A Dissertation Entitled Pituitary Adenylate Cyclase Activating

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A Dissertation

entitled

Pituitary Adenylate Cyclase Activating Polypeptide and Synaptic Plasticity at

Autonomic Cholinergic Synapses

Eric R. Starr

Submitted to the Graduate Faculty as partial fulfillment of the requirements

for the Doctor of Philosophy Degree in Biomedical Sciences

______Joseph F. Margiotta, PhD, Committee Chair

______David R. Giovanucci, PhD, Committee Member

______Scott Molitor, PhD, Committee Member

______Joshua Park, PhD, Committee Member

______Ruili Xie, PhD, Committee Member

______Amanda Byrant-Friedrich, Dr. rer. Nat., Dean College of Graduate Studies

The University of Toledo

August 2017

This document is copyright material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author.

An Abstract of

Pituitary Adenylate Cyclase Activating Polypeptide and Synaptic Plasticity at Autonomic Cholinergic Synapses

Eric R. Starr

Submitted to the Graduate Faculty as partial fulfillment of the requirements of the Doctor of

Philosophy Degree in Biomedical Sciences

The University of Toledo

July 2017

Pituitary adenylate cyclase activating polypeptide (PACAP) is a secretin family

neuropeptide, localized to presynaptic terminals throughout the nervous system. In

autonomic ciliary ganglion (CG) neurons, PACAP exposure engages a PACAP type 1

receptor (PAC1R) signaling cascade that rapidly enhances the function of nicotinic

acetylcholine receptors (nAChR)-mediated synapses both immediately and 24, 48 and 72

hours after PACAP treatment. In this dissertation, I sought to examine the mechanisms

underlying the short-term (ST) and long-term (LT) PACAP-induced synaptic plasticity in

CG neurons. Our lab previously demonstrated that the immediate, ST PACAP-induced

synaptic plasticity, characterized by increases in sEPSC frequency and sEPSC amplitude

is dependent upon canonical AC, cAMP, PKA signaling and neuronal nitric oxide

synthase (NOS1). I participated in collaborative studies to determine how PACAP

stimulates nitric oxide (NO) production and elucidate the mechanisms underlying the ST

PACAP-induced synaptic plasticity. Live-cell imaging revealed that PACAP stimulated

NO production, an effect that required NOS1, PKA, Ca2+ influx, and nAChR activation.

iii

Scavenging of extracellular NO blocked the PACAP-induced synaptic plasticity supporting a role for NO retrograde signaling (post- to presynaptic) on presynaptic ACh release. Both α3*- and α7 nAChRs are potentiated by PKA phosphorylation and were required for both PACAP-induced NO synthesis and increases in sEPSC frequency and sEPSC amplitude. Coimmunoprecipitation experiments show that NOS1 associated with

α7 nAChRs and α3*-nAChRs, suggesting that NOS1-nAChR physical associations could facilitate NO production to enhance ACh release from juxtaposed presynaptic terminals.

The ST PACAP-induced synaptic plasticity was dependent upon localized PKA signaling by PKA anchoring proteins (AKAP). PKA regulatory-subunit overlay assays identified five AKAPs in CG lysates and inhibiting binding of the PKA regulatory subunit to

AKAP blocked the ST PACAP-induced synaptic plasticity. Taken together, our findings indicate that PACAP/PAC1R signaling coordinates nAChRs, NOS1, and AKAP activities to induce targeted, retrograde plasticity at autonomic synapses.

PACAP treatment also induces a LT synaptic plasticity 48 hours after PACAP washout that exhibits a similar increase in sEPSC frequency, but featured more robust increases in sEPSC amplitude. Pharmacological studies verified that PAC1R mediated

AC and PLC signaling cascades was required. However, inhibition of NOS1, PKA and nAChR activiation, three critical effectors underlying the ST PACAP-induced synaptic plasticity had no effect on the LT PACAP-induced synaptic plasticity. Instead inhibition of gene transcription attenuated the LT PACAP-induced synaptic plasticity but had no effect on ST PACAP treatment. In concert with increases in sEPSC frequency and amplitude, the synaptic correlates underlying this LT PACAP-induced plasticity reflect increases in post- and presynaptic strength. The LT PACAP-induced plasticity was

iv accompanied by significant increases in miniature EPSC amplitude (postsynaptic quantal size), α3*-nAChR sensitivity, mEPSC frequency and ACh release (presynaptic quantal content). No detectable differences were observed in excitability suggesting that the physiological hallmarks underlying the LT PACAP-induced plasticity are confined to presynaptic terminals and postsynaptic densities. Analysis of confocal images confirmed these findings, identify that the LT effects of PACAP significantly enhanced the size of individual presynaptic puncta and postsynaptic nAChR clusters labeled with SV2 and nAChR specific antibodies, respectively, as well as the size, density, and number of colocalized pre- and postsynaptic puncta. These results indicate that PACAP induces LT synaptic plasticity via PAC1R induced activation of AC and PLC signaling and gene transcription, an effect results in the structural rearrangement of associated pre- and postsynaptic components. Taken together, our results demonstrate that PACAP induces

ST and LT plasticity through distinguishable biochemical, physiological and synaptic mechanisms.

Acknowledgments

This dissertation would not have been possible had it not been for the guidance and mentoring I received from Dr. Joseph Margiotta. Dr. Margiotta’s excellent guidance and training, as well as his passion for scientific inquiry and drive for scientific discovery have and will always shape my future endeavors. I would also like to thank Samantha

McKee whose support, friendship, and insights have been indispensable in the completion of this dissertation.

Also, this dissertation would not have been possible had it not been for the absolute dedication, encouragement, and love I receive from my fiancé Alison as well as my family. I am so thankful for their continuous optimism, sacrifice, and support during my time at the University of Toledo. Their guidance has always inspired me to never give up and to always keep learning and reach for the stars. As dad always says: “Stay calm;

Stay cool; Don’t drool in school.”

