortical cells to initiate
synthesis and secretion of glucocorticoids (Plotsky, 1989).
Release of glucocorticoids allows the organism to cope with, adapt to, and
recover from stress. Traditionally, glucocorticoids have been thought to act as a
prirnary defensive mechanism, responsible for initiation of physiological actions
needed for immediate survival. In addition, glucocorticoids also have the potential
to initiate a number of adaptive responses requiring genomic activation. lnside
the cell, glucocorticoids bind to high affinity cytosolic receptor and act via
genomic mechanisms to modiFy function (Munck, et al. 1984; Bamberger, et al.
1996). However, it has also been suggested that glucocorticoids may also act on
a membrane bound receptor and in this way rapidly alter cellular events (Brann,
et al. 1995; Bamberger, et al. 1996).
The actions of glucocorticoids display two modes. In the "proactive" mode
glucocorticoids maintain basal activity of the H PA system and control the
sensitivity of the systems to stress. Glucocorticoids promote CO-ordination of
circadian events, such as food intake and sleepiwake cycle, and are involved in
processes underlying selective attention, integration of sensory information and
response selection. In "reactiven mode glucocorticoid feedback teminates stress-
induced HPA activation (De Kloet. et al. 1998), preventing excessive
glucocorticoid exposure which is detrimental to health (Munck, et al. 1 984;
Munck, et al. 1992; Tausk, 1951). It has been known for several decades that
lack of wntrol over the stress response can increase vulnerability to disease
(Seyle, 1952).
1.3 Corticosteroid Receptors and the Hippocampus
Glucocorticoids act on the brain through glucocorticoid receptors (GRs)
and mineralocorticoid receptors (MRs) (McEwen et al, 1986; De Kloet, 1991).
GRs are present throughout the brain but are rnost abundant in hippocampus,
hypothalamic CRH neurons and pituitary corticotrophes. MRs are present at
hypothalamic sites involved in regulation of salt appetite and autonornic
regulation (Broody, 1980; McEwen, 1986; Gomez-Sanchez, 1997). However, the
highest concentration of MRs is detected within the hippocampus, a brain
structure involved in leaming and memory processes. In the hippocampus
corticosterone binds both MR and GR. This is in contrast to the situation in the
perip heral organs. In the kidney, M Rs selectively bind the mineralocorticoid
aldosterone despite the fact corticosterone can bind MRs with equal or greater
affmity than aldosterone and that its circulating concentration is up to 1000-times
greater than that of aldosterone (Seckl. 1997). In the hippocampus the
mineralocorticoid (ie aldosterone) selectivity for MR is lost (Korozowski, 1983;
Reul, 1985; McEwen, 1968; De Kloet, 1975). The selectivity in the kidney is
controlled by the enzyme 1 1 P-hydroxysteroid dehydrogenase, type2 (1 1 p-HSD2).
This enzyme, which is present primarily in the placenta and kidney, deactivates
glucocorticoids by conversion to their inactive metabolites (Ajilore and Sapolsky,
1998). Steroid regulation in the brain is thought to involve 11P-HSDI. This more
ubiquitous isoform. is present in hypothalamus, hippocampus, neocortex and
subthalamus (Moisan, et al. 1992, Lakshmi, et al. 1991 ; Seckl, et al. 1991). 1 1 P-
HSD1 is bidirectional, and thus. it could either act as a dehydrogenase (to
deactivate glucocorticoids) or as a reductase (to regenerate glucocorticoids). At
present, it is believed that 11P-HSD1 acts primarily as a reductase (Ajilore and
Seckl, 1998), thus converting inactive metabolites to active corticosterone. This
speculation rernains somewhat controversial. In the hippocampus, MRs
(Kd-O.5nM) bind glucocorticoids with approximately 10-fold higher affinity than
GRs (Kd-5nM) (Korozowski, 1983; Reul, 1985; McEwen, 1968; De Kloet, 1975).
Under conditions of basal corticosterone plasma levels, MRs will be primarily
occupied, while GRs becorne occupied with increasing concentrations of
corticosterone in the plasma (De Kloet, et al. 1998). Hence, in the hippocampus,
glucocorticoids activate two signaling pathways via MRs and GRs (Reul, 1985).
Occupation of GRs and MRs will have profound effects on neuron activity, some
of which are described below.
Activation of MRs and GRs is associated with modification of neural firing.
Activation of MRs in CA1 hippocampal neurons is associated with small, voltage-
gated calcium currents. Activation of GRs is associated with a n'se in calcium
current amplitude (Karst et al., 1994). GR mediated effects were found to be
dependent on de novo protein synthesis (Kerr et al., 1992) and they affected both
basal and stimulus-induced intracellular calcium levels (Elliot and Sapolsky,
1 992; Elliot and Sapolsky. 1 993). Such alterations in intracellular calcium
homeostasis affected the activation of a slow calcium-dependent potassium-
channels (Storm. 1990). This hyperpolarizes neurons after prolonged firinig.
Hyperpolarization of neurons results in reduction of firing (accommodation) (De
Kloet, et al. 1998). Neural firing is also suppressed by afterhyperpolarization (De
Kloet, et al. 1998). Predominant MR activation was found to result in small
accommodation and after-hyperpolarization amplitude. Additional occupation of
GRs was found to increase accommodation and afterhyperpolarization amplitude
(Joels and De Kloet, 1990). In summary, CA1 hippocampal output to the PVN
neurons is effectively increased with MR activation (at basal corticosterone
levels) and it is reduced when both GR and MR are activated (at stress
corticosterone levels).
Glutamate-mediated responses are also affected by MR and GR
activation. Glutamate is the predominant transmitter molecule in the CA1 area of
the hippocampus. Glucowrticoid activation of MRs is associated with no change
in excitatory transmission, w hile glucocorticoid activation of G Rs is associated
with the depression of glutarnatergic transmission (Joels and Fernhout, 1993).
Additionally, slow inhibitory postsynaptic potentials. mediated by GABAb
receptors, remain unchanged when MRs are activated. GR activation is found to
suppiess these slow inhibitory potentials (De Kloet et al., 1998). Fast inhibitory
postsynaptic potentials are not greatly affected by changes in MWGR activation
(De Kloet et al., 1998). In summary, both excitatory and inhibitory information
rernains unchanged under MR activation. Additional GR activation under
elevated glucocorticoid levels reduces excitatory transmission and hence
negative CA1 hippocampal output to the PVN (De Kloet et al., 1998).
Activation of GRs and MRs also modifies actions of biogenic amines,
noradrenaline and serotonin, in the hippocarnpus. Slow calcium-dependent
potassium conductance is blocked by no rad renaline, via P-ad renergic receptor.
Inhibition of this curent results in increased excitability of the CA1 hippocampal
area (Madison and Nicoll, 1986). Activation of GRs is associated with a reduction
of noradrenaline effect, and hence a decrease in CA1 excitability (De Kloet et al.,
1998). Effects of MR activation are still unknown.
Serotonin has many different effects in the CA1 hippocampal area. One of
the most prominent effects of serotonin is hyperpolarization of the CA1 neurons.
This hyperpolarkation of the membrane is achieved through 5HTla receptors
(Andrade and Nicoll, 1997). Activation of MRs is associated with srnall
afterhyperpolan'zation, whereas additional GR activation increases serotonin-
induced hyperpolarization (Joels and De Kloet, 1 992). The inhibitory effect of
serotonin under conditions of GR activation results in a marked suppression of
excitatory transmission in the CA1 area (Hesen et al., 1998).
1.4 GR and MR Transactivation
lntracellular GRs and MRs are part of a cytoplasmic multiprotein complex.
This complex consists of one receptor molecule. hnro molecules of heat shock
protein (hsp) 90, one hsp70, one hsp56 and one immunophilin molecule (Smith
and Toft, 1993). Activation of the corticosteroid receptors results in rapid
dissociation of this molecular complex, phosphorylation of the receptor at multiple
sites and increased affinity of the ligand-activated receptor for nuclear domains
(Brink, et al. 1992). Activated corticosteroid receptor homodimerizes and
translocates to the nucleus where it interacts with specific DNA sequences or
other transcription factors (Barnberger. et al. 1996). Binding of GRs to a specific
DNA sequence represents the classic model of corticosteroid action. The
receptor homodimer binds to a glucocorticoid response element (GRE), which is
a short palindromic DNA sequence in the promoter region of the glucocorticoid
responsive genes. Once bound to the GRE. the GR homodimer interacts directly
with members of the basic transcription machinery, such as TFIIP. Indirect
interaction via bridging factors, such as steroid receptor coactivator 1 (SRCI), is
also possible. Recent evidence indicates that GRs can act by recruiting multiple
wregulator proteins that may have specific functions during transcriptional
initiation. Activated receptors mobilize members of the SRC family, which is a
group of stnicturally and functionally related transcriptional coactivators.
Interaction with transcriptional cointegrators p300 and CPB is also implicated in
the GR activation. P300 and CPB are proposed to integrate diverse afferent
signals at hormone-regulated promotors (McKenna et al. 1999). Other nuclear
receptor coactivators include BRGI (it couples ATP-hydrolysis to chromatin
rernodeling) and the E3 ubiquitin-protein ligases E6AP and RPFI. Any of the
mentioned interactions (direct or indirect) are sufficient to stabilize the pre-
initiation complex on the promoter and, therefore, enhance transcription by RNA
polymerase II (Barnberger, et al. 1996). In addition, the activated GR can also
bind to negative glucocorticoid response elements. Genes that do not contain
GREs can also be affected by GR activation. One way in which this can be
accomplished by the GR-mediated regulation of transcription factors. These two
mechanisms will be considered in detail. later.
Rapid, short duration effects of GCs are thought to be mediated by
signaling via a membrane bound glucocorticoid receptor (Hua and Chen, 1989).
At present, such a receptor has not been isolated or characterized. though its
existence has been demonstrated indirectly (Hua and Chen, 1989). Within 2
minutes of their application. glucocorticoids can h yperpolarize the membrane
potential of hippocampal neurons in brain slices, and coeliac ganglion neurons.
Bovine aibumin-glucocorticoid conjugates, which cannot enter the cell, produce
the same effect (Hua and Chen, 1989). Considering this finding and the rapidity
of effect, existence of membrane bound receptor is likely.
1.5 The Hippocampus and Regulation of HPA Function
Considerable evidence indicates that the hippocampus inhibits HPA
activity. lnhibitory hippocampal GABA-ergic projections to the hypothalamus
were identified and characterized (Nauta. 1956; Mason. 1958; Herman, 1996).
Electrical stimulation of hippocampal sub-fields CA1 (unilateral) and subiculum in
humans resulted in reduction of 47-hydroxy corticoids (cortisol metabolites) in the
plasma (Rubin et al, 1966). Electrical stimulation of hippocampal sub-fields CA3,
CA4, the dentate gyrus and subiculum significantly reduced stress-stimulated
plasma corticosterone levels (Dunn and Or , 1984). Taken together these studies
suggest that the hippocampus is involved in tonic inhibition of HPA function.
Experiments utilizing hippocampectomy or hippocampal neuron
destruction also support an inhibitory role of the hippocampus. Destruction of
hippocamapal neurons (>50%) by sterotaxic kainate infusion increased plasma
corticosterone levels during restraint stress when compared to control. in rats
(Sapolsky et al. 1984). Total hippocampectomy of the dorsal hippocampus in the
rat resulted in a 4-fold increase in CRH mRNA expression in the PVN relative to
sham-operated animais (Herman et al, 1989). This study confined that the
inhibitory effects of the hippocampus on pituitary adrenocortical function are
mediated via the hypothalamus. Furthemore, hippocampectomy in the rat leads
to an increase in basal and stress-induced levels of ACTH, when wmpared to
sham-controls (Wilson et al, 1980). This finding also confimis that the
hippocampus in hi bits ACTH release. Finally, partial hippocampectomy (only
dorsal but not ventral), in the rat reduced the inhibitory effect of dexamethasone
on basal and stress-induced adrenocortical response (Feldman et al, 1 980). This
study suggests that dorsal hippocampal formation participates in feedback
regulation of the HPA function.
The hippocampus influences the release of adrenaline from brainstem
catecholaminergic nuclei, which innervate the parvocellular field of the PVN
(Meibach, 1977; Swanson, 1977, 1981). This finding suggests that the
hippocampus indirectly infiuences the neurosecretory activity of parvocellular
neurons which directly control ACTH release (Swanson, 1977, 1981 ).
The role of hippocarnpal MRs and GRs in HPA regulation was elegantly
demonstrated by a series of experiments utilizing selective receptor antagonists
in adrenally intact rats. lntraventricular administration of MR antagonist was
demonstrated to increase basal levels of plasma corticosterone as well as stress-
induced levels of corticosterone (Ratka et al, 1989). This effect was observed
both in the moming and the aftemoon phase (in rats, highest levels of
corticosterone are observed in the afternoon phase and lowest corticosterone
levels are observed in the moming phase). Similar observations were made in
the human. Systemic administration of spironolactone (MR antagonist) was found
to increase basal HPA activity (Dodt et al, 1993), though this response was not
noted in al1 studies (Michelson et al.. 1994). Together the data would suggest
that hippocampal MRs mediate the effect of corticosterone in maintaining the
tone of basal HPA activity.
lntraventricular administration of RU38486 to rats (GR antagonist) had no
effect on basal levels of corticosterone in the rnoming phase (Ratka et al, 1989).
lntraventncular RU38486 administration in the aftemoon phase increased HPA
activity. In contrast, administration of the same antagonist directly into the
hippocampus decreased ACTH levels in the aftemoon phase (Van Haarst et al.,
1997). These contradictory findings suggest that hippocampal GRs are prïmarily
involved in decreasing the inhibitory hippocampal outfiow to the PVN (De Kloet et
al, 1998). These findings also indicate that extra-hippocampal GRs, expressed in
PVN neurons, suppress the HPA activity.
In sumrnary, emerging evidence suggests that GR and MR have opposite
roles, with respect to HPA control, at the level of the hippocampus. This
correlates well with the data underiying MR and GR activation, which was
introduced previously. In this scenario. predominant MR activation maintains
hippocampal excitability, and inhibitory polysynaptic projections to the PVN, in
tum, suppress basal HPA activity. However activation of GRs suppresses the
hippocampal output and results in disinhibition of PVN neurons and in increase of
the HPA activity (De Kloet. et al. 1998). The hippocampal input seems to be
overridden at the level of PVN (at elevated corticosterone levels). This is a
relatively new hypothesis and it requires further testing.
1.6 Programming of the HPA Axis by Excess Glucocorticoids
Glucocorticoids are critical for normal brain developrnent, as they exert a
wide range of effects in most regions of the growing brain. Their presence is
necessary for many processes ranging from subcellular reorganization to neuron-
neuron and neuronglia interaction (Gould. 1994; Gould et al, 1991). Predictably,
their absence or prolonged elevation in the fetal brain is detrimental to normal
development and can permanently modty the structure and function of the brain
(Oppenheimer et al. 1997; Bohn. 1984).
It is well established that HPA function can be permanently programmed
during development (Levine, 1957). Available evidence in animal models
indicates that prenatal exposure to glucocorticoids can lead to offspring with
altered HPA activity. In the rat, fetal exposure to synthetic glucocorticoid,
dexamethasone (O.lrnglkg), during the last week of gestation resulted in adult
offspring with elevated basal plasma corticosterone levels (Levitt et al, 1997).
Dexamethasone exposed offspring were also found to have increased blood
pressure. Similarty, dexamethasone treatment (0.05rngIkg) on days I ï , l 8 and 19
of pregnancy resulted in adult offspring which had increased stress-induced
corticosterone levels (Muneoka et al, 1997). Treatment (0.4mglkg) on days 17
and 19 alone was not sufficient to alter HPA function in offspring. However, the
same animals were found to have a difference in the ratio of AVP and CRH in the
extemal zone of the median eminence, suggesting subtle long-term effects on
HPA control (Bakker et al, 1995). These studies demonstrate that glucocorticoids
can program the HPA axis, and that the nature of such programming is
dependent on dose and timing of exposure.
In the guinea-pig, fetal dexamethasone exposure (1 mglkg) on days 50
and 51 of gestation (75% of gestation) resulted in male offspnng (neonate) with
elevated (2-fold) basal plasma cortisol concentrations (Dean et al, 1999).
