The role of amygdala cholecystokinin and parvalbumin expressing neurons

in the acoustic startle reflex in mice

by

Tom E. Curry

A thesis submitted in conformity with the requirements

for the degree of Master of Science

Department of Cell and Systems Biology

University of Toronto

© Copyright by Tom E. Curry 2013

II

The role of CCK and PV expressing amygdalar neurons in the acoustic

startle reflex in mice

Tom E. Curry

Master of Science, Department of Cell and Systems Biology, University of Toronto,

2013

Abstract

Parvalbumin (PV) and cholecystokinin (CCK) proteins are found in the basolateral

amygdala nuclei, particularly in gamma-aminobutyric acid (GABA) interneurons. PV+ neurons

were localized to the basolateral amygdala and they expressed the GABA neuron marker

glutamic acid decarboxylase (GAD). Here, we used Cre recombinase mouse lines to induce

expression of mutant muscarinic inhibitory (hM4D) and excitatory (hM3D) receptors on PV+ or

CCK+ neurons. Activation of the mutant receptors with clozapine-n-oxide (CNO) was used to

measure how amygdala neural changes affect the acoustic startle reflex (ASR). Excitation of

amygdala PV+ neurons potentiated the ASR. Activation of basolateral amygdalar CCK+ neurons

potentiated the ASR and caused seizures, possibly by activating glutamate CCK+ neurons. The

CCK+ subset of GAD neurons were targeted with a new triple transgenic mouse line (Dlx5-

flpe/CCK-Cre/FrePe) to show that most CCK+ neurons were GAD negative. These findings are

compared with optogenetic approaches to target specific neuronal populations.

III

Acknowledgments

Thank you to my supervisor and mentor Dr. John Yeomans. We thank Dr. Junchul Kim for

providing pharmacogenetic tools. Thanks to Sabina Romanescu for immunohistochemistry work.

Thanks also to Robin Nguyen and Shadi Bakir for assistance with pharmacogenetic equipment,

and Dr. Haoran Wang for his guidance. This work was supported by a NSERC grant to J.Y.

IV

Acknowledgments..........................................................................................................................III

1 Introduction .................................................................................................................................1

1.1 Fear- and Anxiety-Related Behaviours ................................................................................2

1.2 Brain Circuits and Fear Behaviour ......................................................................................4

1.3 Parvalbumin and Cholecystokinin .......................................................................................5

1.3.1 Parvalbumin .............................................................................................................5

1.3.2 Cholecystokinin .......................................................................................................7

1.4 Pharmacogenetics ................................................................................................................9

2 Materials and Methods ..............................................................................................................10

2.1 Mice ...................................................................................................................................10

2.2 Pharmacogenetic manipulation of CCK+ and PV+ neurons .............................................11

2.3 Stereotaxic Injections .........................................................................................................11

2.4 Perfusion and Sectioning ...................................................................................................12

2.5 Immunohistochemistry and Histology ...............................................................................12

2.6 Behaviour ...........................................................................................................................14

2.7 Statistical Analysis .............................................................................................................16

3 Results .......................................................................................................................................17

3.1 Expression of hM3D and hM4D in Neurons .....................................................................17

3.2 Acoustic Startle Reflex ......................................................................................................27

4 Discussion .................................................................................................................................31

4.1 Functions of PV+ Neurons in Amygdala ...........................................................................32

4.2 Functions of CCK+ Neurons in Amygdala ........................................................................33

5 Future Directions .......................................................................................................................34

6 Conclusions ...............................................................................................................................36

7 References .................................................................................................................................37

1

1 Introduction

Anxiety disorders are the most common type of psychiatric disease, with many co-

morbidities, including depression and addictions ( Kessler et al., 2005, Koob, 2009, Ressler &

Mayberg, 2007). Anxiolytic drugs, like benzodiazepines, are among the most commonly

prescribed pharmaceuticals in North America (Kessler et al., 2005). The amygdala, a brain

structure important for processing emotionally salient information, has a central role in fear- and

anxiety-related behaviours in humans and rodents (LeDoux, 2003, Lieb, 2005, Pare et al., 2004).

Anxiolytics enhance inhibition of the amygdala via GABA-A receptors, so amygdala neurons

that release the inhibitory neurotransmitter GABA may be important controlling anxiety- and

fear-related behaviours.

The role of amygdalar GABA neurons in fear- and anxiety-related behaviours can be

studied causally using new genetic tools to excite or inhibit neurons. Here, the function of

amygdalar parvalbumin (PV+) and cholecystokinin (CCK+) neurons in the mouse acoustic

startle reflex (ASR) was studied using pharmacogenetic methods (Rogan & Roth, 2011).

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1.1 Fear- and Anxiety-Related Behaviours

Autonomic and behavioural reactions to threatening stimuli promote survival in

dangerous conditions, such as responding to the presence of a predator with increased heart rate

and startle sensitivity (Lang et al., 2000). “Fear”, a psychological term, is used in the animal

literature to summarize the many physiological effects of threatening stimuli, such as foot shock

or predators (Davis, 1992). Fear has an identifiable eliciting stimulus, like perception of a

predator, whereas anxiety is a state of apprehensiveness with predisposed sensitivity to

threatening stimuli. Basic aspects of fear- and anxiety-related behaviours and their brain

correlates are conserved across mammalian species (Grillon, 2002). Stereotyped fear- and

anxiety-related behaviours, such as the startle reflex, are quantifiable and are used as measures of

fear and anxiety in rodents and humans (Grillon, 2002, Lang et al., 1990).