Contents

Abstract iii

Acknowledgments vi

Contents vii

List of Tables xii

List of Figures xiii

List of Abbreviations xv

1 Introduction to Pituitary Adenylate Cyclase Activating Polypeptide 1

1.1 Synaptic Plasticity and Pituitary Adenylate Cyclase Activating

Peptide………………………………………………………………….. 1

1.2 Synthesis and Transport of PACAP in the Nervous System………….... 3

1.3 PACAP Receptors……………………………………………………… 5

1.4 Termination of PACAP Signaling……………………………………… 9

1.5 Is PACAP a Neurotransmitter, Neuromodulator, or Both?...... 12

1.6 PACAP Signaling at Central Synapses………………………………... 14

1.6.1 Hypothalamus…………………………………………………. 14

1.6.2 Hippocampus………………………………………………….. 16

1.6.3 Amygdala……………………………………………………... 23

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1.7 PACAP Signaling at Autonomic Synapses…………………………… 26

1.7.1 Ciliary Ganglion………………………………………………. 26

1.7.2 Cardiac Ganglion……………………………………………… 30

1.7.3 Submandibular Ganglion……………………………………… 31

1.7.4 Superior Cervical Ganglion…………………………………… 33

1.8 Conclusions and Dissertation Hypothesis…………………………….. 35

2 PACAP Induces Plasticity at Autonomic Synapses by nAChR-

Dependent NOS1 Activation and AKAP-Mediated PKA Targeting 39

2.1 Abstract……………………………………………………………….. 39

2.2 Introduction…………………………………………………………… 41

2.3 Methods……………………………………………………………….. 43

2.3.1 Neuronal Preparation…………………………………………. 43

2.3.2 Electrophysiological Recording………………………………. 44

2.3.3 Nitric Oxide Imaging………………………………………….. 45

2.3.4 Detection of Endogenous NOS1………………………………. 47

2.3.5 nAChR-NOS1 Co-Precipitation……………………………….. 48

2.3.6 AKAP5 Detection……………………………………………… 50

2.3.7 Statistics……………………………………………………….. 51

2.4 Results…………………………………………………………………. 51

2.4.1 PACAP Stimulates NOS1 to Increase NO Levels…………….. 51

2.4.2 NO is a Retrograde Messenger………………………………… 54

2.4.3 PACAP-Induced PKA Dependent Up-regulation of α7-

nAChRs Underlies NO Elevation…………………………...... 56

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2.4.4 Concomitant nAChR Activation Underlies PACAP/PAC1R-