Similarly, fetal dexamethasone exposure (1 mglkg) on days 40 and 41, 50 and
51, 60 and 61, resulted in fernale offspring (adult) with elevated basal plasma
cortisol concentrations. However, the sarne treatment produced male offspring
with reduced basal plasma cortisol concentrations indicating a sex-specific
influence of exogenous glucocorticoids (Matthews, et al. 2000). In rhesus
monkeys, daily administration of dexamethasone (4 x 1.25mglkg) starting on
132d of gestation resulted in the young offspring with significantly elevated basal
and stress-induced levels of cortisol (Uno et al, 1994). These three studies
indicate that HPA function can be permanently altered in species that give birth
to neuroanatomically mature young (guinea-pigs and rhesus monkeys), a
situation similar to the human. To date, no studies have examined HPA function
in humans that were exposed to antenatal glucocorticoids during fetal life.
lncreased HPA activity throughout life will elevate tissue exposure to
g lucocorticoids. In humans, elevated tissue exposure to cortisol has been
associated with increased arteriosclerosis, cholesterol concentrations, incidence
of diabetes, immunosupression and cognitive impairment (Lupien et al, 1997;
Brindley et al, 1989; Munck et al. 1994). Similar effects of permanently altered
HPA function have been described in animal studies (Ader et al, 1973). In
contrast, long-ten reduction in HPA activity could have a protective effect
against cortisol tissue over-exposure effects.
HPA programming occurs, at least partially, at the level of the
hippocampus. The hippocampus is very sensitive to endogenous and exogenous
gluwcoricoids during development (Takahashi, 1998; Weinstock, 1997; De Kloet
et al, 1998; Matthews. 2000).
1.6.1 Programming of Hippocampal GRs and MRs by Excess
Glucocorticoid
The hippocampus wntains the highest levels of corticosteroid receptor
observed in any brain region (McEwen et al, 1968; Grlach et al, 1976; Reul et al,
1985; Sutanto and De Kloet. 1987; Matthews, 1998). It has been postulated that
the hippocampus exerts its input to the HPA axis primarily by changing the
sensitivity of glucocorticoid negative feedback (Motta et al, 1970; Jacobson et al,
1991). In support of this staternent, it has been recorded that the loss of
hippocampal corticosteroid receptors disrupts the sensitivity of the HPA axis to
glucocorticoid feed back in hi bition (Sapolsky et al, 1986; Levitt, et al. 1996).
At basal plasma glucocorticoid concentration hippocarnpal MR is occupied
(at approximately 80%) while the GR remains largely unoccupied. This is due to
the higher affinity of the MR for glucocorticoid (De Kloet et al, 1998). However.
during stress-induced elevation of glucocorticoid the GRs become increasingly
occupied (Dallman et al, 1994). Based on these findings it has been postulated
that the MR is primarily involved in feedback regulation during basal secretion of
stress hormone, while the GR is involved in regulation durhg increased
glucocorticoid secretion (Dallman et al, 1994). According to such a postulate, GR
activation is necessary for an increase in inhibitory hippocampal output to the
PVN, which is required to restore the HPA axis to basal state. However, as
described eariier, this view has been recently challenged. According, to De Kloet
and colleagues, GR activation is linked with an increase in HPA activity by
removal of inhibitory output from the hippocampus to the PVN. Under conditions
of GR activation hippocampal neurons were found to be less excitable, their
afterhyperpolarization state longer and their firing frequency lower (De Kloet et al,
1998), al1 of which promote a decrease in hippocampal output. However, this
remains to be tested further. Based on either theory, one can predict that
permanent change in the hippocampal corticosteroid receptor complement wiil
profoundly affect hippocampal inhibitory output to the PVN.
Evidence for a permanent change in corticosteroid receptor complement,
within the hippocarnpus, is provided by animal studies of fetal dexamethasone
exposure. In the rat, daily dexamethasone administration during the last week of
pregnancy reduced concentrations of both GR and MR mRNA in the
hippocampus (Levitt et al, 1996). There was no difference in GR mRNA or MR
mRNA expression in the hypothalamus or in brainstem structures. This reduction
in corticosteroid receptor mRNA was associated with an elevation of basal HPA
act ivity.
Studies undertaken in the guinea pig revealed that prenatal
dexamethasone exposure, during the phase of rapid brain growth (gestational
days 50 and 51 ), results in offspring with an altered hippocarnpal corticoseroid
receptor complement (Dean et al, 2000). Male offspring neonates were found to
have increased GR mRNA levels in the cingulate cortex and hippocampal sub-
field CA3. In wntrast, fernale neonates were found to have decreased GR mRNA
complement in the hippocarnpal sub-fields CA3 and CA4, dentate gyrus and
cingulate cortex (Dean et al, 2000). There was no effect of prenatal
dexamethasone exposure on GR mRNA concentrations in the hypothalamus and
anterior pituitary in either sex. Elevation of GR mRNA levels within the
hippocampus, in the male neonates, suggest an increase in inhibition of
hippocarnpal neurons, and thus, a decrease in the inhibitory tone to the PVN (De
Kloet et al, 1998). This correlates well with the increase in male basal plasma
cortisol concentrations. In the females. the situation is not as clear, as decreases
in the hippocampal GR mRNA did not result in a decrease in basal plasma
cortisol concentrations. Fetal guinea-pig glucocorticoid exposure on gestational
days 40 and 41, 50 and 51, 60 and 61, also results in alteration of hippocarnpal
corticosteroid receptor complement, in adult offspring (Matthews, et al. 2000).
Female adult animals (80 days) were found to have decreased MR mRNA levels
throughout the hippocampus. There were no differences in GR mRNA levels in
the hippocampus, but GR mRNA levels were decreased in the PVN and pituitary.
Male adult animals showed no difference in either GR mRNA or MR mRNA, at
the level of the hippocampus, PVN or pituitary (Matthews, et al. 2000). In adult
females, the decrease in MR mRNA suggests a decrease in inhibitory tone from
the hippocampus. Decreases in GR mRNA at the level of the PVN and the
pituitary suggest a decrease in negative feedback at these sites. 60th of these
speculations correlate well with the increase in the HPA function reported in
female adult animals (Matthews, et al. 2000). The situation in the adult males,
which were reported to have decreased HPA activity. is more difficult to
speculate upon. It is possible that GC exposure in utero has an impact on some
other brain site involved in the HPA regulation or affects other parameters rather
than the GRfMR mRNA expression.
Very little is known about the mechanisms that underlie long-terni
alteration of corticosteroid receptor densities. It is possible that glucocorticoids
can influence the pattern of the GR and MR developrnent at the time of
exposure. In the rat, GR mRNA can be detected in the hippocampus by
gestational day 13 and MR mRNA by gestational day 16 (Cintra et al, 1993; Diaz
et al, 1998). In general, bath GR and MR levels in the rat brain are low
throughout gestation and then increase rapidly after birth (Bohn et al, 1994;
Rosenfeld et al, 1993; Meaney et al, 1985; van Eekelen et al, 1991). In the fetal
guinea-pig, both GR mRNA and MR mRNA can be detected in the hippocampus
by gestational day 40 (Matthews, 1998). Between gestational days 40 and 50,
GR mRNA levels increase dramatically, whereas MR mRNA levels decrease. GR
mRNA levels continue to increase towards terrn (approximately 70 days) while
MR mRNA levels remain low (Matthews, 1998). As one can see, GR and MR
receptor systems develop independently in a tirne-specific manner. thus the
outcome of glucocorticoid exposure may differ depending on timing of exposure.
1.6.2 Mechanisms of Autoregulation
Although animal models clearly demonstrate that developing populations
of GR and MR in the hippocampus can be altered, very little is known about the
mechanisrns by which alteration occurs. One possible mechanism is
autoregulation (glucocorticoids acting on GR and MR genes to change their
expression). Autoregulation of GR was detected in in vitro studies that
demonstrated a reduction in GR in cultured cells after prolonged glucocorticoid
exposure (Cidlowski and Cidlowski, 1981; Svec and Rudis, 1981; Bloomfield,
1981). Dexamethasone administration to HeLa cells resulted in decreased GR
protein levels, as established by tritiated glucocorticoid binding. GR mRNA levels
were also decreased, suggesting a decreased rate of transcription (Cidlowski
and Cidlowski, 1981; Svec and Rudis, 1981). Down-regulation of GR rnRNA
levels in HeLa cells was found to be permanent. This down-regulation was
attributed to a conformational change in the GR promoter (Silva et al, 1994).
Dexamethasone treatment of cells transfected with human GR cDNA produced a
decrease in both GR mRNA and protein. This effect was reported both in
transiently transfected cells (Cos 1, African green monkey kidney cells) and
stably transfected cells (Chinese hamster ovary cells). These studies
demonstrated that human GR cDNA contains sequences critical for homologous
down-reg dation (Bumstein et al, 1 99 1 ).
Elevated concentrations of glucocorticoid within the brain can reduce
corticosteroid receptor expression. The precise mechanism by which such
reduction occurs is not yet known, but several possibilities were postulated
(Bamberger et al., 1996). It is possible that regulation may occur by influencing:
a) intracellular hormone availability, b) interactions between receptor and heat
shock protein, c) receptor phosphorylation, d) nuclear translocation andtor DNA
binding-site modification (kmberger, 1996). lntracellular hormone availability is
regulated by two enzymes, 11 P-hydroxysteroid dehydrogenase (1 1 p-HSD,
isoform 1) and ligand effect modulator 1 (LEMI). II P-HSD metabolizes cortisol
and thus, makes cells cortisol-unresponsive. LEM 1 actively and specifically
exports glucocorticoids from the cell, limiting the glucocorticoid availability (Kralli,
et al. 1995).
Heat shock proteins (hsp) form complexes with glucocorticoid receptorç.
Ligand binding to the complex causes conformational change in the GR, and its
dissociation from hsp molecules. This process can be stirnulated or inhibited by
endogenous regulators, such as a heat-stable protein known as "stimulatof
(Schmidt, et al. 1985). The stimulator enhances the efficiency of GRlhsp
dissociation. The GRlhsp complex can also be stabilized. The modulator, a
phosphoglyceride molecule, binds to both GR and hsp90, thus stabilizing the
inactive GRJhsp wmplex and therefore negatively regulating GR activation
(Robertson. et al. 1995).
The importance of phosphorylation in the regulation of GR activity is not
well understood.
dissociation from
serine residues (H
GR becomes phosphorylated at multiple sites shortly after
the hsp complex. The phosphorylation occurs primarily on
u, et al. 1994). A possible role of phosphorylation in regulation
of GR activation was derived from studies undertaken in rat fibroblast cells. Such
studies reported increases in GR mediated transcription. during progression from
normal to the neoplastic ceil stage, without the increase in the intracellular GR
levels (Vivanco, et al. 1995). The observed increase in the GR mediated
transcription was attributed to changes in receptor phosphorylztion (Vivanco, et
al. 1995). However, studies undertaken in the mouse, reported that selective
mutations of one, multiple or al1 phosphorylation sites on the GR only modestly
decreased transcriptional activity of the receptor (Mason. et al. 1993). Similar
results were reported for the human GR as well (Almlof, et al. 1995). It is possible
that phosphorylation has a role in intracellular localization of the GR, rather than
its transcriptional activity (Bamberger. et al. 1996).
lntracellular distribution of GR is affected by hsp content and receptor
phosphorylation. Translocation of the GR to the nucleus accelerates after
receptor phosphorylation. It is possible that massive phosphorylation results in
unmasking of a nuclear signal (Akner, et al. 1994). Hsp molecules are also
actively involved in GR trafficking. Inhibition of hsp56 by FK506 was found to
increase GR-mediated transcription rate by stimulating nuclear transfer of the
receptor (Sanchez, et al. 1994).
Various nuclear factors can influence the binding of activated GR
complexes to DNA. Binding of GR to DNA can occur within specific GRE, other
specific DNA sequences or naked DNA (Bamberger, et al. 1996). Low molecular
radius (700-3000 Da) factor, isolated from rat hepatoma cells and present in
extracts f o m human HeLa cells, is responsible for about 40% of activated GR
binding to calf thymus DNA. in vitro (Cavanaugh, et al. 1994). At present, it is not
known why only some GRs require this factor for binding. Another nuclear factor
involved in GWDNA binding is ATP-stimulated glucocorticoid-receptor
translocation promoter (ASTP). ASTP is a 93kDa histone-binding protein that
increases GR binding to chromatin in the presence of ATP. Helicase proteins,
such as yeast brm proteins or their human homologues, facilitate binding of GR
to nucleosomal DNA (Bamberger, et al. 1996).
Nuclear factors can also have an inhibitory effect on the GR-mediated
transcription. Purification of 'Yranslocationn inhibitor from rat liver demonstrated
the existence of a factor that inhibits binding of activated GR to rat nuclei
(Dahmer, et al. 1985). Similarly, pyridoxal phosphate, the active fom of vitamin
Be, was cismonstrated to inhibit activated GR binding to the DNA. As a result of
this in hibition, GC-induced transactivation decreased by a bout 50% (Allgood, et
al. 1993). Conversely, inhibition of pyridoxal phosphate was found enhance the
transcriptional effects of glucocorticoids. Cell-specific glucocorticoid sensitivity is
at least partially regulated by pyridoxal phosphate, as levels of this rnolecule Vary
profoundly between different cell types (Allgood. et al. 1993).
At present. the precise involvement of g lucocorticoids in modification of
these mechanisms is unknown. However, it is recognized that glucocorticoids
have the potential to regulate their activity (Bamberger, et al. 1996).
1.6.3 Serotonin and Programming of Corticosteroid Receptors
Serotonin was shown to regulate GR binding levels in primary
hippocarnpal cultures derived frorn fetal rat brain (Mitchell et al, 1990). Incubation
of hippocampal cultures with serotonin increased GR binding to ligand. This
effect was mediated, at least partially, through 5HT2 receptors. Increased GR
binding was found to be long-term as it persisted for 60 days without any
add itional se rot on in exposu re. U nfortu nately, ce1 1-specificity of se rot onin
responses was not addressed in this study. An increase in GR binding has also
been demonstrated in adult rats that were handled as neonates (Anisman et al,
1998; Meaney et al. 1994). Handling exposes the neonates to brief periods of
mild hypothermia. that is associated with thyroid hormone release (Meaney et al,
1994; Mitchell et al, 1990). The increase in GR binding due to neonatal handling
was mimicked by thyroid hormone administration to the neonates (Meaney et al,
1987). Subsequent studies demonstrated that both neonatal handling and thyroid
hormone administration produced an increase in hippocampal serotonin turnover
(Mitchell et al, 1990). As a result. it was proposed that thyroid hormones
stimulate ascending serotoninergic neurons (originating within the Raphe
nucleus) increasing hippocampal serotonin release and therefore increasing GR
expression (Meaney et al, 1994). Gluwcorticoids were found to influence both
serotonergic systems and thyroid hormone turnover. Prenatal glucocorticoid
exposure is known to increase expression of the serotonin transporter system in
the rat brain (Slotkin et al, 1996) suggesting an advance in the activity of the
serotonergic system. In the fetal guinea-pig. dexamethasone exposure was
found to increase both fetal thyroid hormone and GR mRNA levels (Dean and
Matthews, 1999). Taken together these findings suggest that prenatal
glucocorticoids advance maturation of serotonin system, which could mediate
increases in hippocampal GR mRNA complement.
1.7 Multi Drug Resistance
Studies described in this thesis address the direct mechanisms by which
serotonin and glucocorticoids influence corticosteroid receptors in the developing
hippocampus. Very recently it has been suggested that exposure of the brain to
synthetic glucocorticoids is dependent on the expression of rnulti drug resistance
genes within the brain (De Kloet et al., 1998). To date there is no information as
to whether these proteins are present in the developing brain.
Both the synthetic glucocorticoid dexamethasone and the endogenous
glucocorticoid corticosterone (in rats) were found to be substrates of P-
glycoprotein. P-glycoproteins exclude a large number of seemingly unrelated
substrates from the cytoplasrn and are responsible for the phenornenon of multi-
drug resistance. Since p-glycoproteins can exclude dexamethasone from the cell
they can protect against actions of dexamethasone. As a result P-glycoproteins
could be involved in the programming of the HPA axis by synthetic
glucocorticoids (De Kloet, et al. 1998). The protective role of 1 P-glycoprotein
against dexamethasone penetration into the brain has been clearly demonstrated
in adult animais (Meijer et al, 1998). Given this evidence and considering the fact
that one of the primary purposes of this thesis was to examine effects of
dexarnethasone on corticosteroid receptor expression, investigation of P-
glycoprotein mRNA expression in the developing brain became a logical
expansion of this study.
The mammalian P-glycoproteins (P-gps) were initially isolated from cancer
cells that were found to be resistant to rnany foms of anti-cancer therapy.
(Sharom, 1997). Closer investigation of anti-cancer therapy resistance led to
discovery of multi drug resistance syndrome (MDR) and identification of P-gps as
primary candidates for mediating MDR. P-gps confer drug resistance through
active. ATP-dependent extrusion of a wide range of cytotoxic drugs. Dnig
extrusion happens against the concentration gradient and is independent of an
electrochemical transmernbrane potential or hydrogen ion gradient (Ruetz et al,
1994). Further investigation of P-gps generated a rather large list of seemingly
unrelated as well as similar substrates (Sharom. 1997). Such findings
characterized P-gps as unusual transporters whose actions can be both harmful,
as in MDR, and beneficial, as in their protective role against penetration by many
xenobiotic compounds. Human P-gps are encoded by two distinct genes:
MDR1 and MDR3. Rodent P-gps (at least in mouse in rat, other rodent species
are poorly described) are enwded by three distinct genes: mdrla. rndrl b and
mdR. Human MDRI , and mouse mdrl a and mdrl b genes confer multi drug
resistance. These genes are often referred to as mdrl-type P-gps or class I .