The startle response protects mammals from blows by shutting the eyes, dorsiflexion of

the neck, contracting the muscles of the body wall and (Yeomans et al., 2001). Sudden and

intense vestibular, tactile, and acoustic stimuli elicit the startle reflex. Acoustic stimuli over 80

dB at all audible frequencies can elicit startle, and louder noises elicit a more intense ASR

(Yeomans & Frankland, 1995a). The intensity of the ASR is calculated from the amount of

vertical force produced by rodent limb extension 10-200 ms after the eliciting stimulus.

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Fear-inducing stimuli potentiate the startle reflex (Yeomans & Pollard, 1993).

Presentation of a fear-conditioned stimulus (e.g., light cue previously paired with foot shock)

immediately before the startling acoustic stimulus potentiates the startle response. Potentiation

describes startle responses with greater amplitude beginning at lower sound pressure levels.

Conversely, reward-conditioned cues (e.g., light cue paired with food) attenuate the startle

response (Schneider & Spanagel, 2008, Steidl et al., 2001). The potentiated or attenuated

conditioned startle responses return to baseline levels through extinction learning. During

extinction of conditioned startle, the subject learns through repeated exposure that a conditioned

cue (e.g., light) is no longer associated with the unconditioned stimulus (e.g., foot shock).

Acquisition and expression of cue-conditioned startle require an intact amygdala (Campeau &

Davis, 1995).

The elevated plus maze (EPM) and open-field locomotion test (OFT) are used to assess

anxiety levels (Carola et al., 2002). Mice show anxiety-related behaviours in open spaces, so

exploration of open spaces by freely moving mice is characteristic of reduced anxiety levels. The

relative amount of time spent in the “open” aspects of the EPM or open field compared to the

“closed” aspects is used as a measure of anxiety. Stimulating different amygdala nuclei change

anxiety-related behaviours (Davis, 1992, Tye et al., 2011).

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1.2 Brain Circuits and Fear Behaviour

The amygdala nuclei are limbic structures located in the ventral forebrain of mammals

and other vertebrates. The amygdala is important in the processing and storage of emotional

information, such as fear memories. The amygdala is anatomically divided into the cortical,

medial, basolateral (BLA) and central (CeA) nuclei. The BLA is cortex-like, consisting of 75%

glutamatergic projection neurons and 25% GABA interneurons (Sosulina et al., 2010). The CeA

is striatum-like, with over 90% medium spiny GABA neurons found in the lateral CeA (CeL)

and a small minority of glutamate neurons concentrated in the medial subnucleus of the CeA

(CeM) (Swanson & Petrovich, 1998). The BLA receives multimodal sensory information from

the thalamus and cortex (Davis, 1992). BLA neurons project to the CeA, which in turn sends

projections to the hypothalamus, the central gray, and brainstem targets that mediate autonomic

and somatic expression of fear (Ehrlich et al., 2009). Activation of the CeM induces startle

responses in rodents (Petrovich & Swanson, 1997, Yeomans & Pollard, 1993). CeM neurons

project to the deep layers of the superior colliculus, which in turn synapse neurons of the caudal

pontine reticular formation (PnC) (Frankland & Yeomans, 1995, Meloni & Davis, 1999). PnC

neurons synapse on interneurons and the cranial and spinal motor neurons that mediate startle

(Yeomans & Frankland, 1995b).

5

Chemical lesioning of the BLA modestly increases the basal ASR in rats (Wan &

Swerdlow, 1997), but GABA-A receptor agonist muscimol in the BLA reduces the ASR

(Forcelli et al., 2012). Optogenetic inhibition of BLA glutamate neuron cell bodies does not

affect mouse anxiety phenotypes in the EPM and the OFT (Tye et al., 2011). Optogenetic

activation of BLA terminals in the CeM increases, while activation of the BLA terminals in the

CeL decreases, anxiety phenotypes in the EPM and OFT by feed-forward inhibition of the CeM

(Haubensak et al., 2010, Tye et al., 2011). The majority of CeA-projecting BLA neurons project

to either the CeM or the CeL, with less than 10% having terminals in both nuclei (Tye et al.,

2011).

1.3 Parvalbumin and Cholecystokinin

1.3.1 Parvalbumin

PV is a calcium-binding protein whose expression defines a class of GABA interneurons.

PV helps these neurons sustain high firing frequency by sequestering cytotoxic calcium (Baude

et al., 2007). PV interneurons are distributed throughout the cortex and the hippocampus, and PV

projection neurons are especially enriched in the reticular thalamic nucleus and the cerebellum

(Freund, 2003). PV interneurons have extensive axonal arborisation, and hippocampal PV

interneurons can contact thousands of other cells (Klausberger et al., 2003). Also, PV

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interneurons exhibit fast-spiking, firing at frequencies over 100 Hz (Freund, 2003). PV+ GABA

neurons in the BLA modulate principal neuron activity (Bienvenu et al., 2012).