Induced Synaptic Plasticity……………………………………………. 61

2.4.5 nAChRs Associate with NOS1……………………………….. 66

2.4.6 AKAP5 is Present in CG Neurons and Targets PACAP/

PAC1R Signals to Synapses…………………………………… 68

2.5 Discussion……………………………………………………………... 72

3 PACAP Induces Long-term Synaptic Plasticity Through Coordinated

AC and PLC Signaling and Gene Transcription 81

3.1 Introduction……………………………………………………………. 81

3.2 Methods………………………………………………………………... 83

3.2.1 Neuronal Cultures……………………………………………… 83

3.2.2 Electrophysiology……………………………………………… 84

3.2.3 Drug Treatments……………………………………………….. 85

3.2.4 Statistical Evaluation…………………………………………... 86

3.3 Results…………………………………………………………………. 87

3.3.1 PACAP Induces Short-term and Long-term Synaptic

Plasticity……………………………………………………….. 87

3.3.2 PACAP Induces Long-term Synaptic Plasticity via Adenylate

Cyclase/cAMP and Phospholipase C-Mediated Signal

Transduction...... 90

3.3.3 The ST and LT PACAP-Induced Synaptic Plasticity are

Mechanistically Distinct………………………………………. 92

3.3.4 The LT PACAP-Induced Synaptic Plasticity Requires Gene

Transcription but is Independent of PAC1R Internalization…... 97

3.4 Discussion……………………………………………………………… 100

4 The Long-term PACAP-Induced Synaptic Plasicity Features

Increases in Post-and Presynaptic Strength through Dynamic

Synaptic Remodeling. 105

4.1 Introduction…………………………………………………………… 105

4.2 Methods………………………………………………………………. 107

4.2.1 Neuronal Cultures……………………………………………. 107

4.2.2 Drug Treatments……………………………………………… 107

4.2.3 Electrophysiology…………………………………………….. 108

4.2.4 Synaptic Architecture………………………………………… 110

4.2.5 Statistical Evaluation………………………………………… 112

4.3 Results……………………………………………………………….. 113

4.3.1 The LT PACAP-Induced Synaptic Plasticity Features no

Changes in Neuronal Excitability……………………………. 113

4.3.2 The LT PACAP-Induced Synaptic Plasticity Features

Increases in Postsynpatic Strength…………………………… 115

4.3.3 The LT PACAP-Induced Synaptic Plasticity Features

Enhancments in Quantal Content…………………………….. 118

4.3.4 The LT PACAP-induced Synaptic Plasticity is Characterized

by Pre- and Postsynaptic Remodeling……...... 122

4.4 Discussion…………………………………………………………….. 127

5 Discussion of Findings 133

5.1 Summary of Findings………………………………………………… 133

5.2 General Discussion…………………………………………………… 136

References 156

List of Tables

4.1 CG Neuron Excitability Parameters……………………………………… 114

4.2 Parameter Values for Spontaneous mEPSCs and Asynchronous mEPSCs 121

4.3 Structural Correlates of PACAP-induced LT and ST Synaptic Plasticity 125

4.4 The LT and ST Effects of PACAP Exhibit Distinguishable Phsiological

and Synaptic Hallmarks.………………………………………………….. 128

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List of Figures

1.1 Synaptic plasticity at chemical synapses…………………………………… 4

1.2 Canonincal signal transducing pathways coupled to PACAP-

mediated PAC1R, VPAC1,2R activation. ………………………………….. 8

1.3 Simplified summary of PACAP actions at synapses where PACAP is

detected in presynaptic terminals and its receptor isoforms are found on

postsynaptic cells………………………………………………………….. 38

2.1 PACAP increases NO production in CG neruons by activating

endogenous NOS1…………………………………………………………. 53

2.2 NO generated by PACAP signaling in CG neurons acts as a retrograde

messenger to induce synaptic plasticity…………………………………… 55

2.3 PACAP-induced NO elevation assess by increased DAF-FM FLS*

requires PKA, Ca2+ influx and α7 nAChR activation……………………... 59

2.4 nAChR participation in PACAP-induced synaptic plasticity……………... 64

2.5 α7- and α3*-nAChRs interact with NOS1………………………………… 67

2.6 AKAP expression and PKA targeting to nAChR-mediated synapses by

PACAP/PAC1R signaling…………………………………………………. 70

2.7 Proposed model of nAChR-mediated autonomic synapses before

(Left) and after (Right) PACAP exposure to induce plasticity…………… 79

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3.1 PACAP triggers short-term (ST) and long-term (LT) plasticity at

nicotinic synapses…………………………………………………………. 88

3.2 PACAP induces LT synaptic plasticity via PAC1Rs and requires

activation of both AC- and PLC-dependent signaling cascades………….. 93

3.3 The LT PACAP-induced synaptic plasticity is independent of PKA

activation, neuronal activity, and NOS1…………………………………... 98

3.4 The LT but not the ST PACAP-induced synaptic plasticity requires

protein synthesis, while neither requires PAC1R internalization…………. 101

4.1 The LT PACAP-induced synaptic plasticity stabilizes CG neuron

excitability……………………………………………………………….. 116

4.2 The LT PACAP-induced plasticity enhances quantal size and α3*-nAChR

sensitivity………………………………………………………………… 119

4.3 The LT PACAP-induced plasticity enhanced presynaptic quantal

release…………………………………………………………………….. 123

4.4 The LT PACAP-induced synaptic plasticity features dynamic post- and

presynaptic remodeling…………………………………………………... 126

5.1 The ST and LT PACAP-induced synaptic plasticity are mediated

by distinct intracellular signaling mechanisms…………………………… 140

5.2 Summary figure of the LT PACAP-induced synaptic plasticity…………. 154

xiv

List of Abbreviations

αBgt……………….. α-bungarotoxin AC………………… Adenylate cyclase ACh……………….. Acetylcholine Act-D……………. Actinomycin-D αCTx-AuIB……….. α-conotoxin AuIB AKAP……………... A-kinase anchor protein AMPAR…………… α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor ANS……………….. Autonomic nervous system AP2………………... Adaptor protein2

BK channel……….. High conductance, calcium- and voltage-depedent potassium channel

BLA……………….. Basolateral amygdala BNST…………….... Bed nucleus of the stria terminalis

CA………………… Catecholamine CaMK……………... Calcium calmodulin kinase cAMP……………… Cyclic adenosine monophosphate CCh……………….. Carbachol CdG………………... Cardiac ganglion cDNA……………... Complementary deoxyribonucleic acid CeA………………... Central amygdala CG………………… Ciliary ganglion ChAT……………… Choline acetyltransferase CHO………………. Chinese hamster ovary CICR……………… Calcium-induced calcium release CME………………. Clathrin-mediated endocytosis CNS………………... Central nervous system CN VII……………. Cranial nerve 7; Facial nerve CN III……………… Cranial Nerve III; Occulomotor nerve CN X…………….... Cranial nerve 10; Vagus nerve CNQX…………….. 6-cyano-7-nitroquinoxaline-2,3-dione CA1, 3…………….. Cornu ammonis 1, 3 cPTIO……………... 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide CREB…………...... cAMP response element binding protein

DAF-FM…………... 4-amino-5-methylamino-2’7’-difluorofluorescein DAG……………..... Diacyl glycerol DG………………… Dentate gyrus DPP-4……………... Dipeptidyl peptidase IV dTC……………….. d-tubocurarine DOPAC…………… 3,4-Dihydroxyphenylacetic acid

EGTA…………….. Ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic Acid Epac……………….. Exchange factor directly activated by cAMP EPSP………………. Excitatory postsynpatic potential ER…………………. Endoplasmic reticulum ERK……………….. Extracellular signal regulate kinase EDW………………… Edinger Westphal nucleus

GABA…………….. Gamma-aminobutyric acid GLuA1, GLU1R…… Glutamate receptor 1 GluN2……………... NR2 subunit of N-methyl-D-aspartic acid receptor Gαs,q,i………………. G-protein alpha- s, -q, -i subunit g-PCR……………... G-protein coupled receptor

HaChT…………….. High-affinity choline transporter HCN………………. Hyperpolarization-activated cyclic-nucleotide gated channels HCN………………. Hyperpolarization-activated cyclic-nucleotide gated channels HCN………………. Hyperpolarization-activated cyclic-nucleotide gated channels HEK………………. Human embryonic kidney HFS……………….. High frequency stimulation Ht31………………. AKAP fragment encompassing the RII-binding domain Ht31P……………... Inactive proline analog of Ht31 HVA………………. Homovanillic acid

IML……………….. Intermediolateral IP…………………... phosphatidylinositol-4,5-biphosphate IP3…………………. Inositol-1, 4, 5-triphosphate

KO………………… Knock out Kv4.2………………. Voltage gated potassium Channel alpha 4.2 lCeA……………….. Lateral central amydala

xvi

LTD……………….. Long-term depression L-NAME………….. L-NG-Nitroarginine methyl ester LT…………………. Long-term LTP………………... Long-term potentiation