Human MDR3 and mouse mdR do not confer multi drug resistance (Chen et al,
1986; Gross et al, 1988).
1.7.1 Structure and Substrates
P-glycoprotein is a 170 kDa plasma membrane glycoprotein. It belongs to
the ABC superfamily of proteins. which are ATP-dependent transporters.
Structural analysis of P-gps revealed the presence of two membrane-bound
domains, each consisting of six trans-membrane segments (a-helices), and two
ATP cytosolic domains (Sharom 1997). Domains are arranged in toroidai
configuration, when viewed from a bove the membrane, with six-fold symmetry.
Pgp has a diameter of -1Onm with a central pore -5nm in diameter, as reported
by electron microscopy study (Rosenberg et al, 1997). Twelve transmembrane
segments are arranged in two symmetncal halves, with helices six and twelve
being in close proximity (Loo et al, 1996). P-glycoprotein has a large number of
substrates. The largest group of MDR substrates are the numerous anti-cancer
drugs. These include Vhca alkaloids, taxanes, anthracyclines and
epipodophylllotoxins (Sharom 1997). Another large group of MDR substrates
includes other cytotoxic agents, linear and cyclic peptides and certain steroids
(Sharom 1 997). Typical MDR substrates are molecules with molecular weight
greater than 400KDa. that are highly hydrophobic and amphipathic. Typical
substrates usually have planar ring system and often cany a positive charge at
physiological pH. However, many other MDR su bstrates differ from this
description, as they are found to be hydrophilic, linear and uncharged at
physiological pH (Eythan et al, 1994; Loe et al, 1994; Sarkadi et al, 1994;
Sharma et al, 1992; Sharom 1995; Sharom et al, 1996).
1.7.2 Physiological Role of P-gp
A physiological role of P-gp has been pursued since its discovery. Several
studies indicated a function in transport of lipids and steroids. Mouse Pgp class
II, as well as hurnan Pgp class I are highly expressed in the adrenal gland.
Mouse P-gp class I I (also designated mdrl b) is also hig hly expressed in the
pregnant uterus. Human P-gp is found in human placental trophoblasts and
secretory endometrium (Thiebaut et al, 1987; Croop et al, 1989). Such high
expression of Pgp in reproductive and hormone producing organs suggested its
role in reproduction and hormone production. This idea was also supported by
the finding that Pgp could transport hormones cortisol, corticosterone and
aldosterone (Ueda et al, 1992; Bourgeois et al, 1993). On basis of such findings
it was proposed that P-gp wuld play a role in rapid secretion of hormones or
even in protection of hormone producing cells against the high intracelluiar
hormone accumulation. However, such speculations remain controversial.
The greatest insight into the physiological role of P-gp was offered with
creation of MDRla knockout mouse, as well as mouse deficient for al1 of the
MDR genes. Examination of the MDR knockout mouse (-Io) revealed that they
differ from the wild type only in their sensitivity to xenobiotic compounds,
especially those that target central nervous system (Schinkel et al, 1997). All
other aspects of physiological function were found to be identical (Schinkel et al,
1997). It was established that P-gp class I (mdrla) is not essential to basic liver,
kidney or intestinal function, as well as to the functions of the brain, adrenal
gland, ovaries and pregnant uterus. In addition, hematopoietic stem cells ar;d the
tiematological compartment in general are also unaffected by absence of P-gp
class 1. Taken together these findings suggest that function of P-gp class I is
either taken over by some related transporter or that it simply plays no significant
role in these physiological functions (Schinkel et al, 1997).
MDR knockout mice show greatly enhanced susceptibility to a range of
xenobiotic compounds such as: ivermectin. vinblastine, digoxin, cyclosporin A,
loperamide, domperidone and ondanestron (Schinkel et al, 1996). lncreased
penetration of these compounds into the central nervous systern is pronounced.
Exposure of MDR knockouts to ivermectin, a neurotoxic pesticide is lethal.
lverrnectin levels inside the brain of knockout mice were found to be 100 times
higher when compared to wild type (Schinkel, 1995). Sirnilariy, a subpopulation
of CF-1 mice, that do not express mdrla, showed 100% susceptibility to cleft
palate syndrome, when exposed to L-652,280 (the 8,9 Z photoisorner of the
naturally occumng avermectin 1 B) (Lankas et al, 1998). This cornpound is known
to induce cleft palate formation. Heterozygous mice, P-gp (+/-), showed
decreased susceptibility and wild type mice. which express mdrla fully, were
found to be completely insensitive to L-652,280, at the doses tested. The same
study discovered that the degree of chernical exposure of fetuses was inversely
related to the expression of placenta1 P-gp. Such a finding cleariy dernonstrated
the protecüve role of rndrla against potential teratogens (Lankas et al, 1998).
Dexamethasone, a synthetic glucocorticoid used clinically, is also a substrate for
P-gp. It's penetration into the brain of knockout rnice is three fold higher then in
wild type. P-gp could hence offer protection against actions of dexamethasone in
the developing brain (Schinkel et al, 1995).
1.7.3 Expression of MDR
Expression of each MDR gene is highly tissue specific. Mdrl a is highly
expressed in the blood-brain barrier, blood-testes barrier, placenta and intestinal
epithelium (Nakamura et al, 1 997). Mdrl b is highly expressed in the adrenal
gland, pregnant uterus. and ovaries. 80th genes are also expressed in an
overiapping fashion in the liver, kidney, lung and heart tissue
Schinkel et al, 1994).
Expression of MDR has also been examined in
(Croop et al, 1989;
the human fetus.
Expression of MDRI mRNA was observed as eariy as embryonic week 7 (Van
Kalken, 1992). The definitive zone of fetal adrenal gland showed expression of
MDR1 which increased throughout gestation. There was no detectable
expression of MDRl wlhin the fetal zone of fetal adrenal gland. Fetal intestine
showed no expression in eariy gestation, but expression was increased in late
pregnancy. Expression was also detected in main bronchi and pharynx, which do
not express MDRI in adults. Expression of MDRI within kidney and liver was
detectable at eleven weeks of gestation. Expression of MDRl within the brain
capillaries was not observable before the third trimester (Van Kalken. 1992).
Recent studies indicated that expression of MDR could be induced or
suppressed by its substrates. Exposure of Caco-2 cells to verapamil, celiprolol
and vinblastine increased P-gp expression when compared to control. In
contrast, exposure to metkephamid decreased P-gp expression when compared
to control (Andrele et al, 1998). This finding is certainly in agreement with the
development of MDR in tumor cells after exposure to cytotoxins. It would be
interesting to see whether P-gp induction can be observed in normal tissues as
well, after exposure to a substrate.
Chapter 2: Rationale and Objectives
2.1 Clinical Importance
Up to 10% of pregnant women in North America, at risk of preten
delivery. are treated with synthetic glucocorticoids. This is done to prornote fetal
organ maturation (NI H, 1 995). Synthetic glucocorticoid treatment is often
implemented in early gestation and in some cases throughout gestation. At
present, very little is known about the impact of such treatment on brain and
neuroendocrine developrnent (Matthews. 2000).
Epidemiological studies in humans have associated an adverse
intrauterine environment with subsequent development of coronary heart
disease, hypertension, hyperlipidemia and non-insulin dependent diabetes
mellitus (reviewed in Baker, 1991). In addition, sustained fetal exposure to
elevated levels of endogenous glucocorticoids was associated with low birth
weight, developmental delays and neurological disorders (Field, et al. 1985;
Ward. 1991 ; Clernent, et al. 1992). Emerging evidence indicates that alteration in
development of the fetal HPA axis may represent a link between fetal
environment and the development of the adult disease.
2.2 Rationale
Animal studies demonstrated that increased fetal exposure to
glucocorticoids can lead to permanent alteration in HPA function. The alteration
in HPA function was associated, to a great extent, with changes in hippocampal
corticosteroid receptor expression (Levitt, et al. 1996; Dodic, et al. 1 997; Dean, et
al. 2000; Matthews, et al. 2000). The alteration in HPA function was linked to
development of hypertension in adult animal (Levitt, et al. 1996). At present,
there is little information on the direct effects of glucocorticoid exposure on
expression of corticosteroid receptors in fetal hippocam pal neurons. The main
purpose of this study was to examine this question.
Expression of multi drug resistance genes in adult animals was found to
protect against dexamethasone exposure (Meijer, et al. 1997). It has been
suggested that the expression of multi drug resistance genes in fetuses could
have the same effect. Since multi drug resistance gene expression limits cellular
exposure to dexamethasone it probably diminishes the ability of dexamethasone
to change corticosteroid receptor expression. To date there is no information as
to whether these genes are expressed in the developing brain. Therefore,
examination of the multi drug resistance gene expression in the developing brain
was a necessary step in an atternpt to describe the role of muiti drug resistance
genes in the HPA programrning.
2.2 Objectives
The first objective in the mouse study was to set up a mouse hippocampal
culture system that would allow efficient in situ hybridization analysis of GR and
MR mRNA expression levels. The second objective was to examine direct effects
of dexamethasone, corticosterone and serotonin on the expression of GR and
MR mRNA in cultured fetal hippocampal neurons, in vitro (Chapter 3).
Examination of this question in the mouse was necessary in order to compliment
the existing evidence of HPA programming, in species with a postnatal profile of
neuroendocrine developrnent (in species where maturation of neuroendocrine
systems occurs primarily after the birth).
The primary objective of this thesis was to examine direct effects of
dexamethasone, cortisol and serotonin on the expression of GR and MR mRNA
in fetal guinea-pig neurons, in vitro (Chapters 4 and 5). Guinea-pigs have a
prenatal profile of neuroendocrine development (in this species maturation of
neuroendocrine systems occurs primarily prior to birth) and thus, resemble the
human more closely than rodent species such as the rat and mouse.
Examination of GR and MR mRNA expression was carried out at different stages
of developrnent.
The primary objective of the multi drug resistance study (Chapter 6) was
to examine the expression of MDRla gene in the guinea-pig brain throughout
development. The second objective of this study was to determine whether brief
periods (2 days) of dexamethasone exposure in fetal life alters the expression of
the MDRl a gene.
Chapter 3
Cel l Specific Regulation and mRNA
Expression in Mouse Hippocampal Culture
3.1 Introduction
The hippocampus plays a central role in regulation of the hypothalamo-
pituitary-adrenal (HPA) axis (Heman, et al. 1996; Jacobson, et al. 1991). High
levels of glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) are
present within the hippocampus and extensive evidence indicates that
glucocortwid feedback of the HPA axis occurs at this level (De Kloet, et al.
1998).
It is well established that HPA function can be programmed during
development (Levine, et al. 1957). Such programming occurs, at least partially, at
the level of the hippocampus (Levine, et al. 1957; Bakker, et al. 1995; Liu, et al.
1997; Plotsky, et al. 1993; Matthews, 2000). Administration of synthetic
glucocortiwid (dexamethasone) to pregnant rats results in adult offspnng that
exhibit elevated basal plasma corticosterone concentrations. This has been
associated with reduced levels of GR and MR mRNA in the hippocampus (Levitt,
et al. 1996). The mechanism and time course by which GR and MR complement
are pemanently altered remains unknown. Recent studies in the guinea pig
indicate that prenatal dexamethasone exposure has acute effects on
hippocampal GR and MR mRNA levels, in fetuses and neonates (Dean, et al.
1999). However, it is unclear whether glucocorticoids act directly at the
hippocampal receptor or at other sites in the brain to influence GR and MR
Ievels.
Serotonin is known to be involved in maturation and regulation of the HPA
axis (Dinan, 1996). It is well established that serotonin can regulate GR
expression and binding, both in vivo and in vitro (Mitchell, et al. 1990; Mitchel, et
al. 1990). Administration of serotonin to rat hippocampal neuronç, in vitro, causes
long-ten increases in GR expression as well as binding (Mitchel, et al. 1990).
However, it remains unclear whether this change occurs in neurons andlor glia.
3.2 Objectives
In this study, we investigated the direct effects of endogenous
glucocorticoids (corticosterone) and synthetic glucocorticoids (dexamethasone)
as well as serotonin on GR and MR mRNA levels within mouse hippocampal
neurons and glia, in vitro. We utilized a technique that allowed us to examine GR
mRNA and MR mRNA expression in a cell-specific manner.
3.3 Hypothesis
We hypothesize that exposure to dexamethasone will down regulate GR
mRNA expression in fetal mouse hippocampal neurons. MR mRNA expression
will not be affected by the dexamethasone exposure.
Corticosterone treatment will down regulate both GR and MR mRNA
expression in fetal mouse hippocampal neurons.
Exposure to serotonin will up regulate GR mRNA expression in
hippocampal neurons. MR mRNA expression will not be affected by exposure to
serotonin.
These speculations are based on the fact that dexamethasone binds
specifically to the GR with minimal biniding to the MR. In contrast, corticosterone
binds to both GR and MR. and thus, has the potential to influence expression of
both GR and MR mRNA. Effects of serotanin on the expression of GR and MR
mRNA are based on previous findings in related studies.
3.4 Methods
3.4.1 Mouse hippocampal culture
Accurately time-dated pregnant CD1 mice were obtained from
Charles River Laboratories (St-Constant, Quebec, Canada). All animal
experiments were carried out according to protocols approved by the Animal
Care Cornmittee at the University of Toronto, in accordance with the Canadian
Council for Animal Care. Animals were killed on day 18 of pregnancy by crevical
dislocation. Dead animals were placed on a paper towel previously soaked in
ethanol (70%) and sprayed with ethanol (70%). An abdominal incision was made
and skin removed. Another incision was made to the underlying connective
tissue and muscle tissue in order to allow removal of fetuses. Care was taken not
to puncture intestinal organs.
Fetuses were placed into a petri dish (60mm) and transfened to a tissue
culture hood. Fetal membranes were dissected and fetuses removed to fresh
petri dish (60mrn) containing dissection medium (see Appendix 1). Fetuses were
decapitated and fetal skin and dural membranes were removed. Fetal skulls were
cut along the sagital suture and brains were isolated and placed into a fresh petri
dish (60mm). containing dissection medium. Under a dissecting microscope, fetal
brains were separated into hemispheres. This was done without cutting, by
pushing the hemispheres apart. Hippocampi from each hemisphere were
removed using pointed forceps and a scalpel blade (No.10). All hippocampi were
transferred to a clean petri dish (35mm) containing dissection medium.
Dissection medium was replaced with 1.5mI trypsin\EDTA (see Append ix
1). The hippocampi were incubated for 15 min at 37°C. The trypsinlEDTA
solution was removed and hippocampi were placed in a 15ml falcon tube
containing 1.5ml of MEM - HS, FBS (see Appendix 1). Hippocampi were further
dispersed by trituration in MEM-HS, FBS. Hippocampi were passed through a
pasteur pipette (10 times) and then through a flame-polished pasteur pipette (10
times). Dissociated hippocampal cells were suspended in MEM-HS, FBS (6 ml).
The cell suspension (50pL) was placed in each well containing MEM-HS, FBS
(250pL). A total of 8 wells per culture slide were prepared. 6 culture slides per
animal. The day of dissection was counted as day O (DO). On DO + 4 medium
was changed in each well. FUDR (20& 0.02M) was added to each well when
glial cells within the wells were found to be confluent. FUDR addition was
necessary in order to prevent glial cell overgrowth.
On DO+iO treatment was initiated. Treatment groups included 5HT,
corticosterone and dexamethasone. Each treatment was adrninistered at 1 nM,
1 OnM and 1 OOnM concentrations, respectively (see Appendix 2). Each slide had
two control wells, 2 X IOOnM wells, 2 X 10nM wells and 2 X InM wells, for each
treatment respectively. Each treatment was carried out in duplicate and at least
six different experiments were carried out per treatment. Treatment was repeated
for four days. at 24-hour intervals. On D0+14 cultures were fixed with 4%
parafomaldehyde (see Appendix 3) and stored under 95% ethanol, at 4"C, until
processing with in situ hybridization.
3.4.2 IN SITU HYBRlDlZATlON
All solutions used in this section were described in detail in Appendix 3.
3.4.3 Oligonucleotide Probes
The GR oligonucleotide probe used in rnouse cultures was complimentary
to bases 1021-1053 of the coding sequence of mouse GR mRNA (Danielsen et
al, 1986). The MR oligonucleotide probe used was complimentary to bases 2942-
2986 of human MR mRNA (Ariza et al, 1987). The anti-sense GR and MR
oligonucleotide probes were synthesized using an Applied Biosystems DNA
synthesizer (mode1 392).