Benzodiazepines treat a variety of anxiety disorders in humans and effectively block

panic attacks in humans (Malizia et al., 1998) and are used for their amnestic and sedative

properties in traumatic medical procedures (Saari et al., 2011). BLA infusion of benzodiazepines

and other GABA-A agonists is anxiolytic in rats and mice (Davis et al., 1994, Forcelli et al.,

2012, Hodges et al., 1987, Petersen et al., 1985, Sajdyk & Shekhar, 1997). GABA-A receptors

with α1 subunits mediate the amnesic effects of BLA benzodiazepines on aversive learning

tasks in mice (Tomaz et al., 1993). In the BLA, 70% of α1+ neurons were also PV+, and

95% of PV+ neurons were α1+ (McDonald & Mascagni, 2004). BLA benzodiazepines also

disrupt hippocampal-dependent learning, like the inhibitory avoidance task (Tomaz et al.,

1993). BLA PV+ neurons have an important role in the effects of benzodiazepines on fear

behaviour.

BLA PV+ neurons were proposed to function in pacing theta rhythm modulation with the

ventral hippocampus, facilitating memory formation of noxious stimuli (Ehrlich et al., 2009).

However, PV+ neurons do not fire in synchrony with hippocampal theta, nor do they selectively

respond to noxious stimuli (Bienvenu et al., 2012). PV+ neurons tonically inhibit principal

neurons in the BLA (Bissière et al., 2003, Chu et al., 2012).

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1.3.2 Cholecystokinin

CCK is a peptide neurotransmitter in the mammalian CNS that has important functions in

learning, feeding, and nociception (Bowers et al., 2012, Noble et al., 1999, Rotzinger et al.,

2002). Several splice variants derived from the pre-pro-CCK peptide exist in the brain, and the

sulfated octapeptide CCK-8S is among the most abundant peptides in the brain (Bowers et al.,

2012). CCK-8S is found throughout the brain, with higher concentrations in the ventromedial

hypothalamus, amygdala, hippocampus, and the frontal and temporal cortexes (Beinfeld et al.,

1981). Quantification of peptide levels is difficult because anti-CCK antibodies have low

specificity (Wyeth et al., 2012). One-third of BLA neurons produce CCK mRNA in mice, as

determined by fluorescent in situ hybridization (Jasnow et al., 2009). CCK is released from

dense-core vesicles by neurons that also release classical neurotransmitters, like GABA or

dopamine (Rotzinger et al., 2002, Van Megen et al., 1996).

Systemic CCK induces panic in humans with panic disorders and increases fear-related

behaviours in rats (Bradwejn et al., 1990, Frankland et al., 1997, Jerabek et al., 1999, Josselyn et

al., 1995). Knockdown of BLA CCK expression with viral transduction of shRNA decreases

EPM anxiety phenotype and despair-like behaviour in the forced swim test (Del Boca et al.,

2012). Amygdalar infusion of the CCK-B receptor agonist pentagastrin increased the ASR

8

twofold in rats, and this effect of pentagastrin is blocked by pretreatment with systemic CCK-B

receptor antagonist L-365, 260 (Frankland et al., 1997).

The CCK-B receptor is the primary subtype in the mammalian CNS and is widely

expressed, with similar distribution to the CCK peptide (Noble et al., 1999). The CCK-8S,

CCK-8, and CCK-4 fragments show equal affinity for the CCK-B receptor (Bowers et al., 2012).

The CCK-B receptor is Gi/o coupled, and activation of CCK-B increases ADP-ribose cyclase

activity leading to release of intracellular calcium stores through the ryanodine receptor

(Spampanato et al., 2011). In the hippocampus and the amygdala, CCK-B receptors are primarily

localized to fast-spiking GABA neurons (Chung & Moore, 2009), including PV+ neurons (Lee

& Soltesz, 2011). CCK excites PV+ and other fast-spiking neurons in the BLA, leading to

hyperpolarization of BLA principal neurons (Chung & Moore, 2007, Chung & Moore, 2009,

Rainnie et al., 2006, Woodruff & Sah, 2007).CCK does not affect late-spiking interneurons,

which often contain CCK or neuropeptide Y (Ascoli et al., 2008). In the hippocampus, CCK-B

receptors on glutamate neurons can couple to Gq release, which enhances phospholipase C

activity and the production of endocannabinoids (Lee et al., 2011).

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1.4 Pharmacogenetics

Pharmacogenetics, also called pharmacosynthetics, is “a branch of biology which deals

with the creation of pharmacological modulation using artificial components” (Farrell & Roth,

2012). Pharmacogenetic methods can excite or inhibit neurons using transgenic receptors

activated by a synthetic ligand. Mutant G protein-coupled receptors have been evolved by high-

throughput selection to respond to synthetic small molecules but not their natural ligands

(Coward et al., 1998).