M1-4……………… Muscarinice acetylcholine receptors type 1, 2, 3, 4 mAB35……………. Mouse monoclonal antibody specific α1, α3, α5 containing nAChRs mAChRs………….. Muscarinic acetylcholine receptors MAGUK…………... Membrane-associated guanylate kinase MAHMA-NANOate. (Z)-1-(N-Methyl-N-[6-(N-methylammoniohexyl)amino])diazen- 1-ium1,2,-diolate MAPK…………….. Mitogen-activated protein kinase MDL……………… (±)-N-[(1R*,2R*)-2-Phenylcyclopentyl]-azacyclotridec-1-en-2- amine hydrochloride or MDL 12330A hydrochloride

MDM2……………. Mouse double minute 2 homolog mEPSC……………. Miniature excitatory postsynaptic current Musc……………… Muscarine

nAChR……………. Nicotinic acetylcholine receptor NCS……………….. Neuritogenic cAMP sensor NMDAR…………... N-methyl-D-aspartic acid receptor NFкB……………… Nuclear factor kappa-light-chain-enhancer of activated B cells NOS1……………… Nueronal nitrix oxide synthase NO………………… Nitric oxide

p300………………. EP300, E1A binding protein p300 PACAP……………. Pituitary adenylate cyclase activating polypeptide PACAP-/-…………... PACAP deficient PAC1R…………….. Pituitary adenylate cyclase activating polypeptide type 1 receptor PI3K………………. Phosphatidic acid Pit 2……………….. Pitstop 2 PKA……………….. Protein kinase A PKC……………….. Protein kinase C PKI………………... Protein kinase inhibitor-(14-22)-amide, myristoylated PLC……………. Phospholipase C PP…………………. Perforant pathway PP1………………… Protein phosphatase 1 PP2A………………. Protein phosphatase 2A PSD-95, 93………... Postsynaptic density protein-95, 93 PTSD……………… Post-traumatic stress disorder Pyk2………………. Non-receptor tyrosine kinase

xvii

RIIβ……………….. PKA regulatory subunit type II β RGC………………. Retinal ganglion cell Rp-cAMPs………… Cyclic 3',5'-[hydrogen (R)-phosphorothioate] adenosine, triethylammonium salt RSarg……………….. Recording solution with 1 mM L-arginine RShs………………... Recording solution supplemented with horse serum RT…………………. Room temperature

SCG………………... Superior cervical ganglion SCN……………….. Suprachiasmatic nucleus sEPSC……………... Spontaneous excitatory postsynaptic synaptic current sIAHP……………….. Slow afterhyperpolarizing current SMG………………. Submandibular Ganglion Src………………… Sarcoma viral oncogene homolog ST…………………. Short-term SV2………………… Synaptic vesicle associated protein 2

TTX……………….. Tetrototoxin

U7, U73122……….. 1-[6-[[(17β)-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]- 1H-pyrrole-2,5-dione

VDCC……………... Voltage-dependent calcium channel VIP………………… Vasoactive intestinal polypeptide VMN………………. Ventral medial nucleus VPAC1R…………… Vasoactive intestinal polypeptide type 1 receptor VPAC2R…………… Vasoactive intestinal polypeptide type 2 receptor

xviii

Chapter 1

Introduction to Pituitary Adenylate Cyclase Activating

Polypeptide

(This Chapter contains a book chapter copied with permission from Springer

Nature. The Chapter was modified with permission from Eric Starr and Joseph

Margiotta.)

Starr, E. R., and Margiotta, J.F. (2016). PACAP Modulates Distinct Neuronal components to Induce Cell-Specific Plasticity at Central and Autonomic Synapses. In D.

Reglodi, and A. Tamas (Ed.) Pituitary Adenylate Cyclase Activating Polypeptide-PACAP

(83-107). Cham, Switzerland: Springer International Publishing.

1.1 Synaptic Plasticity and Pituitary Adenylate Cyclase Activating

Polypeptide

Neurons communicate with one another at chemical synapses to coordinate sensory information, visceral functioning, cognition, and behavior. Synaptic transmission, initiated via the arrival of an action potential and subsequent activation of voltage-activated calcium channels in presynaptic terminals, occurs following the exocytosis of synaptic vesicles containing neurotransmitters from presynaptic terminals

1 and activation of both ionotropic and metabotropic receptors on postsynaptic targets

(Purves et al. 2012; Nicholls et al. 2012). Neuronal control of synaptic efficacy is plastic and synaptic communication between two adjacent neurons can ultimately become strengthened or weakened (Baudry et al. 1993). Referred to as synaptic plasticity, researchers have primarily focused on examining the mechanisms underlying activity- dependent forms of synaptic plasticity in mediating homeostatic control of chemical synapses (Fig. 1.1). However, the mechanisms underlying other forms of synaptic plasticity that are not exclusively activity-dependent, such as pervasive synaptic modification, are less well understood (Fig. 1.1).

Pervasive synaptic modification can be mediated by activation of metabotropic g- protein coupled receptors (GPCR) or receptor tyrosine kinases by neuropeptides, growth factors and stress factors. One such neuropeptide that induces pervasive synaptic modification is pituitary adenylate cyclase activating polypeptide (PACAP). PACAP is a pluripotent secretin family neuropeptide synthesized by neurons in the central nervous system (CNS) and the autonomic nervous system (ANS; Reviewed in Vaudry et al.

2009). Depending on the context, PACAP signaling has been implicated in neuromodulatory, immunomodulatory, and neurotrophic functions via activation of its associated GPCRs. While impressive in scope, the pluripotent nature of PACAP in the nervous system challenges attempts to develop hypotheses about the significance of endogenous PACAP signaling on visceral regulation, cognition, behavior and neurological disorders it is implicated in such as depression (Hashimoto et al. 2010;

Pinhasov et al. 2011; Pinhasov et al. 2016 ), post-traumatic stress disorder (Ressler et al.