3.4.4 Probe Labeling
Tailing buffer (5X, 2pL, Gibco BRL) was added to each reaction tube. The
oligonucleotide probe (1 PL, 1 UnglpL) was added directly to tailing buffer. 3 5 ~
deoxyadenosine-5'-a-thiotriphosphate (APL, 1300 Cümmol, NEN, Du Pont
Canada Inc.) was added to the reaction mixture. 35~-incorporation into
oligonucleotide probe was achieved with addition of terminal dioxynucleotydyl
transferase (1.5pL, Gibco BRL). Finally, DEPC-treated water (14pL) was added
to the mixture. Reagents were rnixed by gentle passage up and down the pipette.
The mixture was incubated in water bath for 1 h, at 37°C. EDTA (2pL, 0.5M) and
STE (28pL, 1.OM) buffers were added to stop the reaction. The separation of
bound and free probe was achieved by passing the reaction mixture through
sepadex spin columns ( ~ r o b e ~ u a n t ~ ~ G-50 Micro Columns, Amersham
Phamacia Biotech Inc.), which were centrifuged for 3min at 3000rpm.
Dithiolthreitol (2pL) was added to labeled probe to prevent disulfide bond
formation and probe degradation. The radioactivity of the final mixture was
measured using a p-scintillation counter.
3.4.5 Hybridization
Prior to application of labeled probes the plastic charnber sections and
silicone gaskets were removed from the giass slides, which-were allowed to dry
completely. Radio-labeled probe was added to hybridization buffer (Appendixd)
so that final activity in the buffer was 1200 cprn/pL. Each culture-slide was
covered with hybridization buffer (1 80pL). Culture-slides were then covered with
çteriie parafilm. Care was taken to expel air bubbles undemeath the parafilm.
Moisture levels in the incubation chamber were maintained with soak solution
(Appendix3). Slides were incubated overnight at 42OC.
Antisense probes were also incubated with rnouse brain cryo-sections (10
pm). Frozen mouse brain was coronally sectioned (12pm) using a cryostat
(JUNG CM3000, Leica instruments GrnbH, Nussloch. Germany). Sections were
thaw-mounted on poly-L-lysine wated glass microscopie slides. Sections were
stored at -80°C. Prior to hybridization the sections were fixed in 4%
parafomaldehyde solution (5rnin). Slides were washed in phosphate buffer
saline (PBS) (Wlmin) and dehydrated in 70% ethanol (5min) and 95% ethanol
(1 min).
3.4.6 Washing
Culture-slides were placed in 1XSSC solution and the parafilm was gently
rernoved. Slides were washed in lXSSC (room temperature, for 20min) and then
in 1XSSC for 30min at 55OC. Finally, slides were rinsed in IXSSC, 0.1 %XSSC,
ethanol (70%) and ethanol (95%), at room temperature.
3.4.7 Emulsions
Emulsion autoradiography was camed out in the dark room.
Radiosensitive photographic emulsion (Ilford Scientific, K.5) was allowed to rnelt
prior to use (42OC water bath). Emulsion solution was diluted to 50% with
ultrapure (millipore) water. Emulsion was placed in specially designed glass
beaker which aIlowed emulsion thickness to be constant across the slides.
Ernulsion-coated slides were allowed to dry in the dark for 1 h prior to their
storage at 4OC. Exposure time for MR mRNA was 8 weeks and for GR mRNA
was 10 weeks.
3.4.8 Developing and staining emulsions
Emulsion coated slides were placed in developer (Kodak D520) for 5 min
and then transferred into stop bath (Kodak) for 1 minute. Finally, slides were
moved into liquid fixative (Kodak) bath for a further 5 min. Slides were then
rinsed extensively in ultra water prier to counter staining.
Developed ernulsion-coated slides were counter stained with methylene blue
(0.01%). Methylene blue is a positively charged (cationic) dye which combines
strongly with negatively charged cellular constituents such as nucleic acids and
acidic polysaccharides. It is an excellent general dye used to stain cells and
increase their contrast so that they can be more easily seen in the bright-field
microscope (Brock, et al. 1994). Slides were processed in the following order:
PBS (2min), methylene blue (OBI%, made in PBS, 10-20s), ultrapure water
(20s), ethanol (50%, 2min), ethanol (70%, Gmin), ethanol (go%, 7min) and xylene
(3min). After xylene exposure each slide was mounted with mounting compound
(Tissue Tek). Slides were allowed to dry ovemight.
3.4.9 Cell Viability
Viability of cells in culture was determined by trypan blue staining. Mouse
cultures were prepared and treated as previously described. Treatment included
only the highest doses (100nM). After the treatment, trypan blue (04%, 20pL)
was added to each well of a culture-slide. Culture-slides were allowed to stand
for 2 minutes prior to counting.
3.5 Data analysis
Autoradiography revealed high levels of GR and MR mRNA in the
hippocampus as well as GR mRNA distributed in the areas of the brain (Fig.3.1).
This is consistent with the previously reported distribution of these receptors
(Matthews, 1998; Levitt, et al. 1996). A sense probe dernonstrated no
hybridization in an adjacent section known to contain GR and MR mRNA
(Fig.3.1).
Analysis of trypan blue stained cells was camed out at 50X magnification.
On average, up to 100 cells per well were examined for presence of trypan blue.
In total, less than 5% of cells were identified as trypan blue positive. To confirm
the validity of trypan blue test, cells were allowed to stand under the microscope
after the initial count was done. After approximately 10min drastic increase in the
number of trypan blue positive cells was observed, thus confirming the validity of
the test.
The laboratory has extensive experience in using liquid emulsion
autoradiography and great care was taken to ensure that emulsion thickness was
consistent across the slides. Care was also taken to avoid saturation of silver
emulsion, which leads to non-linearity of analysis. Analysis of GR mRNA and MR
mRNA in pyramidal neurons was camed out at 200X rnagnification. On average
30-80 grains were identified over positive neurons. Mean grain count per neuron
was calculated for each treatment (6 separate experiments per treatment).
Approximately 15 mRNA positive neurons were randornly sampled and analyzed
in each well (2 wellsltreatment/analysis). Positive neurons were selected on
basis of MR mRNA expression and cellular morphology. Within the CNS, MR
mRNA is expressed only in neurons (De Kloet et al., 1998). In addition selected
cells were found to have typical neuron cell morphology, such as the large
nucleus, large and visible nucleolus and multiple processes (Banker and Goslin,
1991). MR mRNA expressing cells were also found to retain more methylene
blue stain. In case of neurons that were analyzed for GR mRNA expression,
selection was based only on cellular morphology and methylene blue staining.
Random sampling was achieved by selecting neurons for analysis at 50X
magnification. At this magnification level silver emulsion grain expression
between neurons is not distinguishable, and thus selection is blind with respect to
silver emulsion grain expression. Grain counting was also performed in a blinded
fashion, with respect to treatment group. AH neurons within the view field were
included in analysis, even if grain expression in specific neuron was not detected.
Cell counting revealed approximately 100 neurons/well. Grain counting over
regions of confluent glial cells was undertaken using a cornputen'zed image
analysis system (Imaging Research, St. Catherines, Ontario, Canada)(Matthews,
et al. 1995; Matthews and Challis, 1995). Group data are presented as
MeansS.E.M. Statistical analysis was performed using Statistica (Release 5, 97
Edition, Oklahoma, USA). The effects of treatment on GR and MR mRNA were
determined using a one-way ANOVA followed by the Duncan's method of post-
hoc comparkon. Significance was set at pe0.05.
3.6 Results
Glucocorticoid receptor mRNA levels were high in the hippocarnpal
pyramidal neurons as well as in glia. High levels of mineralocortirnid receptor
mRNA were detected in the pyramidal neurons. with no expression in glial cells.
This distribution was consistent with that obsewed in whole brain sections
following hybridization with the same probes (Fig.3.la and b).
Dexamethasone exposure (1 0nM and 100nM) significantly ( ~ ~ 0 . 0 5 ) down-
regulated GR mRNA levels within neurons (Fig.3.2a). while GR mRNA levels
were no different between dexamethasone treatment (1 nM) and control wells. In
wntrast, dexamethasone treatment (100nM) had no significant effect on MR
mRNA levels (Fig.3.2b).
Treatment of hippocampal cultures with corticosterone at either 100nM or
1 OnM significantly (p<0.015) down-regulated GR mRNA expression (Fig.3.3a).
Exposure to corticosterone (InM) did not significantly alter GR mRNA levels.
Corticosterone administration (1 00nM) had no significant impact on MR mRNA
levels (fig.3.3b).
Serotonin exposure (1 00nM) resulted in significant (pc0 .Os) u p-regulation
of GR mRNA levels within neurons, where as exposure at lOnM and InM was
without effect (Fig .3.4a). MR mRNA expression was u naffected by serotonin
exposure (100nM) (Fig.3.4b). Analysis of glial cells revealed no significant effect
of any treatment (dexamethasone. corticosterone, serotonin) on GR mRNA
levels (Table 1 ).
Exposure to any of the treatments was found to have no effect on
proportion of GWMR mRNA positive to negative cells. Analysis of al1 treatments
revealed that more than 95% of examined cells were G W M R mRNA positive.
3.7 Discussion
In the present study we have demonstrated, for the first time, that
glucocorticoid exposure modifies GR mRNA levels in a cell-specific manner. Both
synt hetic glucocorticoid (dexamethasone) and endogenou s glucocorticoid
(corticosterone) decreased GR mRNA levels in hippocampal neurons but not
glial cells. We have also identified that se rot onin exposure (1 OOnM) increases
GR mRNA levels in neurons but not glial cells.
It has been previously demonstrated that fetal exposure to glucocorticoids
leads to long-term changes in HPA activity in rats, guinea pigs and primates
(Bakker, et al. 1995; Levitt, et al. 1996; Dean, et al. 1999; Muneoka, et al. 1997;
Uno, et al. 1990). Exposure of fetal rats to glucocorticoids over the last week of
gestation results in adult offspring with increased basal adrenocortical activity
and this was associated with increased blood pressure (Levitt, et al. 1996).
Analysis of prepubertal rat offspring following exposure to glucocorticoids on day
l7, l8 and 19 of pregnancy revealed increased corticosterone responses to
stress (Muneoka, et al. 1997). Elegant studies in the rat have demonstrated that
increased basal corticosterone in adult offspring, that had previously besn
exposed to glucocorticoids in utero, is associated with a reduction in
hippocampal GR and MR mRNA levels (Levitt, et al. 1996; Dean, et al. 1999). No
differences in corticosteroid receptor mRNA were noted in other brain regions.
Together these data indicate that the 'programmed' increase in HPA activity is
linked to a reduction in glucocorticoid feedback sensitivity. Results from the
present study would suggest that there are acute effects of synthetic
glucocorticoids on GR production in fetal hippocampal neurons but no direct
effect on MR populations. However, the relatively limited life span of mouse fetal
neurons in culture (approximately 21 days) makes it difficult to establish whether
the acute direct effects of glucocorticoids on GR synthetic processes are
permanent.
Changes in GR mRNA levels following synthetic glucocorticoid exposure
have been reporteci in other in vitro culture systems (Cidlowski, et al. 1 981 ; Svec,
and Rudis. 1981; Webster and Cidlowski. 1994). One such study demonstrated
that dexamethasone can perrnanently down-regulate expression of GR mRNA in
HeLa cells. This permanent down-regulation was associated with modification
(methylation) in the GR promoter (Silva, et al. 1994). It is possible that
glucocorticoids induce similar changes to the GR promotor in developing
neurons, though this rernains to be detenined.
The failure of glucocorticoids to modify MR mRNA levels in the present
study would impiy that the effects of glucocorticoid exposure on MR mRNA levels
demonstrated in adult rat offspring (Levitt, et al. 1996) occur through an indirect
mechanism. Dexamethasone binds mainly to GR (Kd-5nM) and has very low
afinity for the MR (Kd-5OnM) (Veldhius, et al. 1982). Therefore, it is perhaps not
surprising that dexamethasone was found to have direct effects on GR mRNA
levels but no effect on MR mRNA (at 1 and IOnM concentrations). However, it is
possible that dexamethasone activated MRs at 1 OOnM concentration. Absence of
effect on MR mRNA expression at IOOnM concentration irnplies that
dexamethasone dose not regulate MR mRNA expression directly. Corticosterone
has 10-fold higher affinity towards MR than GR (Rupprecht, et al. 1993; Anna,
etal. 1988; Evans and Ariza. 1989; Cato, et al. 1991). Hence, the absence of
any significant direct effect of corticosterone or dexamethasone exposure on MR
mRNA supports the suggestion that glucocorticoids exert their influence on MR
synthetic processes via indirect pathways.
It is accepted that prenatal programming of the HPA axis occurs, to a
large part, at the level of hippocampal GR and MR (Matthews, 2000). Both the
hippocampal GR and MR act to suppress HPA activity (Jacobson and Sapolsky,
1 991 ). Evidence indicates that the MR is involved in feed back regulation of basal
HPA function while the GR is important during periods of HPA activation (De
Kloet et al., 1998). If similar GR autoregulation, to that which we report in vitro,
occurs in vivo, then this may itself have very significant consequences for normal
hippocampal development. Longitudinal studies examining acute as well as long-
term effects of fetal glucocorticoid exposure are clearly warranted.
A role of serotonin in the regulation of GR development in primary
hippocampal cultures, derived frorn fetal rat brain, has previously been
described. Meaney and colleagues demonstrated that 4 days of serotonin
exposure was sufficient to pemanently increase GR binding. It has subsequently
been esta blis hed that this effect is mediated via high-affinity 5HT2 receptors
(Kd-5nM) (Mitchel, et al. 1990). However, it has remained unclear whether the
increase in GR levels occurred in neurons, glia or both. In the present study, we
clearly demonstrate that increases in GR mRNA following serotonin exposure are
neuron-specific. A possible functional limitation in this part of the study was the
low stability of 5HT in solution, as 5HT is known to oxidize within one to two
hours in solution (Mitchel et al., 1990). In the future this limitation can be
overcorne by use of specific 5HT2 receptor agonists, which are much more stable
in solution. Use of 5HT2 receptor agonists/antagonist in a sirnilar study would
also confirm the specificity of 5HT receptor involved in mediation of the response.
Meaney and colleag ues also elegantl y demonstrated that central
serotonergic systems were responsible for the increase in GR binding observed
in rats that were handled as neonates. Neonatal handling is associated with
thyroid hormone release (Mitchell. et al. 1990; Meaney, et al. 1994) and thyroid
hormone administration to neonatal rats increased hippocampal serotonin
turnover (Meaney, et al. 1987). However, thyroid hormone administration had no
effect on hippocampal GR or MR mRNA levels, in vitro (Meaney, et al. 1994). It
has subsequently been proposed that a thyroid-serotonin pathway plays an
integral role in regulating GR levels during development. Dexamethasone has
been found to promote maturation of serotonergic systems (Slotkin, et al. 1996),
and in the guinea pig, dexamethasone administration was found to induce an
acute increase in fetal thyroid hormone, as well as hippocampal GR mRNA levels
(Dean, et al. 1999). Based on these findings one can conclude that serotonin
plays an important role in the programming of hippocampal corticosteroid
receptors. Our findings certainly support such a view, and indicate a direct effect
of serotonin on hippocampal pyramidal neurons. Therefore, it appears that
glucocorticoids can act both to suppress and increase GR mRNA expression. By
acting directly on hippocarnpal pyramidal neurons glucocorticoids down-regulate
GR mRNA expression, as demonstrated by the present study. However,
glucocorticoids also have the potential to indirectly increase GR expression
within hippocampal pyramidal neurons through actions on thyroid hormone and
on maturation of the serotonergic system (Dean, et al. 1999; Slotkin, et al. 1996).
Therefore, it is likely that the outcome following glucocorticoid treatment depends
on the developmental stage of the central newous system at the time of
exposure. If serotonergic systems are immature at the time of glucocorticoid
exposure, one would predict a decrease in hippocampal GR complement.
However, if serotonergic systems are mature at the time of glucocorticoid
exposure, the autoregulatory effect (decrease in GR mRNA) may be ovemdden
by serotonergic ddve, resulting in a net increase in GR rnRNA complement.
In summary, our results indicate that exposure to glucocorticoids has a
direct cell-specific effect on GR mRNA expression in hippocampal neurons.
Furthenore, we have shown that exposure to serotonin increases the GR
mRNA complement within hippocampal neurons. We speculate that both
glucocorticoids and serotonin exert their influence on the developing
hippocampus, but that the net effect of such treatme~t is dependent on the
maturational stage of both systems, at the tirne of exposure.
Figure 3.1: A) Digitized image of GR mRNA and MR mRNA (8) in coronal sections of an adult
mouse brain following in situ hybridization. C) Fetal hippocampal neurons in culture. D)
Autoradiographic image of a coronal section incubated with a sense probe.
Figure 3.l b: Silver emulsion grains over rnouse hippocampal neurons. Black dots represent clusters of GR mRNA.