In behaving mice, expression of transgenic receptors is restricted to genetically specified

neurons using intracranial injection of Cre-inducible viral vectors and Cre mouse lines. Two

muscarinic receptors (hM3 and hM4) were mutated and engineered (hM3D and hM4D) by two

point-mutations in order to be activated by clozapine N-oxide (CNO) at concentrations where

CNO is otherwise pharmacologically inert (Dong et al., 2010). The newly designed receptors,

hM3D and hM4D, are insensitive to the endogenous ligand acetylcholine at physiological

concentrations (Armbruster et al., 2007, Nawaratne et al., 2008). The hM3D receptor activates

excitatory Gαq signaling, and the hM4D activates inhibitory Gαi signaling (Rogan & Roth,

2011). Neurons can be activated or inhibited in behaving mice with systemic injection of CNO.

CNO is administered to mice intraperitoneally at a concentration of 1-10 mg/kg. CNO has a

plasma half-life of 110 minutes in the mouse.

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2 Materials and Methods

2.1 Mice

Male and female CCK-IRES-Cre, PV-2A-Cre, GAD65-IRES-Cre mice (129/Sv ×

C57BL/6) and WT C57BL/6 littermates or age-matched controls were used for all behavioural

tests. Dlx5-Flpe/CCK-Cre/FrePe triple transgenic mice were used to label forebrain CCK+

GABA neurons. Dlx5-Flpe/CCK-Cre/R26c-of-Halo mice were used to drive expression of

halorhodopsin in forebrain CCK+ GABA neurons.

All testing was conducted between 1000 h and 1400 h. Mice were group-housed at the

Ramsay Wright Zoological Building of the University of Toronto and were maintained in a light-

and humidity controlled room (12:12 h light:dark cycle with lights on at 0900 h) at 22±1◦C.

Laboratory Rodent Diet 5001 (Purina LabDiet, St. Louis, MO, USA) and water were available ad

libitum except during behavioural testing. All experimental protocols using mice were approved

by the University of Toronto Animal Care Committee, in accordance with the Canadian Council

on Animal Care guidelines.

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2.2 Pharmacogenetic manipulation of CCK+ and PV+ neurons

CNO activation of excitatory hM3D and inhibitory hM4D mutant muscarinic receptors

was used to control CCK+ and PV+ neuron activity. Expression of the receptor genes was

restricted to amygdalar GABA+ and PV+ neurons with stereotaxic injection of Cre-recombinase-

dependent adeno-associated virus (AAV-hsyn-FLEX-hM3D-mCherry and AAV-hsyn-FLEX-

hM4D-mCherry) (obtained from the University of North Carolina Vector Core) into GAD65-Cre

and PV-Cre mice.

2.3 Stereotaxic Injections

Eight to 10 week old mice were anesthetized with isoflurane (1.5-3% in oxygen),

positioned in a stereotaxic frame, and received Anaphine (3 mg/kg) and Baytril (2.5 mg/kg)

intraperitoneally. AAV-GFP, AAV-mCherryhM3D, or AAV-mCherryhM4D (6.14 X 1016

pfu/ml) was injected in the BLA (-1.34 mm AP; ±2.75 mm ML; -4.75 mm DV) bilaterally with a

Hamilton needle syringe (0.3 µL; 1µL/min) through a steel needle injector (28 gauge). The

injector was removed 10 min after each injection. Postoperatively, mice received Anaphine (0.2

mg/kg) for 2 days.

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2.4 Perfusion and Sectioning

After behavioural testing, mice were anesthetized with intraperitoneal (i.p.) Somnotol

(86.5 mg/kg sodium pentobarbital) and transcardially perfused with phosphate buffered saline

(PSB) and then 10% formalin. Following perfusion, mice were decapitated, the top of the skull

was removed, and brains were post-fixed overnight in 10% formalin. Twenty-four hours later,

brains were immersed in a 30% PBS-sucrose solution for 24 hr. Brains were cryosectioned

coronally at -20°C at a thickness of 40μm. Sections were transferred to section storage buffer

(30% sucrose, 30% ethylene glycol, 40% PBS at pH 7.4).

2.5 Immunohistochemistry and Histology

Brain slices were stained with fluorescently labeled antibodies for PV, CCK8S, GAD65,

and cFos expression. Brain sections with the amygdala (AP: -0.58mm to -2.5mm) were washed

in PBS for 5 min. Sections were collected in 1.5mL Eppendorf tubes and permeabilized in 0.1%

Triton-X in PBS solution (PBS-T) for 30 min on a pivoting shaker at room temperature. Next,

the slices were blocked with 16% normal goat serum in 0.1% PBS-T for 30 mins on a shaker at

room temperature. The slices were incubated with primary antibodies for 48h for CCK8 (rabbit

polyclonal anti-body; Sigma-Aldrich, St. Louis, MO; 1:800 dilution) and for 78h for anti-

parvalbumin (rabbit polyclonal anti-body; Sigma-Aldrich; 1:800 dilution) or anti-cFos (rabbit

polyclonal anti-body; 1:2000; Santa Cruz, Dallas, TX). The sections were then washed in PBS,

13

and then incubated for 2h in secondary antibodies (AlexaFluor488 or AlexaFluor568 goat anti-

rabbit IgG; 1:1000; Calbiochem, Billerica, MA). The sections were again washed with PBS and

then incubated for 5 min with 1:10,000 4',6-diamidino-2-phenylindole, DAPI stain (Sigma-

Aldrich, St. Louis, MO). The slices were washed 3 times for 5 min with PBS and mounted on

glass slides.