2011), anorexia (Kocho-Schellenberg et al. 2014; Iemolo et al. 2015) schizophrenia

(Ishiguro et al. 2001; Hashimoto et al. 2007; Katayama et al. 2009; Moody et al. 2011), and migraine (Schytz et al. 2009; Vécsei et al. 2014). At synapses PACAP is a potent neuromodulator capable of inducing both short-term and long-term synaptic plasticity by altering pre- and postsynaptic components as well as the excitable properties of neurons

(Reviewed in Starr and Margiotta, 2016). Despite the relevance of synaptic activity on visceral functioning and behavior, the mechanisms underlying PACAP-induced synaptic plasticity at chemical synapses is still an emerging picture.

1.2 Synthesis and Transport of PACAP in the Nervous System.

PACAP was discovered in anterior pituitary cells in the ovine hypothalamus and is expressed as either a full length 38 amino acid polypeptide (PACAP38) or a truncated

27 amino acid polypeptide (PACAP27) (Miyata et al. 1989, 1990; Vaudry et al. 2009).

The first 27 N-terminal amino acids are critical for the biological actions of PACAP, and have almost been completely conserved across species, suggesting that PACAP exerts essential biological functions (Sherwood et al. 2000). The active sequence shares 68% identity with that of vasoactive intestinal polypeptide (VIP) and both peptides activate the same type of receptors. Within neurons, PACAP is synthesized through gene transcription from its associated gene (ADCYAP1) and is stored at presynaptic terminals in dense core vesicles (Chiba et al. 1996; Légrádi et al. 1997; Reimer et al. 1999; Seki et al. 2000; Vaudry et al. 2009). To date, no PACAP transporters have been identified on either neurons or in astrocytes, indicating that PACAP expression in presynaptic terminals is primarily mediated by gene transcription and retrograde axonal transport.

Figure 1.1: Synaptic plasticity at chemical synapses. Each set of synapses reflects a functional chemical synapse in which a presynaptic terminal (Axon) converges near (synapse) a postsynaptic dendrite. In the first 4 sets of images, synaptic plasticity is triggered by fluctuations in neuronal activity which can positively or negatively affect overall output. Each set of synapses reflects a mechanism by which activity-dependent synaptic plasticity can occur. Synaptic plasticity can be a product of alterations in the amount of receptors expressed on postsynaptic terminals or the amount of transmitter released from presynaptic terminals (Synaptic strength), the formation of new synapses (Synapse number), changes in the properties or number of voltage-dependent ion channels underlying action potential propagation (Neuronal excitability), and even reflect changes in the type of receptors and neurotransmitter expressed within post-and presynaptic terminals respectively (Neurotransmitter respecification). Pervasive synaptic modification is contingent upon activation of metabotropic receptors via nonclassical neurotransmitters such as neuropeptides, stress factors and growth factors. Unlike the previous types of synaptic plasticity, pervasive synaptic modification is not necessarily dependent on neuronal activity.

However, PACAP signaling in the nervous system is not only confined to release from presynaptic sites. PACAP is readily transported across the blood brain barrier via the saturatable peptide transport system-6 (Banks et al. 1993; Nonaka et al. 2002, 2005,

2012). This capacity for PACAP to be transported across the blood brain barrier has led to increased interest in a therapeutic application for PACAP in the treatment of neurodgenerative disesases such as Parkinson’s Disease and Alzheimer’s Disease

(Reviewed in Reglodi et al. 2011; Yang et al. 2015).

1.3 PACAP Receptors

PACAP is recognized by three distinct G-protein coupled receptors (GPCRs) that are distinguished according their affinity for PACAP and VIP and their potencies for linking peptide bonding to activation of canonical AC and PLC signaling cascades (Fig.

1.2; Rawlings S, 1994; Harmar et al. 2012). Type 1 receptors exhibit a greater affinity for

PACAP (Kd≈0.5 nM) than VIP (Kd > 500 μM) and are referred to as the PAC1 receptor

(PAC1R) (Spengler et al. 1993; Hashimoto et al. 1993; Vaudry et al. 2009; Blechman and

Levkowitz, 2013). The PAC1R is a seven transmembrane protein that shares similar homology with secretin (50%), VIP (54%) and glucagon (34%), and growth hormone releasing hormone receptors (40%) (Spengler et al. 1993). Modeling of ligand-receptor binding proposes that PACAP binds to the N-terminus or transmembrane domains of the

PAC1R (Furness et al. 2012). At PAC1Rs, PACAP triggers adenylate cyclase (AC) synthesis of cyclic adenosine monophosphate (cAMP) at ≈ 100-1000 greater potency than VIP. Similarly, PACAP-induced activation of the PAC1R activates phospholipase C

(PLC) to hydrolyze phosphatidylinositol-4,5-biphosphate (IP) into inositol-1,4,5-

5 triphosphate (IP3) and sn-1,2-diacylglycerol (DAG) with 100-1000 greater potency than

VIP (Buscail et al. 1990; Rawlings S, 1994; Margiotta and Pardi, 1995; Pardi and

Margiotta, 1999; Rampelbergh et al. 1997; Dickson and Finlayson, 2009; Blechman and

Levkowitz, 2013). Therefore, the PAC1R predominately couples to both canonical Gαs and Gαq signaling pathways which can result in activation of protein kinase C (PKC), protein kinase A (PKA), neuritogenic cAMP sensor (NCS) and exchange protein activated by cAMP (ePAC) and subsequently activate other downstream effectors (Fig.

1.2; Reviewed in Blechman and Levkowitz, 2013). One study has identified that the

PAC1R can couple to Gαi (Shi et al. 2010), however the effects of PAC1R-induced

2+ activation of Gαi on cAMP synthesis, [Ca ]in release, and IP turnover was not directly examined.