Figure 3.2
A) Analysis of GR mRNA levels following dexamethasone (0, 1. 10, 100nM) exposure (4 days, n=8). Results are expressed as meantS.E.M. *Indicates statistical difference (pc0.01) from control.
B) Analysis of MR mRNA levels following dexamethasone (O, 1, I O , 100nM) exposure (4 days. n=8). Results are expressed as m e a n ~ S .E.M.
Figure 3.2
O 1 10 100
Treatments (nM)
Treatments (nM)
Figure 3.3
A) Analysis of GR mRNA levels following corticosterone (0. 1 , 1 0, 100nM) exposure (4 days, n=8). Results are expressed as meanf S E M . 'Indicates statistical difference (pcO.01) from control.
B) Analysis of MR mRNA levels following corticosterone (0. 1, 10, 100nM) exposure (4 days, n=8). Results are expressed as meanfS.E.M.
Figure 3.3
O 1 10 1 O0
Treatment (nM)
0 1 O0
Treatrnent (nM)
Figure 3.4
A) Analysis of GR mRNA levels following serotonin (O, 1, 10, 100nM) exposure (4 days. n=8). Result: are expressed as rnean+S.E.M. *lndicates statistical difference (pc0.01) from control.
B) Analysis of MR mRNA levels following serotonin (O, 1, 10, 100nM) exposure (4 days, n=8). Result: are expressed as rnean2S.E.M.
Figure 3.4
O 10 1 O0
Treatment (nM)
Treatment (nM)
Table 1
Table 1. Analysis of GR mRNA levels in glial cells. following dexamethasone,
corticosterone and serotonin treatment (n=8). Dexamethasone, corticosterone
and serotonin were administered at 1 OOnM, 10nM and 1 nM concentrations.
Controls (OnM) received vehicle alone.
Treatments (n=8)
Corticosterone
Results are expressed as meankS.E.M number of silver emulsion grains per
4x1 oJ mm2.
Dexamethasone Serotonin
Treatment concentrations OnM
275.2k5.4 201.3k29.0 229.6k11.4
1 nM 250.019.7 192.7k32.1 228.7+1 0.6
10nM 255.0I8.6 189.3Q6.7 228.4t11.2
100nM 256.9k9.7 191.7k27.7 232.1 k7.4
Chapter 4
Regulation of GR and MR mRNA During Development:
Guinea-pig Studies, Gestationai Age 40 Days
4.1 Introduction
Long-terni modifications in hippocampal GR and MR levels have been
associated with permanent alteration of basal and stress-induced HPA function
(Bakker, et al. 1995; Jacobson. et al. 1991; Levitt, et al. 1996; Liu, et al. 1997;
Plotsky, et al. 1993). Studies undertaken in rats have dernonstrated that fetal
exposure to synthetic glucocorticoids can lead to offspring that express
decreased levels of GR and MR mRNA in the hippocampus and dentate gyrus.
These offspring also had elevated basal plasma corticosterone concentrations
and were found to display hypertension (Levitt, et al. 1996; Langley-Evans,
1997). Chronically elevated plasma glucocorticoid concentrations are associated
with increased susceptibility to hypertension, as well as irnmunosuppression and
diabetes (Baxter, et al. 1987; Munck, et al. 1984; Walker and Williams, 1992).
Studies undertaken in rats have generated important information. Unfortunately,
this information is sornewhat diff~cult to correlate to the situation in the human,
due to the postnatal profile of neuroendocrine maturation in the rat (Matthews.
2000).
Neuroendocrine maturation in the guinea-pig occurs during late fetal life
(Matthews, 1998), which is similar to the pattern of neuroendocrine development
in primates (Dobbing and Sands, 1973; Dobbing and Sands, 1979). Studies
undertaken in guinea-pigs demonstrated that fetal exposure to synthetic
glucocorticoids during critical periods of brain development. modifies the
developing GR and MR systems in the hippocampus. The alteration of GR and
MR expression within the hippocampus was highly sex-specific. In addition.
modifications at the hippocampal level were accompanied by altered basal
plasma cortisol levels. These alterations were also highly sex-specific (Dean and
Matthews. 1999; Matthews, et al. 2000). It is unclear whether glucocorticoids act
directly at the receptor or at other sites in the brain to influence GR and MR
levels.
Serotonin is known to be involved in maturation and regulation of the HPA
axis (Dinan, 1996). It is well established that serotonin can regulate GR
expression and binding, both in vivo and in vitro (Mitchell. et al. 1990; Mitchel, et
al. 1990). Administration of serotonin to rat hippocampal neurons, in vitro, causes
long-terni increaseç in GR expression as well as binding (Mitchel, et al. 1990).
However, it remains unclear whether this change occurs in neurons andlor glia.
Work described in the previous chapter demonstrated that changes in GR mRNA
were neuron specific.
In Chapter 2 we described glucocorticoid and serotonin-induced changes
in the glucocorticoid receptor mRNA in the mouse. In this chapter we
investigated similar questions but in the more advanced model.
4.2 Objectives
The objective of this study was to investigate the direct effects of
endogenous (cortisol) and synthetic glucocorticoid (dexamethasone) as well as
serotonin on GR and MR mRNA levels within fetal guinea-pig hippocampal
neurons, in vitro. Based on evidence from in vivo studies, it was decided to
investigate males and females separately. We used a technique that allowed us
to examine GR mRNA and MR mRNA expression in a cell-specific manner.
4.3 Hypothesis
We hypothesized that exposure to cortisol and dexamethasone will down
regulate GR mRNA expression in the fetal guinea-pig hippocampal neurons. We
expected that exposure to serotonin would up regulate GR mRNA expression in
hippocampal neurons. We hypothesized that exposure to cortisol will down
regulate MR mRNA expression, while dexamethasone and serotonin treatments
will not affect expression of MR mRNA within fetal hippocampal neurons.
Although corticosterone did not affect MR mRNA expression in the previous
study (Chapter 3), here we anticipated an effect due to species-specific
differences. Our hypothesis is based on the same rationale as described in
Chapter 3.
4.4 Methods
4.4.1 Development of Fetal Guinea-pig Culture System
At the onset of my studies, methods for producing cultures of hippocampal
neurons from fetal guinea-pigs were not developed. Therefore. the first step
towards establishing a working protocol involved modification of existing culture
methods, from mouse studies. Applying the mouse culture protocol to fetal
guinea-pig culturing proved to be unsatisfactory, as this did not produce viable
neurons. Further modification of the existing mouse culture protocol involved
modification of culture media, such that culture media contained human brain
extract N2 (Gibco, see Appendixl) and/or neme growth factor (mNGF,
Appendixl). However, combinations again failed to produce viable neurons.
Magistretti et al (1 996), successfully developed adult guinea-pig cortical neuron
culture using supplemental basic fibroblast growth factor (bFGF) . Addition of
bFGF to Our system was not possible due to the prohibitive high cost of bFGF
and the high volume of media required for our experiments.
Examination of fetal guinea-pig hippocarnpal cultures revealed that
supporting non-neural cells were absent from the preparations. Glial cells are
thought to be a source of neurotropic substances and, in practice, most protocols
for culturing CNS neurons depend on high ceIl densities and the rapid
proliferation of endogenous glia to support neuronal survival and development
(Banker and Goslin. 1991). In order to resolve this problem, we modified an
existing Ban ker culture technique (Cultu ring Nerve Cells, 1 991 , pz61 ) which
wmbined mouse cerebellar cells acting to condition the medium for the guinea-
pig hippocampal cells. The two-type culture system was achieved by seeding
cerebellar cells on the bottom of a petri dish (35mm), and placing guinea-pig cells
on glass cover-slips (25mm diameter, Appendixl ). Cover-slips were mounted
with small paraffin legs, which allowed the cover-slips to be elevated and out of
contact with cerebellar cells. This system produced the first viable fetal guinea-
pig hippocampal neurons in culture. The first group of hippocampal cultures,
derived from gestational day 40 fetuses was undertaken using this system.
4.4.2 Mouse Cerebellar Culture
Accurately time-dated CD1 mouse neonates were obtained from Charles
River Laboratories (St-Constant, Quebec, Canada). Six day old neonates were
euthanized by decapitation. Neonatal heads were disinfected in 70% ethanol. for
5 minutes. Neonatal brains were removed using standard dissection methods
(described in Chapter 2) and placed in dissection medium (see Appendix 1).
Neonatal cerebella were dissected from neonatal brains. At this stage of mouçe
development. cerebella were large enough so that dissection could be perfoned
without the dissection microscope. The cerebella were cut into small pieces.
Dissection medium was removed and replaced with trypsinlEDTA ( l x ) solution
(see Appendix 1) and tissue pieces were incubated for 15 minutes at 37°C.
TrypsinlEDTA solution was removed and tissue was placed in MEM (1.5ml)
containing fetal bovine serum (FBS) and home serum (HS) (see Appendix 1).
Tissue was further dissociated by trituration as described in Chapter 3.
Dissociated tissue was suspended in MEM containing FBS and HS (25ml).
Cell suspension (0.5ml) was then placed on each collagen-coated 35mm
petri dish containing MEM with FBS and HS (Iml). 50 petri disheç were
generated in total. Each cerebellar culture was allowed to develop for at least five
days prior to initiating guinea-pig culture. 24 hours pnor to initiating guinea-pig
culture, medium was removed from each cerebellar culture and replaced with
MEM containing N2 and mNGF (2.0ml, see Appendix 1).
4.4.3 Guinea Pig Hippocampal Culture, Gestational Day 40
Accurately time-dated pregnant guinea pigs were generated according to
established methods (Elvidge, 1972). Animals were sacrificed on day 40 of
gestation, by decapitation. Fetuses were rapidly removed and decapitated. Fetal
brains were isolated and placed on ice. Gender of fetuses was established by
visual identification of testes or ovaries, upon dissection of the lower abdomen.
Fetal brains from each litter were grouped according to sex and hippocampi
isolated from each brain. Each brain was positioned dorsally and the cerebellum
and frontal cortex were removed. A cut was made on each side of the brain,
between rnidbrain and the two hemispheres. Care was taken not to cut too
deeply, as this would damage the hippocampus. The midbrain was gently
pushed forward and hemispheres were flattened, allowing visualization of the
hippocampus. Each side of the hippocampus was dissected with careful attention
to rernove any non-hippocampal brain tissue. Each fetal hippocampus was
placed in dissection medium (see Appendix 1) and was cut into smaller pieces.
Dissection medium was replaced with trypsinIEDTA (lx). Tissue was incubated
at 37'C for 20 minutes. Trypsin EDTA was removed and tissue pieces placed in
15ml falwn tubes mntaining MEM-N2 (1.5ml, see appendixl). Tissue was
rnechanically processed with trituration, first passing it through a Pasteur pipette
(10 times) and then through flame-polished Pasteur pipette (10 tirnes).
Dissociated tissue was combined with MEM-N2 (1.5m1, final suspension volume
3ml). Cell suspension (120 PL) was placed on each poly-D-lysine-pretreated
cover slip and allowed to stand in the incubator (37'C) for 40 minutes. Cover
slips were transferred into petri dishes (35mrn) containing mouse cerebellar
cultures. The day of dissection was counted as day 0.
Twenty four hours after the dissection FUDR (200pL, see Appendix 1 ) was
added to each petri dish (as described in Chapter 3). Cultures were then allowed
to incubate for 4 days. On day four the medium was changed. On day six the
cover slips and medium from the culture dishes were transferred into separate
petri dishes (35mm), such that guinea-pig's hippocampal neurons remained in
cerebellar conditioned media but not in the same dishes as cerebellar cells.
Treatment was initiated on day 7. Treatment groups included
dexamethasone, corticosterone and serotonin. Each treatment group was
administered at InM, 10nM and 100nM concentrations, respectively (see
appendix2). Treatments were repeated for 4 days. 24 hours apart. 24 hours after
the last treatment, cultures were fixed with 4% parafomaldehyde (see
appendix3). Cultures were kept under ethanol (95%), at 4"C, until processing
with in situ hybridization.
4.4.4 IN SITU HYBRIDIZATION
All solutions used in this section were described in detail in Appendix3.
4.4.5 Oligonucleotide Probes
The GR oligonucleotide probe used in guinea pig cultures was
complimentary to bases 1-45 of guinea pig GR mRNA (Keightley et al, 1994).
The MR oligonucleotide probe was complimentary to bases 2942-2986 of human
MR mRNA (Ariza, et al. 1987). Probes were labeled with s~~ as described in
Chapter 3. 80th of the probes have been characterized previously (Matthews.
1998).
4.4.6 Hybridization
Radio-labeled probe was added to hybridization buffer (Appendix 3) so
that final activity in the buffer was 1000 cpmi;iL. Sterile parafilm slips (5cm X
15cm) were placed in incubation chamber. Hybridization buffer (100pL) was
dotted on parafilm. Cover-slips were placed on parafilm face (cells) down. Care
was taken to expel air bubbles undemeath the cover-slips. Moisture levels in the
incubation chamber were maintained with soak solution (Appendix 3). Cover-
slips were incubated overnight at 42OC.
4.4.7 Washing, Emulsions, Developing and Counter-staining Emulsions
Identical to those described in Chapter 3 (see Methods), with the following
exceptions:
1. Radiosensitive photographic emulsion used was l lford Scientific K.5D. This is
a pre-diluted form of the same emulsion.
2. Counter staining: PBS (2min), methylene blue (0.01%, made in PBS, Imin),
ultrapure water (20s), ethanol (50%, 1 min), ethanol (70%, 2min), ethanol
(95%, 1 min), xylene (3rnin).
4.5 Data analysis
Analysis of GR mRNA and MR mRNA in pyramidal neurons was carried
out at 200X magnification. On average 20-50 grains were identified over positive
neurons and mean grain count per neuron was calculated for each treatment (at
least 4 separate experiments per treatment). Approximately 15 mRNA positive
pyramidal neurons were randomly sampled and analyzed in each well (1
well/treatrnentlanalysis). Cell counting revealed approximately 50 neurons/well.
Non-neural cells were present at very low density or were absent due to the
nature of our culture technique. As a result it was not possible to investigate
mRNA levels in non-neuronal cells.
Group data for each sex are presented as MeanskS.E.M. Additionally,
data for both sexes were pooled and examined together. Data were statistically
analyzed using Statistica (Release 5, 97 Edition, Oklahoma, USA). The effects of
treatment and sex on GR and MR mRNA were deterrnined using a two-way
ANOVA followed by the Duncan's method of post-hoc comparison.
In the case of lower treatment concentrations (1 and 10nM, GR mRNA
analysis) our n value was too small for analysis to be statistically viable. In those
cases we report trends between data.
MR mRNA analysis was possible only on grouped data from cortisol
treated cells due to a low n value. Analysis of MR mRNA in response to
dexamethasone and serotonin, was not possible even after grouping data. due to
limited material. The reduced MR analysis resulted from extreme difficulty in
handling ernulsion covered wver slips in the dark-room. This led to a heavy loss
of cover slips in Our first (MR mRNA) experiment. Given the time frame of these
studies (1 5 months), it was not possible to repeat these treatments.
4.6 ResuIts
High levels of glucocorticoid receptor mRNA were detected in fetal
hippocampal neurons. Mineralocorticoid receptor mRNA levels were also high in
the fetal hippocampal neurons. Treatment of hippocarnpal cultures with cortisol,
at 1 OOnM, significantly (pc0.05) down-regulated GR mRNA expression in both
sexes (Fig. 4.1a). Exposure to 1 or lOnM did not alter GR mRNA when male and
female data was combined. There was no difference in MR mRNA between
cortisol treatrnent (100nM) and controls (fig.4.l b).
Dexamethasone exposure (100nM) significantly (pc0.05) down-regulated
GR mRNA levels within hippocampal neurons, in both males and females (Fig.
4.2). GR mRNA levels were not different between dexamethasone (1 and 10nM)
treatment groups and control. Analysis of MR mRNA was not possible due to
problems outlined above.
Serotonin exposure (1 00n M) resulted in significant (~~0.05) up-regulation
of GR mRNA levels within neurons (Fig.4.3). Analysis of MR rnRNA was not
possible due to low n value.
4.7 Discussion
The present study has revealed, for the first time, that glucocorticoid
exposure changes GR mRNA levels in neurons. Both synthetic glucocorticoid
(dexarnethasone) and endogenous glucocorticoid (cortisol) exposure (1 OOnM)
decreased GR mRNA levels in hippocampal neurons, removed from male and
female fetuses at d40 of gestation. Serotonin exposure (100nM) increased GR
mRNA levels in the hippocampal neurons in both sexes.