Brain sections (40 µm) were imaged with an Olympus FSX100 fluorescence microscope

(Olympus America Inc., NJ) at 4.2X objective. Further magnification and contrast adjustments

were performed digitally with CellSens software (Olympus).

Because CCK is expressed in both GAD+ and GAD- neurons, Dlx5-Flpe/CCK-Cre/FrePe

mice were used to visualize the number of recombined CCK+ GABA interneurons expressing

eGFP (Figure 1).

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2.6 Behaviour

All behavioural tests were conducted 14 - 28 days after surgery. When relevant,

behavioural tests were performed an experimenter blind to mouse genotype. On day 1, mice

received i.p. injections of CNO (0.5 mg/kg) in a 10% DMSO-saline solution (10 mL/kg) or a

DMSO-saline control injection immediately prior to testing. On day 8, mice received the

opposite treatment (CNO or vehicle). Treatment order was counterbalanced so that on day 1 half

Figure 1. FrePe dual-recombinase-based genetic fate mapping with Flpe/FRT and

Cre/loxP system in the Dlx5-Flpe/CCK-Cre/FrePe mouse. Flpe-recombinase drives

expression of mCherry in all forebrain GABA neurons that have not expressed CCK and

Cre-recombinase drives eGFP expression in forebrain neurons that have expressed both

GABA and CCK. Figure courtesy of Junchul Kim.

15

of the mice received the vehicle and the other half received CNO. Results are presented as mean

± SEM.

Acoustic Startle Reflex

Startle response was measured with the Animal Acoustic Startle System (Med

Associates, Whitehall, PA, USA). Four startle chambers with internal dimensions of 64 cm x 60

cm x 38 cm containing a PHM255A Stimulus package with a dual speaker were used. The

mouse holding cage (4 cm x 5 cm x 5 cm) was positioned 5 cm in front of the pulse speaker and

the background speaker produced a white noise of 65 dB throughout behavioural testing. The

mouse holding cage was fixed to a PHM-250A load cell platform transducer. The load cells were

connected to a SG-6503AS Startle Interface Cabinet with 28 Volts DC. The interface cabinet

was controlled with a desktop computer running Research Startle Control and Data Collection

Software (Med Associates Inc., SOF-825, St. Albans, VT., USA). Mice were habituated to the

startle chambers with 70 dB ambient noise for 10 minutes per day for 2 days. Immediately

following the second habituation, acoustic startle sweeps were run. Startle sweeps consisted of 2

ms white noise stimuli, ranging from 75 to 120 dB in 5 dB steps, as measured by a Quest 2100

SPL meter (Quest Technologies, Oconomowoc, WI, USV). Stimuli were presented in a

pseudorandomized order every 30 sec during 30 min blocks. Each compete startle test was

composed of 3 consecutive 30 minute blocks. In total, 18 presentations of each of 10 sound

16

pressure levels comprised a complete startle test. Startle response was measured as the peak

value (unitless) detected between 10 ms-250 ms after the acoustic stimulus presentation.

The CCK-B receptor blocker L365 260 (3 mg/kg) diluted with 25% DMSO in saline was

injected i.p. 30 min before CNO injection. The ASR was tested as described above with vehicle-

injected controls.

2.7 Statistical Analysis

A two-way ANOVA with repeated measures was used to assess the effect of CNO

treatment on the ASR compared to mice treated with a vehicle control. The means for the ASR

(10 trials) at each of the 10 sound pressure levels for mice (n=4) were analyzed. For each mouse,

the variance within the 10 trials at each sound pressure level was not considered in the analysis.

The Mauchly test was used to assess sphericity. The Bonferroni post hoc test was used to

compare treatment differences at each sound pressure level.

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3 Results

3.1 Expression of hM3D and hM4D in Neurons

To control CCK+ and PV+ neuronal populations in the amygdala, AAV-hsyn-

FLEX-hM3D-mCherry was injected into the BLA of PV-Cre and CCK-Cre mice.

500µm

B

C

Figure 2. The amygdala nuclei and PV+ neurons. A. Coronal section of the amygdala - Bregma -

1.34 (Paxinos & Franklin, 2004; ASt – amygdalostriatum, BLA – basolateral amygdala, CeL –

central lateral amygdala, CeM – central medial amygdala, GP – globus pallidus, LA – lateral

amygdala). B. BLA neurons expressing mCherry in an Epe (Cre-responsive reporter) mouse. C.

mCherry reports hM3D on virally transduced BLA PV+ neurons.

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In PV-Cre mice, mCherry-tagged hM3D (Figure 3) and hM4D (Figure 4) receptors were

expressed by IHC labeled PV+ neurons. The mCherry reporter was expressed in dendrites,

axons, and cell bodies of PV+ neurons (Figure 6). No staining was observed in WT mice infected

with the same viruses (not shown). Therefore, hM3D and hM4D receptors were expressed in the

amygdala of PV-Cre mice in a Cre-dependent manner.