Twenty variants of the PAC1R have been cloned and exhibit alterations in intracellular loops, transmembrane domains, and N-terminal domains (Reviewed in

Dickson and Finlayson, 2009; Blechman and Lekowitz, 2013). Several PAC1R splice variants display preferential signaling mechanisms (Dautzenberg et al. 1999, Dickson and

Finlayson, 2009; Blechman and Levkowitz, 2013). In new born rat colliculi, Spengler et al. (1993) cloned four splice variants of the PAC1R that exhibited variability within the third intracellular loop region. Specifically, the variants were characterized by the absence (null), or presence one of two cassettes of 28 amino acids (hip1, hop1) or 27 amino acids (hop 2). The most prominent of all four receptors in the brain were the null and hop1 variants (Spengler et al. 1993, Journot et al. 1994). Quantification of cAMP and inositol phosphate production in transiently transfected LLC PK1 cells revealed that inclusion of the hip domain impaired PAC1R-induced activation of PLC while largely

6 enhancing cAMP accumulation. Additionally, PAC1Rs containing both the hip and hop1 domain exhibited increased sensitivity to PACAP38 than to PACAP27, and were characterized by increases in both cAMP synthesis and PLC signaling (Spengler et al.

1993; Journot et al. 1995). Despite the wide array of splice variants, the relevance of these variants on PACAPs endogenous functions is still somewhat elusive.

In addition to the PAC1R, PACAP also binds to the VPAC receptors (VPAC1R and VPAC2R). Expressed as seven transmembrane GPCRs, all VPACRs are categorized as type 2 receptors as they exhibit high-affinity for both VIP and PACAP (Kd ≈ 1 nM)

(Gottschall et al. 1990; Lam et al. 1990; Vaudry et al. 2009). Unlike the PAC1R, PACAP- induced activation of the VPACRs exhibits a similar potency in stimulating intracellular cAMP production as VIP (Lutz et al. 1993, Harmar et al. 2012). While VPACRs predominately couple to AC they can couple to PLC signal transduction and Gαi

(Sreedharan et al. 1994; Mackenzie et al. 1996; Rampelbergh et al. 1997; Langer and

Robberecht, et al. 2005; Dickson and Finlayson, 2009). In COS7 cells transfected with cDNAs coding for the VPAC1R or VPAC2R, stimulation with 3μM VIP resulted in a 2.55 and 2.00-fold increase in IP3 accumulation respectively (Mackenzie et al. 1996).

Interestingly, inhibition of Gαi with pertussis toxin reduced IP3 accumulation in VPAC1R transfected cells by 46% and by 38% in VPAC2R transfected cells suggesting that PLC activation was mediated, in part by stimulation of Gαi signaling (Mackenzie et al. 1996).

Consequently, while stimulation of VPAC1R and the VPAC2R primarily is associated with canonical AC signaling (Fig. 1.2; Reviewed in Vaudry et al. 2009; Dickson and

Finlayson, 2011) results from these studies confirms that these receptors can also couple their activation to Gαi as well. Several splice variants of the VPACRs have also been

Fig. 1.2: Canonical signal transducing pathways coupled to VIP/PACAP-mediated PAC1R, VPAC1,2R activation. Both the PAC1R and the VPAC1,2Rs predominately couple to Gαs (green) and Gαq (purple) mediated signal transducing pathways. The stimulatory Gα subunit (Gαs) selectively activates the transmembrane bound enzyme adenylate cyclase (AC) which functionally synthesizes the second messenger cyclic-adenosine monophosphate (cAMP) from adenosine triphosphate. cAMP can activate protein kinase A (PKA), neuritogenic cAMP sensor (NCS; Blechman and Levkowitz, 2013), and exchange protein activated by cAMP (ePAC; Ster et al. 2007, Ster et al. 2009) all of which can activate other downstream signaling cascades. Activation of the Gαq subunit selectively activates phospholipase C Beta (PLCβ), a membrane localized enzyme that hydrolyzes phosphatidylinositol-4,5-biphosphate (IP) into inositol-1,4,5-triphosphate (IP3) and sn-1,2-diacylglycerol (DAG). Both IP3 and DAG act as second messengers. DAG activates PKC while IP3 binds to IP3 receptors on the endoplasmic reticulum to 2+ cause release of intracellular Ca . Both the PAC1R and VPAC1,2R have been shown to also couple to the inhibitory Gα subunit (Gαi, not pictured) which functionally inhibits AC and enhances PLC signaling (Section 1.3).

8 reported however the effects of these variants on VPACR mediated signaling cascades has yet to be elucidated (Reviewed in Dickson and Finlayson, 2009).

1.4 Termination of PACAP Signaling

PACAP signaling can be terminated through desensitization and internalization of

PACAPs receptors as well as by enzymatic degradation of PACAP. Desensitization of

GPCRs is triggered by phosphorylation of intracellular loops by kinases such as PKA,

PKC, and g-protein receptor activated dependent kinases (GRKs) (Reviewed in

Gainetdinov et al. 2004; Sorkin and Zastrow, 2009). Phosphorylation of the GPCR triggers β-arrestin to bind to the receptor, impeding further activation of g-protein- mediated second messenger cascades. Additionally, β-arrestin triggers internalization of

GPCRs, bonding adaptor proteins (e.g. AP2) which functionally recruit the formation of clathrin coats. The GPCR/AP2/clathrin coat complex causes an invagination in the plasma membrane that is cleaved by the membrane localized enzyme dynamin following hydrolysis of ATP. Referred to as clathrin-mediated endocytosis (CME), the internalized

GPCR complex is transported to endosomes where the receptors are either degraded or recycled back to the plasma membrane (Reviewed in Gainetdinov et al. 2004; Sorkin and

Zastrow 2009). GPCRs can also be internalized via clathrin-independent endocytosis as well, however the mechanisms are still being elucidated (Scarcelli and Donaldson, 2009).