Previous studies in the guinea-pig have demonstrated that fetal exposure
to glucocorticoids leads to long-term changes in HPA function (Dean, et al. 2000;
Matthews, et al. 2000). Exposure of fetal guinea-pigs to dexamethasone on days
50 and 51 of gestation results in prepubertal male offspring with increased basal
adrenocortical activity (Dean, et al. 2000). The increase in basal adrenocortical
activity is associated with an increase in hippocampal GR mRNA levels (Dean, et
al. 2000). In addition, our laboratory recently demonstrated that fetal exposure to
dexamethasone on days 40, 41, 50, 51, 60 and 61 of gestation results in adult
female offspring with increased basal adrenocortical activity and adult male
offspring with decreased basal adrenocortical activity. Changes in adult female
offspring were associated with a decrease in hippocampal MR mRNA and a
decrease in PVN and pituitary GR mRNA (Matthews, et al. 2000). Taken together
with evidence from rat studies (Levitt, et al. 1996) these data suggest that the
"programming" of HPA activity is linked to a change in glucocorticoid feedback
sensitivity.
Results from the present study suggest that there are acute effects of
glucocorticoids on GR production in cultured fetal hippocampal neurons but no
direct effect on MR production. It is difficult to establish whether the acute effects
of glucocorticoids on GR synthetic processes are permanent. due to limited life
span of fetal neurons in culture.
Down-regulation of GR mRNA expression following synthetic
glucocorticoid exposure has been reported in other in vitro culture systems using
immortalized cell lines (Cidlowski. et al. 1981 ; Svec and Rudis, 1981; Webster
and Cidlowski. 1994). Permanent down regulation of GR mRNA levels in HeLa
cells was associated with modification in the GR prornoter (Silva, et al. 1994). It
rernains to be investigated whether the same mechanism is responsible for
down-regulation of GR mRNA in developing neurons.
Dean and Matthews (1 999) demonstrated that fetal dexamethasone
exposure in the guinea-pig. on days 50 and 51 of gestation. results in an
increase of hippocampal GR and MR mRNA levels in the fernale fetus, on day
52. The same study found no change in hippocampal GR and MR mRNA in the
male fetus. The acute effects of dexamethasone in that study differ frorn the
present findings. The reasons for this discrepancy are unknown at present.
However, it is possible that in in vivo studies dexamethasone also affects the
maturing serotonergic system, which is involved in the hippocampal GR
regulation. Dexamethasone has been found to promote maturation of
serotonergic systems (Slotkin, et al. 1996). ln the guinea-pig, dexamethasone
exposure was found to induce an acute increase in fetal thyroid hormone (Dean
and Matthews, 1999), which is known to increase hippocampal serotonin
turnover in the rat (Meaney, et al. 1987). If this hypothesis is correct then it
appears that direct effects of gluwwrticoids on the hippocampal neuron GR
mRNA levels are masked or ovemdden by the effects of increased serotonergic
activity. However, this remains to be investigated further. One possible way of
examining this issue would be to treat neurons with dexamethasone/serotonin
combination and analyze subsequent changes in GR mRNA. However, it is also
possible that glucocorticoids may affect other additional neurotransmitter
systems that modify GR compliment. It has been demonstrated that alterations in
hippocampal norepinephrine can mode@ corticosteroid receptor levels
(Barbazanges, et al. 1996). Slotkin and wlleagues demonstrated that
norepinephrine turnover in the cerebellum and forebrain is reduced following
dexamethasone exposure in utero (Slotkin, et al. 1992). Norepinephrine content
within the hippocampus and neocortex was also reduced following
dexamethasone exposure in ufero (Muneoka, et al. 1 997). Longitudinal studies
have shown that in utero glucocortiwid exposure results in premature maturation
of norepinephrine systems in the brain stem, forebrain and cerebellurn (Slotkin,
et al. 1992) and results in overexpression of norepinephrine transporter (Slotkin,
et al. 1991). It is clear that dexamethasone profoundly influences the
development of norepinephrine system. Unfortunately, the long-terni
consequences (on HPA function) of the norepinephrine system manipulation are
not known. Future studies examining long term-effects of norepinephrine system
on the HPA function are required. To conclude, it is possible that differences
observed in in vivo situation result from the norepinephrine system input.
Previous studies reported sex-differences in hippocampal GR and MR
mRNA levels, as a result of dexarnethasone treatrnent (Dean and Matthews,
1999; Dean, et al. 2000; Matthews, et al. 2000). It was suggested that male and
female HPA axis and corticosteroid receptor systems are at different stages of
development at the time of exposure, and thus, respond differently (Dean, et al.
2000). In the present study, sex-differences in the hippocampal GR mRNA, in
response to direct dexamethasone exposure, were not observed. GR mRNA
levels were found to decrease in response to dexamethasone exposure. in both
male and female derived hippocampal neurons. There are several possibilities
that may explain this observation. In the male fetal neurons, circulating
testosterone is converted to estrogen. This is mediated by the enzyme P450
aromatase (MacLusky, et al. 1987). Activity of P450 is believed to be responsible
for sexual differentiation observed in the hippocarnpus and CNS in general
(MacLusky, et al. 1987). The effects of estrogen in the developing CNS have
been suggested to be mediated by estrogen receptors (O'Keefe and Handa.
1990). Therefore, in the male fetal neurons, sex hormones and dexamethasone
could act in synchrony to modrfy GWMR levels within the hippocampal neuron.
Both sex hormones and glucocorticoids act through similar transcriptional
machinery (Barnberger, et al. 1996) and it is possible that they interact. It would
be interesting to conduct a study where hippocarnpal neurons in culture are
treated with combinations of both testosterone/dexamethasone or
estrogenldexamethasone. This would establish whether the observed differences
between in vivo and in vitro studies in the male fetuses, are due to the absence
of sex hormones in the latter system. The second possibility involves sex
hormone modification of certain systems, which like the serotonergic system,
influence the hippocampal GWMR populations. Presence of sex hormones could
increase or suppress the maturation of the neurotransmitter system in question.
Alteration in maturation could change the activity of this system, and therefore
influence development of hippocampal GR and MR populations. This remains to
be investigated. Finally. the observed differences between the two systems could
be explained by the differences in dexamethasone concentration. It is possible
that 100nM dexarnethasone applied in in vitro system is higher than
concentration of dexamethasone experienced at the hippocampus in in vivo
studies. Also, distribution of dexamethasone in in vivo studies could be affected
by the presence of P-glycoproteins within blood-brain bamer, which have the
potential to exclude dexamethasone from the brain (Meijer, et al. 1998).
The present study did not detect glucowrticoid-induced modifications in
hippocampal MR mRNA levels. This finding implies that the long-term effects of
glucowrticoid exposure demonstrated in female adult offspring (Matthews, et al.
2000) occurç through an indirect mechanism. Corticosterone has 10-fold higher
affinity towards MR than GR (Rupprecht, et al. 1993; Anna, et al. 1988; Evans
and Anna. 1 989; Cato, et al. 1 991 ).
Prenatal programming of the HPA axis occurs, at least partially, at the
level of hippocarnpal GR and MR (Matthews. 2000). Hippocampal GR and MR
are involved in regulation of HPA activity (De Kloet. et al. 1998). Evidence
indicates that the MR is involved in feedback regulation of basal HPA function
while the GR is important during periods of HPA activation (De Kloet, et al.
1998). If similar GR autoregulation, to that which we report in vitro, occurs in vivo,
then this may itself have very significant consequences for normal hippocampal
development.
The role of serotonin in the regulation of GR development in primary
hippocampal culture, as weil as importance of thyroid-serotonin system in
regulating hippocampal GR levels during development. was addressed in
Chapter 2. In the present study. four days of serotonin exposure (100nM)
increased GR mRNA levels in the hippocampal neurons. It remains to be
detemined whether this increase is permanent. This finding could exptain the
acute increase in GR mRNA, which is documented in the female guinea-pig
fetuses. on 52"d day of gestation (Dean and Matthews, 1999). The same study
reported acute increase in fetal thyroid hormone in response to dexamethasone
administration. It is likely that the serotonergic system is mature by the time of
exposure, and is thus, able to respond to increased thyroid hormone secretion by
increasing serotonin tumover. lncreased serotonin tumover could, in tum,
increase hippocampal GR mRNA levels. This effect was rewrded in the rats
(Meaney, et al. 1987). and discussed in Chapter 2. The findings of this study
support a view that serotonin plays an important role in the prograrnming of
hippocampal corticosteroid receptors, and indicate a direct effect of serotonin on
hippocampal neurons.
To conclude, results of this study indicate that exposure to glucocorticoids
has a direct effect on GR mRNA expression in fetal guinea-pig (gestational day
40) hippocampal neurons. In addition, we have shown that exposure to serotonin
increases the GR mRNA complement within hippocampal neurons.
Findings described in this chapter were recorded in neurons which were
denved from fetuses at 40 days of gestation. In the guinea-pig, neurogenesis is
almost completed by gestational day 40 (Dobbing and Sands. 1979). Studies
undertaken in vivo reported changes in hippocampal GR and MR mRNA in
fetuses at 50 days of gestation (Dean and Matthews. 1999). It is established that
the most rapid period of brain growth in the guinea-pig occurs by gestational day
50. The rapid phase of brain growth is charactenzed by rapid neuronal
proliferation, synaptogenesis, dendritic aborization and increase in brain weight
(Dobbing and Sands. 1979). The period of rapid brain growth is also associated
with rapid neuroendocrine development (Dobbing and Sands.1979). Differences
in responses to dexamethasone between in vivo and in vitro studies could be
attributed to different stages of neuronal development. In the hippocampus, GR
mRNA is expressed at higher levels at gestational day 50 than at gestational day
40 (Matthews. 1998). It is possible that neurons, at 50 days of gestation, are
more responsive to dexamethasone due to higher GR levels. This possibility was
investigated in Chapter 5.
Figure 4.1 :
A) analysis of GR mRNA following cortisol (O. 1. 10, 1OOnM) exposure (4 days, n=6). Results are
expressed as mean+S.E.M. Yndicates statisücal difference from control (pcO.01).
(Numbers under bars represent the n value for each group; c=combined data; f= females only.
n=6; m= males only, n=6)
5) Analysis of MR mRNA following cortisol (O, 1 OOnM) exposure (4days, n=6). Results are
expressed as meankS.E.M. Analysis was perfomed on grouped data (males + females) only.
Figure 4.1
c m f c m f c m f c m f
O 1 10 100
Treatrnent (nM)
O 1 O0 Treatment (n M)
Figure 4.2:
Analysis of GR mRNA levels following dexamethasone (O, 1, 10, 100nM) exposure (4 days).
Results are expressed as meankS.E.M. 'Indicates statistical defierence from control (p<0.01).
(Numbers under bars represent the n value for each group; C=cornbined data; m=males alone.
n=6; f=fernales alone, n=6)
Figure 4.3:
Analysis of GR mRNA levels following serotonin (O, 1, 10, 100nM) exposure (4 days). Results are
expressed as mean2S.E.M. *Indicates statistical difference from wntrol (p<0.01).
(Numbers under bars represent the n value for each group; C=combined data; m=males alone,
n=6; Hernales alone, n=6)
Figure 4.2
1 .*. -.
... *.- W . . . -.
S . . ... ... ... *:.:.: . *. S . . .*. ...
t r . ... ... ... ... ... 2
c m f c r n f c m f c m f
O 1 I O 100
Treatment (nM)
Figure 4.3
c r n f c m f c m f c rn f
1 I O IO0 Treatment (nM)
CHAPTER 5
Regulation of
Development:
Days
5.1 Introduction
Hippocampal GR and MR mRNA During
Guinea-pig Studies, Gestational Age 50
Studies in Chapter 4 described corticosteroid mRNA alterations in fetuses
at 40 days of gestation. In the guinea-pig, days 40 and 50 of gestation are
considered cntical times for brain development (Dobbing and Sands. 1979).
Exposure to glucocorticoids in the human would likely occur during similar critical
times in human brain development. Guinea-pigs give birth to neuroanatomically
and neuroendorinologically mature Young. Peak brain growth. in the guinea pig,
occurs between 50 and 52 days of gestation (-75% of gestation; gestation length
- 70 days) (Dobbing and Sands, 1970; Dobbing and Sands. 1979). The rapid
phase of brain growth is characterized by rapid neuronal proliferation,
synaptogenesis, dendritic aborization and increase in brain weight (Dobbing and
Sands. 1979). The period of rapid brain growth is also associated with rapid
neuroendocrine development (Dobbing and Sands.1979). Neuroendocrine
maturation and development of central GR and MR systems occurs during Iate
fetal life in this species (Matthews, 1998). GR and MR populations go through
rapid and neuron-specific changes during this period. These changes can be
acutely and pemianently modified by fetal glucowrticoid exposure (Dean and
Matthews. 1999; Dean, et al. 2000; Matthews, et al. 2000). Cleariy, gestational
day 50 represents a critical period in the development of a guinea pig brain. This
period is wnsidered to be particularly vulnerable to drugs and other
pathophysiological insults (Dobbing and Sands. 'l979).
5.2 Objectives
Acute changes in hippocampal GR and MR mRNA levels at gestational
day 52, as a result of fetal dexamethasone exposure on days 50 and 51 of
gestation, were demonstrated previously, in vivo (Dean and Matthews, 1999). It
remained unclear whether these changes resulted from direct actions of
glucocoriicoids on the hippocampal neurons. The main objective of this study
was to investigate the direct effects dexarnethasone on hippocampal GR and MR
mRNA levels, in vitro. Direct effects of serotonin on hippocarnpal GR mRNA and
MR mRNA were also examined. Effects of cortisol were not examined in this
study.
5.3 Hypothesis
We hypothesize that exposure to dexamethasone will down regulate GR
mRNA expression in fetal guinea-pig hippocampal neurons. Exposure to
serotonin will up regulate GR mRNA expression in hippocampal neurons. We
hypothesize that exposure to dexamethasone or serotonin will not affezt
expression of MR mRNA within fetal hippocampal neurons. The rationale for this
hypothesis is identical to one described in Chapters 3 and 4.
5.4 Methods
5.4.1 Development of guinea-pig culture system
During the processing of fetal guinea-pig cultures it was establiçhed that
further optimization of the system was required. Manipulation of cells in this
system, such as media exchange or treatment was time consuming and it
increased the risk of contamination during handling. In addition, the cover-slips
were difficult to handle dunng N, situ hybridization analysis. We also had to
design and manufacture storage containers and cover-slip racks in the lab. The
greatest difficulty was experienced during emulsion application and exposure,
during which many of the slides adhered to each other and hence becarne
unusa ble.
We proceeded to further optimize our culture system, especially focusing
on final in situ handling. We adopted the use of tissue culture inserts (IOmm, Life
Technologies, Toronto, Canada), which allowed us to use the chamber slide
system similar to that desct-ibed for the mouse studies. This resolved al1 technical
difficulties associated with final in situ processing and analysis. In this system,
mouse cerebellar cells were grown on the insert and guinea-pig cells were grown
on the glass bottom of the chamber slide. This system produced viable fetal
guinea-pig (gestational day 50) neurons and was very easy to use.
5.4.2 Mouse Cerebellar Culture, On Culture lnserts
Preparation of the mouse cerebellar cultures was identical to that
described in Chapter 4, with the following difference:
1. Cell suspension (0.1 -0.2rnl) was placed in each culture insert.
5.4.3 Guinea Pig Hippocampal Culture, gestational day 50
Preparation of the guinea-pig hip pocam pal cells was identical to that
descnbed in Chapter 4, with the following differences:
1. Cell suspension (250pL) was placed in each PDL-precoated well. Each well
previously contained MEM-N2, mNGF (1 750pL). Culture-inserts containing
mouse cerebellar culture were inserted into the each well. Total of 16
welIs/sexfanimal were prepared.
2. Each culture slide well contained cuIture insert and 1 ml of MEM-FBS, HS.
5.4.4 IN SITU HYBRIDIZATION
All solutions used in this section were described in detail in Appendix3. In
situ hybridization and subsequent ernulsion autoradiography was identical to that
descnbed in Chapter 3. The same anti-sense oligonucleotide probes as those
described in Chapter 4 were used.
5.5 Data analysis
Analysis was sirnilar to that carried out in Chapters 3 and 4. Analysis of
GR mRNA and MR mRNA in pyramidal neurons was carried out at 200X
magnification. On average 20-50 grains were identiied over positive neurons and
mean grain count per neuron was calculated for each treatrnent (4 separate
experiments per treatment). Approximately 30 mRNA positive pyramidal neurons
were randomly sampled and analyzed in each well (1 well/treatment/analysis).
Cell wunting revealed approximately 70 neurons/well. Non-neural cells were
present at very low density or were absent due to the nature of Our culture setu p.
Given the lack of sex difference observed in Chapter 4, statistical analysis
of pooled (male + fernale) cultures was undertaken though male and female data
are also presented. Group data for each sex are presented as MeanskS.E.M.
Data were statistically analyzed using Statistica (Release 5, 97 Edition,
Oklahoma, USA). The effects of treatment on GR and MR mRNA were
detemined using a two-way ANOVA followed by the Duncan's method of post-
hoc corn parison.