200µm

200µm

Figure 3. Representative hM3D-mCherry expression (red) and PV antibody (green) in

the lateral amygdala of transduced PV-Cre mice (n = 4). Double-staining indicates that

PV+ neurons are concentrated in the BLA of the amygdala.

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Figure 4. Representative hM4D-mCherry expression (red) and PV IHC (green) in the lateral

amygdala of transduced PV-Cre mice (n = 4). Double-staining indicates that the majority of

PV+ neurons also express the hM4D marker mCherry.

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Figure 5. Basolateral amygdala neurons double-labelled with PV IHC (green) and mCherry

fused to hM3D (red puncta). Both anti-PV fluorscence and mCherry fluorscence are strongest in

the somata compared to neuronal processes.

Staining for cFos expression was used to determine whether hM3D receptor activation

excited PV+ neurons and other neurons. A low degree of co-localization between cFos and

hM3D was observed in mice perfused 1h after CNO treatment (Figures 7 & 8). cFos was

observed in the central medial nucleus

21

D

Figure 7. Amygdala of PV-Cre mouse with hM3D perfused 1 hr after CNO treatment: A.

hM3D-mCherry expression B. cFos IHC C. merged image shows a low level of co-

expression in the amygdala of PV-Cre mouse perfused 1h after CNO treatment. D. cFos

IHC in BLA of non-transduced WT mouse perfused 2h after CNO treatment.

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Figure 8. PV+ amygdala neurons expressing hM3D-mCherry (red) in mice treated with CNO.

Few transduced PV+ neurons were also positive for the activity marker cFos (green), like the

yellow-circled neuron. Most PV+ neurons were negative for the cFos marker, like the neuron

circled in white.

Six of 6 GAD65-Cre mice bilaterally transduced with hM3D or hM4D showed significant

viral expression of mCherry in the medial nuclei of the amygdala and in the striatum (Figure 9).

50µm

23

Figure 9. Representative hM3D- and hM4D-mCherry (red) expression in the amygdala of

transduced GAD65-Cre mice. mCherry was consistently observed in medial nuclei of the

amygdala.

Expression of Cre-inducible viral hM3D was observed in non-GABA neurons. Figure 10

shows that all infected lateral amygdala neurons expressed mCherry. Figure 11 shows a lateral

viral injection miss, indicating that infected cortical neurons also expressed mCherry. CCK and

GAD65 mRNA expression profiles (Figure 12) indicate that CCK mRNA is produced by many

non-GABA neurons.

200µm

24

Figure 10. Representative (red) expression in lateral amygdala of transduced CCK-Cre mice.

mCherry was expressed in non-GABA neurons. The density of mCherry expression in the BLA

suggests that transgenes are expressed in non-GABA neurons.

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Figure 12. CCK FISH and CaMKII genetic reporter. A. Expression of CCK mRNA on one

section from the Allen Brain Atlas (Dong, 2008) and B. GFP reporting glutamate neurons in a

Epe/CaMKII-Cre mouse.

A

Figure 11. hM3D-mCherry expression in cortex of mouse with viral injection lateral to BLA

target. This lateral miss shows that many cortical pyramidal neurons are transduced by the

virus.

500µm

26

To determine what proportion of lateral amygdala GABA neurons expressed CCK, Dlx5-

Flpe/CCK-Cre/FrePe triple transgenic mice were used to report CCK+ GABA neurons with

eGFP and the remaining GABA population with mCherry (Figure 13).

Figure 13. Amygdala of Dlx5-Flpe/CCK-Cre/FrePe reporter mouse. A. CCK+ GABA neurons

expressing eGFP (green) were found in the lateral amygdala nuclei and the central amygdala.

Central amygdala CCK+ GABA neurons have extensive local processes. B. Non-CCK GABA

are in the lateral amygdala nuclei and a dense population was observed in the central nuclei. C.

Merged image with green CCK+ GABA neurons and red non-CCK GABA neurons.

27

To visualize CCK peptide in the amygdala, anti-CCK8S staining was used (Figure 14). The

highest concentration of CCK8S was observed in the central nuclei, where the antibody strongly

labeled neuronal processes.

Figure 14. Amygdala anti-CCK8S (RFP) staining shows CCK peptide concentrated in local

processes of central amygdala neurons. Genetically labeled CCK GABA neurons (YFP green)

were most concentrated in the amygdalostriatal transition area (AStr).

3.2 Acoustic Startle Reflex

To determine whether PV+ neurons in the amygdala affects the ASR, hM3D- or hM4D-

transduced PV-Cre mice were given CNO (0.5mg/kg) in saline (10 ml/kg) immediately before

behavioural testing. CNO treatment in hM3D mice potentiated ASR compared to vehicle control

200µm

28

treatment (F1,3 = 12.392, P<0.05) (Figure 15). CNO treatment did not significantly affect the

ASR of PV-Cre mice with amygdalar injection of AAV-hM4D (Figure 16).