PAC1Rs exhibit desensitization following repeated exposure to PACAP, an effect mediated by GRK and PKC (Shintani et al. 2000; Dautzenberg and Hauger, 2001).

PAC1Rs are internalized within minutes of activation (Shintani et al. 2000; Germano et al. 2001; Dautzenberg and Hauger, 2001; Merriam et al. 2013). In NIH/3T3 fibroblasts

25 transfected with cDNAs encoding the PAC1R, radioligand application of I -PACAP-27 resulted in rapid internalization, with ≈ 50% of PAC1Rs being internalized 3 minutes after application of I25-PACAP-27 and reaching a plateau after 60 min (Germano et al.

2001). In HEK 293 cells overexpressing PAC1Rs as well as in cardiac ganglion neurons, which endogenously express PAC1Rs, inhibition of either clathrin coat formation with

Pitstop 2 or dynamin with dynasore blocks PAC1Rs internalization suggesting that

PAC1Rs s are internalized primarily through CME (Merriam et al. 2013; May et al.

2014). It is unknown whether internalized PAC1Rs are degraded or recycled back to the plasma membrane. Interestingly, in HEK 293 cells, inhibition of PAC1R internalization attenuated PACAP-induced activation of MAPK suggesting that PAC1R internalization is linked to the activation of intracellular signaling pathways (May et al. 2014). A similar effect was observed in dissociated superior cervical ganglion neuronal cultures in which attenuation of PAC1R internalization blocked PACAP-induced phosphorylation of AKT

(May et al. 2010). Taken together, these findings raise important questions regarding the role of PAC1R internalization in activating additional signal transducing cascades.

Both VPACRs are also rapidly phosphorylated, desensitized and internalized within minutes after activation (Shetzline et al. 2002; Langlet et al. 2004, 2005).

Biochemical experiments have confirmed that desensitization and internalization of the

VPAC1R is primarily mediated by GRK signaling (Shetzline et al. 2002). In HEK cells transfected with cDNAs encoding an epitope tagged VPAC1R, stimulation with 100 nM

VIP caused a rapid internalization of epitope tagged VPAC1Rs, with 50% of VPAC1Rs being internalizaed between 15-20 min. Confocal microscopy revealed that stimulation with VIP induced translocation of β-arrestin to the plasma membrane, with β-arrestin

10 being colocalized with VPAC1Rs on the plasma membrane and in endocytotic vesicles

(Shetzline et al. 2002). Subsequent co-immunoprecipitation confirmed the formation of a

β-arrestin/VPAC1R complex suggesting that internalization of VPAC1Rs on HEK cells was mediated by β-arrestin. The mechanisms underlying VPAC2R internalization remain unknown. Following internalization both VPACRs are trafficked to endosomes.

However, internalized VPAC1Rs and VPAC2Rs exhibit unique intracellular trafficking patterns. Two hours following ectopic application of 1μM VIP to CHO cells expressing either wild type VPAC1R or VPAC2R, recycled VPAC2Rs were present on the plasma membrane but no recovery was observed for phosphorylated VPAC1Rs suggesting that phosphorylation and internalization of VPAC1Rs occurs through a non-reversible pathway (Langlet et al. 2004, 2005; Reviewed in Dickson et al. 2009).

PACAP signaling can also be terminated through enzymatic degradation. In blood circulation, PACAP exhibits poor metabolic stability with a half-life of 2-10 min (Zhu et al. 2003; Li et al. 2007). One enzyme that breaks down PACAP is dipeptidyl peptidase

IV (DPP-4) (Zhu et al. 2003). DPP-4 is a cell surface serine peptidase that is distributed throughout the endothelial lining of the vasculature as well as circulates in a soluble form

(Mentlein, 1999). Inhibition of DPP-4 extends the half-life of intraveneously injected

PACAP38 in mice (Ahrén and Hughes, 2005). Despite these findings it is still unknown whether there are additional enzymes that terminate PACAP signaling. This is particularly important in the nervous system, where the neuromodulatory actions of

PACAP can induce drastic changes in the synaptic properties of neurons within minutes.

1.5 Is PACAP a Neurotransmitter, Neuromodulator, or Both?

Given that PACAP and its receptors are expressed throughout the central and autonomic nervous system (Vaudrey et al. 2009) the very nature of PACAP as a neurotransmitter or neuromodulator remains debated. The three minimal criteria for a neurotransmitter are its presence in and activity-dependent release from presynaptic nerve terminals (1, 2) and the expression of cognate receptors on adjacent postsynaptic cells (3)

(Purves et al. 2012). Neuromodulator criteria in some cases include, while in other cases exclude, those of neurotransmitters but the main distinctions are that neuromodulators can alter the presynaptic release or postsynaptic efficacy of conventional neurotransmitters, and can have effects that are spatially and temporally more widespread, impacting extrasynaptic receptors, even those on nonadjacent cells, and evoking changes that can persist for minutes, hours, or even days (Iverson, 1978;

Burrows, 1996; Pugh et al. 2009; Smith and Eiden, 2012). The existence of dual transmission where peptides such as PACAP and conventional neurotransmitters such as acetylcholine (ACh) are released from the same nerve terminal under different stimulus conditions (reviewed by Smith and Eiden, 2012) complicates a clear neurotransmitter/neuromodulator distinction. At all the nerve–nerve synapses considered below, including those on hypothalamic, hippocampal, amygdala, and autonomic neurons, PACAP signals via GPCRs on postsynaptic neurons (satisfying neurotransmitter criterion 3 above) and acts as a neuromodulator because it alters membrane properties or the release or efficacy conventional neurotransmitters. In many of the cases cited below

PACAP and conventional transmitters are also co-expressed in the same presynaptic terminal (satisfying criterion 1 above). For most, it is inferred or indirectly determined

12 that PACAP is also co-released (criterion 2). In parasympathetic cardiac and ciliary ganglia, criterion 2 has been satisfied directly by demonstrating activity-dependent release of PACAP from ACh containing nerve terminals (Tompkins et al. 2007; Pugh et al. 2009) qualifying it as a definitive neurotransmitter in these systems.