5.6 Results
High levels of glucocorticoid receptor mRNA were detected in
hippocampal neurons removed at 50 days of gestation. Mineralocorticoid
receptor mRNA levels were also high in the hippocarnpal neurons.
Dexarnethasone treatment (1 00nM) significantly ( ~ ~ 0 . 0 5 ) down-regulated
GR mRNA levels within neurons (Fig. 5.la). Dexamethasone treatment at lower
concentrations (1 and 10nM) did not change GR mRNA levels in neurons
(Fig.5.1 a). MR mRNA levels were not affected by dexarnethasone treatment, at
any of the tested concentrations (1, 10 and 1 OOnM)(Fig.5.1 b).
Treatment of hippocampal cultures with serotonin, at 1 OOnM, significantly
( ~ ~ 0 . 0 5 ) upregulated GR mRNA expression (Fig.5.2a). Exposure to serotonin. at
1 and IOnM, did not alter GR mRNA levels (Fig.5.2a). Serotonin administration
had no impact on MR mRNA levels, at any of the concentrations tested
5.7 Discussion
Results from this study are consistent with those reported in neurons
derived from 40 days old fetuses (Chapter 3). Dexamethasone exposure
decreased GR mRNA levels in hippocampal neurons, while serotonin exposure
increased GR mRNA levels. In the present study we describe acute effects on
GR mRNA. It remains to be established whether synthetic processes are affected
permanently.
Effects of dexamethasone in this study contrast those reported in in
vivo situation. Dexamethasone exposure, in vivo, induced up-regulation of GR
mRNA within fernale hippocampal neurons and it had no effect on GR mRNA
expression within male hippocampal neurons (Dean and Matthews. 1999).
Discrepancies between the two studies can be explained by two possibilities. It is
possible that presence of serotonergic system in vivo, as well as the presence of
noradrenergic systems that also modifies GR compliment, counter-acted the
negative input of glucocorticoids. As it was discussed in the previous chapter,
glucocorticoids enhance the maturation of serotonergic system, whose activity is
known to cause up-regulation of GR mRNA within the hippocampus. Based on
the findings in neurons derived from animals at 40 and 50 days of gestation, one
can suggest that actions of serotonergic system, h vitro, are more potent than
the direct actions of glucocorticoids. It would be interesting to test this proposal in
vitro, in neurons derived from animals at 50 days of gestation. Information
generated at this gestational age, in combination with similar information from 40
days of gestation. would greatly enhance Our understanding of serotonergic
system input in HPA programming during development. The second possibility
for observed discrepancies in two studies is glucocorticoid concentration
difference in the hippocampus. It is possible that glucocorticoid concentration in
the hippocampus, in vivo, is too low to effect GR mRNA expression. In in vivo
studies distribution of dexamethasone could be affected by the presence of P-
glycoproteins within blood-brain bamer. which have the potential to exclude
dexamethasone from the brain (Meijer. et al. 1998).
Dexamethasone has a very low affinity for MR and it selectively binds GR
(Veldhius, et al. 1982). Therefore. it is not surprising that the dexamethasone
treatment failed to affect MR mRNA levels. Serotonin exposure did not modify
MR mRNA levels in neurons. This was similar to the situation in neurons derived
from gd40 fetuses. The absence of serotonin effect on MR protein levels, in vitro,
was recorded in rat studies (Mitchell, et al. 1990). Taken together, these latter
findings suggest that development of rnineralocorticoid receptor system is not
influenced by serotonin at 50 days of gestation in the guinea-pig. Serotonin
administration u p-regulated GR mRNA expression. This finding is in agreement
with results previously reported in mouse studies and gestational day 40 guinea-
pig neurons. Taken together with studies done in rats (Meaney, et al. 1987;
Meaney et al. 1994), these findings support the involvement of thyroid-serotonin
system involvement in HPA programming, as discussed in Chapter 2.
Results described in gestational day 50 neurons were identical to those in
neurons that were derived from animals at 40 days of gestation. There was no
difference in response to dexamethasone or serotonin despite the major
differences in hippocampal development at these times. Based on these findings
one can propose that the main direct effect of glucocorticoids, on fetal neurons, is
to down-regulate expression of GR. It would be interesting to test this further, in
neurons derived from animals that are closer to term. However, such neurons
would be difficult to culture due to advanced level of neuroanatomical maturity in
the guinea-pig fetuses which are close to ten .
In surnmary, results of this study indicate that exposure to glucocorticoids
has a direct effect on GR mRNA expression in fetal guinea-pig hippocampal
neurons. Furthenore, this study has shown that exposure to serotonin increases
the GR mRNA complement within hippocampal neurons. One can speculate that
both glucocorticoids and serotonin exert their influence on the developing
hippocampus, but that the net effect of such treatment is dependent on the
maturational stage of both systems, at the time of exposure. However, the main
direct effect of glucocorticoids, on fetal neurons, is to down-regulate expression
of GR.
Figure 5.1:
A) Analysis of GR mRNA following dexamethasone (O, 1, 10, 100nM) exposure (4 days). Results
are expressed as rnean~S .E.M . 'Indicates statistical difference from control(p<O.Ol).
(Numbers under bars represent the n value for each group; C=combined males+females; m=males
alone; n=3; Hemales alone; n=2)
B) Analysis of MR mRNA following dexarnethasone (O, 1, 10, 100nM) exposure (4 days). Results
are expressed as rneankS.E.M.
(Numbers under bars represent the n value for each group; C=combined males+females; m=males
alone; n=3; Memales alone; n=2)
Figure 5.2:
A) Analysis of GR mRNA following serotonin (O, 1, 10, 1 OOnM) exposure (4 days). Results are
expressed as mean6.E.M. 'Indicates statistical difference frorn control(p<O.Ol).
(Numbers under bars represent the n value for each group; C=combined males and females;
m=males alone; n=3; f=fernales alone; n=2)
B) Analysis of MR mRNA following dexamethasone (O, 1, 10, 100nM) exposure (4 days). Results
are expressed as meantS.E.M.
(Nurnbers under bars represent the n value for each group; C=combined males and females;
m=males alone; n=3; Hernales alone; n=2)
Figure 5.2
A 40 *
s mg L 30 m u
a 2 20 ar E P! I O C3
O I 1
c ; ? 9 1 5 ' 31 2l
c m f c m f c m f
1 I O 1 O0
Treatment (nM)
L
c r n f
O
c r n f c m f c m f c m f
O 1 10 100 Treatment (nM)
94
CHAPTER 6
Multi-drug Resistance Studies
6.1 Introduction
The hippocampus is one of the most sensitive targets for the action of
endogenous and synthetic glucocorticoids. Excess glucocorticoid is detrimental
to neuron development (Uno, et al. 1994; Uno, et al. 1990; Matthews. 2000). In
adults, it has been çuggested that dexamethasone (a synthetic glucocorticoid)
penetrates poorly into the brain due to expression of the multidrug resistance
gene (MDRl a) (Meijer, et al. 1998). Drug-transporting P-glycoprotein, a product
of the multi-drug resistance gene, functions as an exclusion pump for synthetic
glucocorticoids and other chemotherapeutic agents (De Kloet, et al. 1998;
Schinkel, et al. 1995; Meijer, et al. 1998). Approximately 10% of pregnant women
in North America, at risk of pre-term delivery, are treated with synthetic
glucocorticoids in late gestation to promote fetal lung maturation (NIH, 1995).
However, little is known about the effects of dexamethasone on fetal brain and
there is no information concerning the expression of MDRI a in the developing
brain. It is likely that exposure of the fetal brain to synthetic glucocorticoids could
be regulated by expression of the MDRla gene.
6.2 Objectives
The primary objective of this study was to examine the expression of
MDRl a gene in the guinea-pig brain throughout development. The secondary
objective of this study was to detemine whether two-day dexamethasone
exposure in fetal life alters the expression of MDRl a gene.
6.3 Hypothesis
The MDRla gene is expressed in the fetal brain but expression changes
during development. Glucocorticoid exposure duting fetal life can alter the
expression of MDRl a gene.
The first part of Our hypothesis was based on the fact that MDR
expression in adult anirnals was found to protect against dexamethasone
exposure (Meijer et al. 1997). It is possible that the MDR expression in fetuses
could have the same effect. If the MDR expression is to protect the brain during
critical petiods of development, the expression of this gene should have a simiiar
pattern to that of a guinea-pig brain development. The second part of our
hypothesis was based on findings of in vitro studies which demonstrated that
certain P-gp su bstrates, like vinblastine or rnetkephamid, induced or su ppressed
MDR expression (Meijer, et al. 1997).
6.4 Methods
6.4.1 Animai Work
All animal tissues used in this section were generated previously by S.G.
Matthews and F. Dean. Accurately tirne-dated guinea pigs (Elvidge, 1972; Ree
and Hounslow, 1971) were killed on days 40-45, 50-55 and 60-65 of gestation.
Fetal brains were rapidly removed and frozen. A group of 7 day old neonates and
adult animals were also killed and brains were collected. Each group comprised
six to seven animals.
An additional group of pregnant guinea pigs was subcutaneously injected
with dexamethasone (Imglkg of body weight; n=9) or vehicle (200pL;
n=lO)(Dean and Matthews, 1999). Injections were given at 0800h on days 50
and 51 of gestation. Pregnant guinea pigs were killed on day 52 and the fetal
brains were rapidly removed and frozen.
6.4.2 Tissue Preparation and Sectioning
Frozen guinea-pig brains were coronally sectioned (12pm) using a
cryostat (JUNG CM3000, Leica instruments GmbH, Nussloch, Germany).
Sections were thaw-mounted on poly-L-lysine coated glass rnicroscopic slides.
Sections were stored at -80°C. Prior to hybridization the sections were fked in
4% paraformaldehyde solution (5min). Slides were washed in phosphate buffer
saline (PBS) (2Xlmin) and dehydrated in 70% ethanol (5min) and 95% ethanol
('lmin). This animal work was camed out by F. Dean and S.G. Matthews.
6.4.3 IN SITU HYBRIDIZATION
The author of this thesis did al1 in situ hybridization work and subsequent
analysis. ln situ hybndization was identical to that described in chapter 3, with the
following exceptions:
1. MDRla oligonucleotide probe hybridized tu guinea pig brain cryo-sections
was complementary to bases 202-246 of Chineçe hamster MDR mRNA. This
sequence is homologous across many rodent species, including that of the
guinea-pig (Croop et al, 1989).
6.5 Autoradiography and Analysis
Slides were dried and exposed to autoradiographic film (Biomax, Eastman
Kodak Co.. Rochester, N.Y., USA) in a light proof cassette. standards
(American Radiochernical Inc., St. Louis, M.O., USA) were exposed along with
the slides to ensure analysis within the linear range of the autoradiographic film.
Cassettes were stored at room temperature. Films were exposed for 28 days and
developed using an automatic processor (Kodak M35A- OMAT processor).
Analysis of autoradiographic signals on film was camed out using a
computerized image analysis system (Imaging Research Inc., St. Catherines,
Ontario, Canada).
MDR mRNA levels were measured in the hippocampus (CA1, CA2, CA3
and CA4 subfields), dentate gyrus, thalarnic nuclei and cortex. Autoradiographic
signal was measured in each area (8 sections per area).
Statistical analysis was perfotmed using Statistica (Release 5, 97 Edition,
Oklahoma, USA). Data are presented as MeansIS.E.M. and were analyzed
using two-way ANOVA followed by the Duncan's method of post-hoc
comparison. Statistical significance was set at pc0.05.
6.6 Results
MDR1 a mRNA was detected in several regions of the fetal guinea pig
brain. High levels of expression were detected in the CAIICA2 regions of the
hippocampus, CA3 region of the hippocampus, dentate gyrus and thalamic nuclei
(Fig . 6.1 ). Silver emulsion autoradiogra phy performed on coronal sections
revealed cell-specific expression of MDRla mRNA (Fig. 6.2).
Within the CAlICA2 region of the hippocampus, expression of MDRl a
mRNA was detected at al1 gestational groups as well as in neonatal and adult
anirnals. The highest expression was observed in fetuses at 50-55 days of
gestation. Expression of MDRla mRNA progressively decreased towards term. A
significant decrease (pc0.05) in MDRl a mRNA expression was detected
between animals at 60-65 days of gestation and seven day old neonatal animals
(Fig.6.3).
In the CA3 region of the hippocampus, the highest expression of MDRla
mRNA was detected in fetuses at 40-45 and 50-55 days of gestation. Expression
of MDRl a mRNA progressively decreased towards term. There was a significant
reduction ( ~ ~ 0 . 0 5 ) in MDRla mRNA between 50-55 days of gestation and 60-65
days of gestation. MDRla mRNA levels rernained relatively stable into the
adulthood (Fig.6.4).
In the CA4 region of the hippcampus, the highest expression of MDRla
mRNA was detected in fetuses at 40-45 and 50-55 days of gestation. Expression
of the MDRla mRNA significantly decreased ( ~ ~ 0 . 0 5 ) between 50-55 days of
gestation and 60-65 days of gestation. After 60-65 days of gestation the
expression remained relatively constant at term and through to adulthood (Fig.
6.5).
In contrast, in the dentate gyrus MDRla mRNA levels were detected at
low levels by 40-45 days of gestation. MUR1 a mRNA levels then progressively
increased towards tem. Significant up-regulation ( ~ 4 . 0 5 ) in the MDRla mRNA
levels was detected between 50-55 days of gestation and 60-65 days of
gestation (Fig. 6.6). MDR mRNA levels remained at this increased level in
neonates as well as adult animals.
In the cortex the pattern of MDRla mRNA expression was similar to that
observed in the hippocampus. The highest levels of expression of the MDRla
mRNA were observed at 40-45 days of gestation. Significant (pc0.05) reduction
in the MDRla mRNA expression were detected between 4045 days of gestation
and 50-55 days of gestation as well as between 60-65 days of gestation and 7
day old neonates (Fig. 6.7).
In the anteroventaral thalamic nuciei MDRla mRNA was detected at
gestational days 40-45, 50-55 and 60-65. Expression decreased drarnatically
after t e n and in some animals wuld not be detected (Fig. 6.8).
Finally, no significant differences were observed in limbic regions,
between animals that were exposed to dexamethasone and animals that
received vehicle alone (Fig. 6.9).
Discussion
Using the MDRla probe, we have described the expression of MDRla
mRNA in the developing brain. We have also shown that levels of MDRla mRNA
change during development in a region-specific manner.
The pattern of MDRla mRNA expression coincides with the pattern of the
guinea pig brain development. The highest levels of MDRla mRNA expression
within the hippocampus and cortex were observed on gestational days 40 and
50. In the guinea-pig, neurogenesis in these areas is completed by day 40 of
gestation, while the most rapid brain growth is observed between days 50 and 55
(Dobbing and Sands, 1970; Dobbing and Sands, 1979). Given the evidence, one
can suggest that MDRla expression is high in order to ensure maximal
protection against xenobiotic substances, during these critical periods of brain
development.
In the dentate gynis, the highest levels of MDRla mRNA expression were
observed juçt prior to term (65 days of gestation) and in neonatal and adult
animals. Development of dentate gynis is known lag behind the hippocampus
(Dobbing and Sands, 1970; Dobbing and Sands, 1979). Hence, it is not
surprising that peak MDRl a expression was observed at a later gestational date.
It is interesting that MDRia mRNA was expressed at very similar levels in
neonatal and adult animals. This may be explained by the fact that neurogenesis
at low levels, in the dentate gynis, is observed in adult animals (Gould, et al.
1994; Gould, et al. 1991). Continued high expression of MDRl a mRNA within the
dentate gyrus may act to protect neurogenesis from detrimental influences of
xenobiotic agents.
An unexpected finding of this study was the high expression of MDRl a
mRNA within the specific anteroventral thalamic nuclei. In fact, the highest
expression of MDRla mRNA, in gd50 fetal brainç, was recorded in this structure
(see Fig. 10). More perplexing was the dramatic decrease in expression after
t e n and subsequent disappearance of expression in some animals. The high
levels of MDRla mRNA expression in this thalamic nucleus may be partially
explained by its close proximity to the lateral ventricle. High MDRla expression
could protect against the xenobiotic compounds penetrating from cerebrospinal
fluid. However, it is not known how this structure is involved in development.
Such a high level of protection suggests an important role, but this remains to be
investigated .
Since the synthetic glucocorticoids are substrates for the P-glycoprotein
cellular extrusion pump, it is possible that they could induce their own resistance.
The induction of resistance by other P-glycoprotein substrates had been
described in vitro (Anderle, et al. 1998). In this study, two day dexamethasone
exposure (I mglkg of body weight) had no significant effect on the expression of
MDRla gene, in day 50 fetuses. Based on this finding one can speculate that
dexamethasone cannot induce its own resistance. However, this remains to be
confirmed in studies where dexamethasone would be administered in a repeated
fas hion.