Figure 15. hM3D-mediated excitation of PV amygdala neurons potentiated the ASR in mice

with CNO treatment compared to vehicle treatment (±SEM, n = 4). *P < 0.05, *P<0.01

29

Figure 16. CNO treatment did not affect the ASR of PV-Cre mice with BLA injection of AAV-

hM4D compared to treatment with vehicle (±SEM, n = 4).

CNO treatment potentiated the ASR and caused seizures in 3 CCK-Cre mice with amygdala

injetions of AAV-hM3D (Figure 17). Testing in all 3 mice was stopped prematurely due to

seizures.

30

Figure 17. Individual ASR traces from CCK-Cre mice A, B, and C with amygdala AAV-

hM3D. Error bars are ±SEM, n = 10

A B

C

31

4 Discussion

In these studies, the role of genetically defined classes of amygdala GABA neurons in the

mouse ASR was measured. GABA neuron populations were controlled by AAV transduction of

Cre-inducible genes for the excitatory hM3D and inhibitory hM4D receptors. PV-Cre mice with

BLA injections of AAV-hM3D or AAV-hM4D showed robust expression of the receptor marker

mCherry in PV+ neurons. CNO activation of hM3D receptors in PV-Cre mice potentiated the

ASR. CNO treatment in hM4D transduced PV-Cre mice did not affect the ASR significantly.

In CCK-Cre mice with BLA injection of AAV-hM3D, viral expression was restricted to

the lateral amygdala nuclei and the adjacent cortex. Many non-GABA neurons were virally

transduced in the CCK-Cre mouse line, as determined by a comparison to an intersectional

reporter mouse that only labeled neurons expressing both a GABA marker and CCK peptide.

CCK peptide stained most densely in the processes of central amygdala neurons, and cell bodies

were not discernible with this anti-CCK antibody.

CNO-mediated excitation of transduced neurons in CCK-Cre mice resulted in seizures,

consistent with activation of CCK+ glutamate neurons. Together, the results indicate that the

function of amygdala GABA neurons in fear behaviours may be studied causally using

pharmacogenetic strategies. The potent effect of BLA PV+ neuron excitation observed here

suggests that this small population may exert large effects on fear-related behavior.

32

4.1 Functions of PV+ Neurons in Amygdala

hM3D-mediated excitation of PV+ neurons was observed to potentiate the ASR (Figure

16). Previously, disinhibition of PV+ neurons by genetic deletion of GABA-A receptor γ2

subunits was found to potentiate the ASR (Leppa et al., 2011). PV+ neurons tonically inhibit

principal neurons in the BLA, and dopamine inhibits GABA release from PV neuron terminals

via the D2 dopamine receptor (Bissière et al., 2003, Chu et al., 2012). BLA dopamine facilitates

fear memory formation and extinction (Bissière et al., 2003), and PV neuron D2 receptors may

function to disinhibit principal neurons. Therefore, activating PV+ neurons could interfere with

BLA function in tuning the central amygdala response to threatening stimuli. Perhaps, in this

study, activating PV+ neurons interfered with within-session habituation to startling acoustic

stimuli, resulting in an increased mean startle response. However, there was no apparent

treatment trend in the level of startle responses at different time points in the ASR tests.

hM4D-mediated inhibition of amygdala PV+ neurons did not affect the ASR (Figure 17).

BLA neurons expressed mCherry in IHC-labeled PV+ neurons at levels similar to those

observed in hM3D-transduced mice. Also, hM4D-mediated inhibition of hippocampal PV+

neurons did not affect behavioural phenotypes, whereas optogenetic inhibition of these

hippocampal neurons had a strong effect on behaviour (Robin Nguyen, personal

correspondence).

33

We have not measured cellular changes in PV+ amygdalar neurons, so our assumption of

“excitation” and “inhibition” is made on previous studies of hippocampal pyramidal neurons

(Armbruster et al., 2007, Nawaratne et al., 2008). In vitro, CNO activation of hM4D receptors

hyperpolarized hippocampal PV+ neurons (Vivek Mahadevan and Melanie Woodin, personal

correspondence). In a hippocampal slice preparation with hM4D-expressing PV+ neurons, CNO

was superfused at concentrations that approximate brain levels achieved by IP injections, and

PV resting membrane potential was hyperpolarized. Therefore, hM4D receptor activation may be

insufficient to inhibit PV+ neurons in vivo and thus does not affect the ASR. Also, even if partial

inhibition of PV+ neurons was achieved using hM4D activation, there may be redundant

inhibition of principal neurons from other GABA neuron populations (Ascoli et al., 2008)

4.2 Functions of CCK+ Neurons in Amygdala

CNO administered to CCK-Cre mice with amygdala injection of AAV-hM3D potentiated

the ASR and induced seizures (Figure 18). Viral expression of mCherry-labeled hM3D receptors

was observed throughout the BLA (Figure 10), consistent with CCK mRNA expression in

roughly one third of BLA neurons (Jasnow et al., 2009). CCK peptide, however, was observed in

far fewer neurons of the BLA (Mascagni & McDonald, 2003), and CCK stained most densely in

processes of central amygdala neurons in our study (Figure 11). Because viral hM3D and hM4D

transgenes are under a constitutively active promoter, Cre-mediated activation of transcription

results in high levels of transgene expression. Transgene expression levels are not representative

34

of CCK peptide levels, because these proteins are regulated differently. In this study,

pharmacogenic excitation equally affected neurons at all levels of CCK expression. It is unlikely

that the population activated in our study is functionally distinct from all other BLA neurons.