It was previously reported that penetration of dexamethasone into the
brain of MDRla knockout mouse (-1-) is 3-fold higher than that of a wild type
(Schinkel, et al. 1995). The enhanced uptake of dexamethasone in the MDRla (-
/-) brain disappeared when the dose of dexamethasone was raised to lmglkg
mouse, from the previous dose of 0.2mglkg mouse (Schinkel, et al. 1995). This
indicated a limited capacity of the P-glycoprotein transport system. Exposure to
dexamethasone at 100nM (this is approximately 0.014mg) induced down
regulation in GR mRNA within mouse hippocampal neurons (Chapter3). This
dose is higher then that at which the P-glycoprotein saturation is reached
(0.004mg in mouse weighing 209). Therefore, changes in GR mRNA within
mouse neurons due to dexamethasone (1 00nM) exposure could be explained by
P-glycoprotein transport saturation. Absence of effect at lower concentrations of
dexamethasone (1 an 10nM) could be explained by the P-glycoprotein-mediated
exclusion of dexamethasone.
In conclusion, the MDRl a gene is widely expressed in the developing
brain, in a region-specific manner. MDRla gene expression changes during
development in a highly region-specific manner. We speculate that the local
expression of MDRla gene can protect against exogenous glucocorticoid
exposure at low to moderate levels. Expression of the MDRla gene allows
exclusion of dexamethasone from specific regions of the fetal brain and this
could have a protecüve effect during critical periods of development.
Figure 6.1: Color-enhanced image of MDRla in coronal sections of a fetal guinea pig. following in situ hybridizaüon. MDRla mRNA was present throughout the brain with the folowing regions showing high levels of expression: CAIICA2 region of the hippocampus (CAI), CA3 area of the hippocampus (CA3). dentate gynis (DG) and thalamic nuclei (T).
Legend: dark blue=background; pink=low to moderate expression; red=high expression
Figure 6.2: Images of coronal sections that underwent silver emulsion autoradiography. MDRla mRNA is represented by black clusters of dots.
rY *- )I Thalamus $
5 al-
*
Hippocampus \ - 2 3 . i-
h - : . 4 <; <:s - - ' - W .
B. Vessel - ~ t ' . - a . - a7
Figure 6.3: MDRla mRNA levels within CAlICA.2 region of the hippocampus. Highest expression was observed at gd50-55, which corresponds to period of the most rapid brain growth in the guinea pig. Expression significantly (~~0.05) decreased between gestational ages 60-65 and 7 day old neonatal animals.
Figure 6.4: MDRl a mRNA expression within CA3 region of the hippocampus. Expression levels were highest at gd40-45. Expression significantly (~~0.05) decreased between gestational ages 50-55 and 60-65.Expression remained at deceased levels into the adulthood.
Figure 6.3
40-45 50-55 60-65 7 AduIts
GestationalINeonatal age (days)
Figure 6.4
40-45 50-55 60-65 7 Adults
GestationalINeonatal age (days)
107
Figure 6.5: MDRla mRNA levels in CA4 region of the hippocampus. Highest expression was observed at gd50, which decreased progressively towards tem. Expression significantly ( ~ 4 . 0 5 ) decreased between gestational ages 50-55 and 60-65.
Figure 6.6: MDRl a mRNA expression within dentate gyms. In contrast to the hipocarnpus, mRNA levels peaked at terni and remained at the same level throughout neonatal life. Expression significantly (pe0.05) increased between gestational ages 50-55 and 60-65.
Figure 6.5
40-45 50-55 60-65 7 Adults
GestationalINeonatal age (days)
Figure 6.6
40-45 50-55 60-65 7 Adults
GestationallNeonatal age (days)
1 O9
Figure 6.7: MDRla mRNA expression within the cortex. Expression levels were uniform between the layers. Highest levels were detected at gd40 and then decreased progressively towards term. Expression significantly (p<0.05) decreased between gestational age 4045 and 50-55, and gestational age 60-65 and 7 day old neonates.
Figure 6.8: MDRla rnRNA expression within the anteroventral thalamic nuclei Expression was detected at gd40. gd50 and gd60. However, expression decreased drarnatically after term and could not be detected.
Figure 6.7
40-45 50-55 60-65 7 Adults
Gestational/Neonatal age (days)
Figure 6.8
40-45 50-55 60-65 7 Adult
GestationalINeonatal age (days)
111
Figure 6.9: MDRla mRNA expression in the brain regions of fetal guinea pigs (gd50). No significant differences were observed between animals that received dexamethasone (n=9) and anirnals that received vehicle alone (n=10).
Cortex
Chapter 7
Summary and Conclusions
This study has demonstrated. for the first time, the direct effects of
endogenous and synthetic glucocorticoids on GR and MF? mRNA in fetal
hippocampal neurons. in vitro. In addition. this study examined the effects of
serotonin on expression of GR and MR mRNA in fetal hippocampal neurons, h
vitro. Examination of GR and MR mRNA was undertaken in neurons which were
derived from species with both prenatal (guinea-pig) and postnatal (mouse)
profiles of neuroendocrine development.
Studies undertaken in the mouse demonstrated that exposure to
endogenous (corticosterone) and synthetic (dexamethasone) glucocorticoids
down regulate the GR mRNA expression. Mechanisms by which this takes place
probably involve autoregulation of GR gene transcription. One possible way to
address the mechanism is to implement simultaneous GR antagonist and
glucocorticoid administration, in the same system. In this scenario, one would
expect to see no change in GR mRNA expression due to glucocorticoid
exposure. If dfierences were to be detected then one could conclude that
glucocorticoids can influence GR expression indirectly.
No differences in MR mRNA expression were detected due to either
endogenous or synthetic glucocorticoid exposure. Therefore it was concluded
that the down regulation of MR mRNA expression recorded in vivo (Levitt, et al.
1996) is probably mediated through indirect mechanisms. Serotonin exposure, in
vitro, was found to increase GR mRNA levels. This confirmed the importance of
thyroid-serotonin axis involvement in the HPA programming. Taken together,
studies undertaken in the mouse have expanded and complimented the
understanding of HPA programming in the postnatal developer model.
The guinea-pig represents a model in which extensive neuroendocrine
development occurs in ulero, a situation similar to the human. Studies in the
guinea-pig examined the direct effects of cortisol (endogenous glucocortiwid)
and dexamethasone (synthetic glucocortiwid) and serotonin on the expression of
GR and MR mRNA in fetal neurons, in vitro. This was done in a sex-specific
manner, at two gestational ages (gd40 and gd50) which are considered critical in
the guinea-pig brain development. 60th cortisol and dexamethasone exposure
down regulated GR mRNA expression in fetal neurons of both sexes. MR mRNA
expression was unaffected by glucocorticoid exposure. Serotonin treatment up
regulated GR mRNA expression in neurons of both sexes and did not affect MR
mRNA. These effects were recorded at both gestational ages considered.
Discrepancies from in vivo studies (Dean and Matthews. 1999; Dean, et al. 2000;
Matthews, et al. 2000) were explained by the presence of serotonergic system in
in vivo situation. Absence of sexual dimorphism in response ta glucocorticoids in
vitro may be explained by absence of sex hormones. Differential timing of brain
development, particularly the serotonergic system, may also be implicated in the
previously described sex differences, in vivo.
A possible functional limitation of this study was the lack of GR and MR
protein binding consideration. It would be very useful to detemine how levels of
GR and MR protein correspond to the changes in GR and MR mRNA levels. The
next step in future studies of this type would be to examine how GR and MR
protein levels change in response to glucocorticoids and serotonin. Another
functional limitation of this study is the stability of mRNA. It is possible that
observed differences in mRNA levels in this study reflect degradation of mRNA
rather than down-regulation. Examination of GWMR protein levels would also
help to resolve this issue. Another future consideration involves examination of
glucocorticoid and serotonin effects on neurons derived from fetuses at 60 days
of gestation. By this gestational time the brain of the guinea-pig displays high
neuroanatomical maturity and the process of myelination is occurring (Dobing
and Sands. 1979; Dobing and Sands. 1973). Examination of glucocorticoid
impact on hippocampal GR and MR mRNA levels at this time would offer better
insight into HPA programing.
We have also deterrnined the pattern of MDRl a mRNA expression in the
guinea pig brain. The MDRla gene was widely expressed in the developing brain
and the expression changed during development in a highly region-specific
manner. In the future studies, role of MDRla genes in the HPA programming can
be examined by using MDR -1- mouse. MDR knockout mice wuld be treated with
dexamethasone during pregnancy and their offspring allowed to reach adulthood.
Examination of wrticosteroid receptor levels in the hippocampus and
corticosterone levels in such adult animals would more definitively answer the
question of MDR gene involvement in HPA programming.
In conclusion, this study suggests that both glucocorticoids and serotonin
can direcüy influence GR mRNA and MR mRNA in developing hippocampal
neurons. Since glucocorticoids can promote maturation of the serotonergic
systern. the net effects of glucocorticoids during development on hippocampal
GR mRNA in vivo, likely depends on a balance between direct glucocorticoid
effect and indirect serotonin modulation. However. it is possible that other
aspects of brain function may be modified by prenatal glucocorticoid exposure
and that these may in tum rnodrfy corticosteroid receptor levels in the
hippocampus.
Appendix 7: Culture solutions
Minimal Essential Medium (MEM)
MEM was purchased frorn TCMP at Faculty of Medicine, University of
Toronto. 0.0259 of NaHCOJ and 3.59 of glucose was added to 500 ml of MEM.
Osmolality was adjusted with additional glucose, to 320-325 mOsM, using
osmorneter. 1 ml of rehydrated insulin (GIBCO, BRL, Insulin, Crystalinne, Bovine,
Zinc, lyophilized, cat#13007-018) was added to 500ml of MEM.
Minimal Essential Medium and NZ
MEM-N2 was prepared by adding 0.011g of sodium pyruvate (a-
Ketoproprionic acid; 2-Oxoproprionic acid; Sodium Salt; C3H303Na, FW 1 10.0;
SIGMA, P-5280, lot 68H6195). O.lg of chicken egg albumin per 100ml of MEM.
Both compounds were dissolved and MEM was filtered through 115mI
NALGENE filter, into sterile bottle. 1 ml of N2 (100X) was added per 100 ml of
MEM.
Minimal Esenfial Medium - N2 and NGF
400pL of Neural Growth Factor (NGF) solution, 40ng/pL, was added to
each ZOOml of MEM-NP. NGF was purchased from Alornone Labs.
Minimal Essential Medium containing Fetal Bovine Serum and Horse Serum
160ml of MEM was filtered through 115ml Nalgene filter, into sterile boffle.
20ml of Horse Serum (Horse Serum, heat inactivated; GIBCO: Cat. No.26050,
Lot No 1025190). and 20ml of Fetal Bovine Serurn (GIBCO: Cat. No. 26140-087,
Lot. No. 1022442) were added.
Dissection Medium
Hank's Basic Salt Solution (BSS) containing 15m HEPES was purchased
from TCMP at Faculty of Medicine, University of Toronto. 2.59 of glucose and 39
of sucrose was added to 500ml of Hank's BSS containing 15mM HEPES.
Osmolality was adjusted to 320-325 mOsm by adding glucose.
Fluorodeoxyuridine (FUDR)
0.0049 of 5-Fluoro-Zdeoxyuridine (SIGMA, F-0503) and 0.01 g of uridine
(SGMA. U-3750) were dissolved in 20ml of MEM. Solution was filtered through
1 15ml Nalgene filter.
Appendix 2:
Derivation of Treatments for Mouse Hippocarnpal Cultures
Treatment : Dexarnethasone
Stock preparation: 0.00479 of dexarnethasone was measured (Sa-Fluoro-16a-
methylprednisolone, C22H29F05, FW 392.5, SIGMA. D-1756) and dissolved in
469.59pL of 100% ethanol (concentration 25.5mM). IOpL of this solution was put
in 49990pL of PBS (concentration 5.1pM). This solution was separated into
200pL stocks. which were stored at 4'C.
Treatment: 6.5pL from stock directly to the well. This was 100nM treatment. IOpL
from stock to 90pL of MEM-FBS,HS. 6.5pL of this solution was added to each
well. This was
10nM treatment. 10pL from sock to 990pL of MEM-FBS.HS. 6.5pL of this
solution was added to each well. This was 1 nM treatment.
Calculations:
Stock: 0.01 g DW392.5/0.001L = 25.5 mM
1OpL x 25500pM = C x 50000pL; C = 5.1 VM
100 nM treatrnent: 6.5~1 x 5.1 pL = 326~ (final volume of well) x C
C = 101.5 nM
10 nM treatment: 10pL x 5.1 pM = 90pL x C; C=O.Sl pM
6.5vL x 0.51 pM = 326.5~1 x C
C = 10.15 nM
1 nM treatment: 10vL x 5.1pM = 990yL x C; C = 0.051pM
Final well volune: 50pL (cell suspension) + 250pL (volume of MEM-FBS-HS) +
20pL
(FUDR) + 6.5pL (treatment) = 326.5pL final well volume.
Similar calculations were perfonned for corticosterone and serotonin treatments.
Appendix 3: Molecular biology solutions
All of the following solutions were made with Molecular Biology Grade
reagents.
Diethyl pyrocarbonate (DEPC) treatment:
All DEPC work was camed out in a fume hood. DEPC (0.5mlllL, Omnipur,
EM Science, North Amencan associates of Merk KgaA, Darmstadt, Genany)
was added directly to each treated solution. Solution was allowed to stand in the
hood for at least 3 hours before being autoclaved.
Hybridization buffer:
Hybridization buffer was prepared a day prior to in situ hybridization (ISH).
Salmon spem was added on the day of ISH. Hybridization buffer was prepared
in a 50ml stefile polypropylene tube in following order:
25ml
1 Oml
2.5ml
0.5ml
2.5ml
1 .Oml
1 .Oml
50pL
100% deionized formamide (see below)
20X saline sodium citrate (SSC)
0.5M sodium phosphate
0.1 M sodium pyrophosphate
A00X Denhardts solution
Chlorofom-phenol extracted salmon spem DNA (1 Omglml)
Polyadenylic acid (5mgiml)
Sodium heparin (1 20mglml)
Dextran sulfate, sodium salt (Phamacia LKB)
Final volume was adjusted to 50ml with DEPC-H20. Solution was mked
vigorously, protected from light and stored at -4OC.
Deionized formamide:
Formamide (Gibco)
"AmberliteV monobed resin MB-1 (20-50 mesh; Bio-Rad
Laboratories, CA, USA)
Foramide was added to resin and the mixture was stirred at room
temperature for 2h. The mixture was filtered through a sterile filter paper and
stored at -20°C in 25ml aliquots.
Saline Sodium Citrate (20X SSC):
175.39 NaCl
88.209 Sodium citrate
50th reagents were dissolved in H20 (1 .OL) and pH was adjusted to 7.0
with HCI. Solution was then DEPC-treated and autoclaved.
Sodium Phosphate (0.5M):
Na2HP04 (0.5M)
NaH2P04 (0.5M)
The solutions were mixed and pH was adjusted to 7.0. The solution was
filtered, DEPC-treated and autoclaved.
Denhardts solution:
Polyvinylpynolidine (PVP)
Bovine semrn albumin
Ficoll
DEPC-H20
Solution was filtered and stored in aliquots (6ml) at -20°C.
Salmon Spem DNA:
Salmon sperm (10mglml) was purchased from Sigma in a ready to use
form.
Polyadenylic acid:
1 OOmg Polyadenylic acid (sodium salt)]
Ployadenylic acid was dissolved in DEPC-H20 (20ml), aliquoted (1 ml) and
stored at -20°C.
Sodium Heparin:
Freeze dtied sodium heparin was dissolved in DEPC-H20 (120mglml) and
stored in aliquots (50pL) at -20°C.
Soak Solution:
DEPC-H20
2oxssc
50% formamide
Terminal Deoxynucleotydyl Transferase (TdT):
TdT was purchased fom Gibco-BRL (FPLC pure, Calf Thymus)
5X Tailing Buffer:
Tailing buffer was supplied with the TdT (Gibco-BRL).
EDTA (0.5M):
EDTA (93.059) was dissolved in water. NaOH (log) was added and pH
was adjusted to 8.0. Final volume was adjusted to 500ml. The solution was
DEPC treated, autoclaved and stored at room temperature.
Phoshpate Buffered Saline (PBS):
8.0g/L NaCl
0.2gIL KCI
1.44gJL Na2HP04
0.24gtL NaH2P02
Paraforrnaldehyde:
Parafonaldehyde (409) was dissolved in DEPC-PBS (IL) by heating to
60°C in a fume hood. Dissolution was facilitated by NaOH addition, in a drop
wise rnanner. The pH was adjusted to 7.0 with HCI. The solution was filtered and
stored at 4°C.
Tissue fixation:
All tissues as well as cultured cells were fixed as follows:
Paraformaldehyde
PBS
PBS
5min
1 min
1 min
70% EtOH
95% EtOH
Smin
1 min
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