Therefore, hM3D-mediated activation of the BLA enhances the ASR and induces seizures.

CCK expression in GABA neurons may define a functionally distinct group of neurons,

as is the case in the hippocampus. Genetic labeling of CCK+ GABA neurons using an

intersectional reporter mouse line showed a small number of CCK+ GABA neuronal soma with

extensive neuronal processes in the amygdalostriatal area. However, our IHC results suggest that

the central nucleus contains the greatest amount of CCK peptide in the amygdala (Figure 14).

These CCK+ processes in the central amygdala may arise from CCK+ dopaminergic projections

(van Megen et al., 1996).

5 Future Directions

Using optogenetics, we can assess whether inhibiting amygdalar CCK+ GABA neurons

is anxiolytic. The Dlx5-Flpe/CCK-Cre /R26c-of-halo mouse line was developed to restrict

expression of receptor transgenes to the CCK+ subset of GABA neurons in the forebrain.

Experiments using the excitatory channelrhodopsin can measure the effect of neurotransmitter

release from CCK+ GABA neurons on fear-related behaviours. Because the receptors are

expressed congenitally, only transient transcription of loci with Cre and Flpe recombinases is

35

sufficient to activate transgene expression. Therefore, all neuronal lineages with a history of

GABA and CCK co-expression may express the rhodopsins, and CCK staining may be necessary

to confirm neuronal selectivity of transgene expression.

CCK+ GABA neurons express CB1 receptors on their terminals in the amygdala

hippocampus and cortex (Brown et al, 2003). In future experiments, cannabinoids may be used

to block or enhance the effects of CCK+ GABA neuron excitation or inhibition on fear- and

anxiety-related behaviours. Cannabinoids affect anxiety states and fear learning in mice (Jasnow

et al., 2009). CB1 knockout mice were deficient in tests of fear extinction but acquired fear

memories normally (Marsicano et al., 2002) . Systemic CB1 receptor blockade with rimonabant

led to dose-dependent decreases in extinction of fear-potentiated startle without affecting

baseline startle (Chhatwal et al., 2008). However, in the same study, the CB1 receptor agonist

WIN did not affect fear extinction. Interestingly, the endocannabinoid breakdown and reuptake

inhibitor AM404 enhanced fear extinction without affecting baseline startle (Chhatwal et al.,

2004). None of these manipulations affected basal startle responses, suggesting that test of fear

learning may be more sensitive to CCK+ GABA neuron manipulation than tests of

unconditioned fear. CB1 receptors are found on 80-90% of CCK+ neurons in the rat amygdala

(Katona et al., 2001, Mascagni & Mcdonald, 2003), and CB1 receptors presynaptically inhibit

CCK+ interneuron terminals through Gi/o coupling that inhibits adenylyl cyclase (Mcdonald &

Mascagni, 2001). Post-synaptic glutamate neurons produce the endocannbinoids (eCB) 2-

36

arachidonoyl glyceride (2-AG) in an activity dependent manner, and 2-AG passively crosses the

cell membrane to the synaptic cleft (Lee et al., 2011). Therefore, presynaptic CB1 receptors

target inhibition of GABA release at specific synapses, without somatically inhibiting

interneurons that synapse a multitude of other neurons.

Moving forward, the combination of drug treatments with optogenetic or

pharmacogenetic manipulations of GABA interneurons in the amygdala will permit a deeper

understanding of their role in fear behaviours. Because interneurons release transmitter locally,

optogenetic stimulation and drug injection can be administered through a cannula at the same

brain site. This approach may be especially important for parsing the contributions of CCK and

GABA release from the same neurons on fear behaviours. For example, intra-amygdala

pretreatment with the CCK-B receptor blocker L365 260 followed by optogenetic activation of

CCK+ GABA neurons may uncover the effects of GABA release alone on fear behaviours.

6 Conclusions

In summary, exciting BLA PV+ GABA neurons potentiates the ASR in mice. Also, our

findings show the distribution of CCK peptide in amygdala and provide a framework for targeted

control of CCK+ GABA neurons using an optogenetic strategy. Importantly, our results using a

the Dlx5-Flpe/CCK-Cre/FrePe reporter mouse suggests that the distinct concentration of CCK

peptide in the central amygdala is not produced by CCK+ GABA neurons (Figure 13). Exploring

37

the function of PV+ and CCK+ GABA neurons may be important in understanding fear and

anxiety disorders and their pharmacological treatments. Therefore, future mouse experiments

will use drugs, like cannabinoids and benzodiazepines, to test how selectively altering PV and

CCK GABA interneuron activity affects fear-related behaviours. Our work provides a proof of

concept for the use of new genetic tools to target control of this powerful interneuron population.

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