In the adrenal gland, PACAP satisfies the three criteria as a neurotransmitter since it is expressed in presynaptic splanchnic nerve terminals (Hamelink et al. 2002) and likely coreleased with ACh onto postsynaptic chromaffin cells that express PACAP receptors and nicotinic ACh receptors (nAChRs). Splanchnic nerve stimulation as well as exogenous ACh and PACAP stimulate catecholamine (CA) secretion from chromaffin cells but CA secretion occurs even when nAChRs are desensitized or blocked suggesting

ACh and PACAP operate independently as dual neurotransmitters (Kuri et al. 2009;

Smith and Eiden, 2012). Acting as a neurotransmitter, PACAP is thought to stimulate its selective high affinity receptor (PAC1R) and associated phospholipase-C (PLC) signaling pathways (See Below), leading to voltage-dependent Ca2+ channel (VDCC) activation and subsequent CA secretion (Smith and Eiden, 2012). The adrenal gland may be a unique case, however, since chromaffin cells unlike neurons are specialized for CA secretion, rather than synaptic communication with a postsynaptic target, and may be maximally increased by PACAP-induced Ca2+ mobilization, masking any contribution secretion from accompanying modulatory effects on nAChR mediated transmission (Starr and Margiotta, 2016). Thus, except for the specialized adrenal-chromaffin synapse where it apparently acts solely as a neurotransmitter, PACAP fulfills criteria as a neuromodulator and neurotransmitter at nerve–nerve synapses, with the caveat that for the latter case the release criterion has been verified in a limited number of cases.

1.6 PACAP Signaling at Central Synapses

1.6.1. Hypothalamus

The hypothalamus contains the densest PACAP immunopositive neurons, fibers, and binding sites in the central nervous system (Reviewed in Vaudry et al. 2009).

Consequently, it is not surprising that PACAP signaling induces synaptic plasticity in several regions in the hypothalamus including the suprachiasmatic nucleus and the ventromedial nucleus.

The suprachiasmatic nucleus (SCN) is a region in the hypothalamus that synchronizes hypothalamic neuronal activity to the circadian daily cycle of light and dark

(Colwell, 2011; Schmidt et al. 2011; Pickard and Sollars, 2012). The SCN receives excitatory inputs from photosensitive retinal ganglion cells (RGCs) containing melanopsin as a photopigment. PACAP is co-expressed with glutamate in RGC projections (Hannibal et al. 2001) and postsynaptic SCN neurons express ionotropic glutamate receptors (N-methyl-D-aspartate and α-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid receptors; NMDARs and AMPARs), as well as PAC1Rs and

VPAC2Rs (Hannibal et al. 2001; Michel et al. 2006, Schmidt et al. 2011). Evidence for

PACAP release from RGC axon terminals is indirect (Hannibal, 2002) but exogenous

PACAP does potently modulate glutamate-mediated transmission in the SCN (Michel et al. 2006). Here, both the frequency and amplitude of AMPAR-mediated spontaneous miniature excitatory postsynaptic currents (mEPSCs) recorded from SCN neurons increased minutes after 10 nM PACAP application. The effects are consistent with

PACAP enhancing presynaptic glutamate release and postsynaptic AMPAR and

NMDAR sensitivity, with the latter mediated via PAC1R activation of AC and PKA.

These findings indicate that PACAP is a potent neuromodulator and a possible neurotransmitter at RGC→SCN hypothalamic synapses. Interestingly, PACAP phase shifts the glutamate-induced circadian rhythm in SCN neurons (Harrington et al. 1999) and the circadian system is perturbed in mice deficient in PAC1R or PACAP (Hannibal et al. 2001; Kawaguchi et al. 2003) suggesting that such modulation may actively influence synchronization of SCN neurons to the light dark cycle in vivo.

The ventromedial nucleus (VMN) is another region in the hypothalamus where transmission is modulated by PACAP signaling. As the “satiety center” of the diencephalon, the VMN contains several groups of bilateral neurons that coordinate food intake, thermoregulation, energy expenditure, and sexual behavior (Reviewed in

McClellan et al. 2006; Meister, 2006; Choi et al. 2013). Excitatory activity in the VMN is primarily mediated by glutamatergic and GABAergic inputs and VMN neurons express all glutamate and gamma-aminobutyric acid receptor subtypes (Tappaz et al. 1977;

Meeker et al. 1994; Bäckberg et al. 2003; Bäckberg et al. 2004; Tong et al. 2007) .

PACAP is expressed in presynaptic terminals to the VMN, and VMN neurons express both PAC1R and the VPAC2R mRNA (Usdin et al. 1994; Sheward et al. 1996; Sheward et al. 1998; Hashimoto et al. 1996; Shioda et al. 1997; Dürr et al., 2007). In rats, bilateral microinjections of PACAP (50 pmol/0.25μl/side) to the VMN reduces feeding behavior for up to 6 hours, an effect accompanied significant elevations in core body temperature and locomotion for similar durations of time (Resch et al. 2011, 2013, 2014). The reduction in food intake and increases in core body temperature and locomotion occurred via a PAC1R-dependent mechanism and required Src-mediated phosphorylation of

Y1336 on the GluN2 subunit on NMDARs, an effect associated with enrichment in

NMDAR expression synaptically and extrasynaptically within hippocampal slices

(Goebel-Goody et al., 2009; Resch et al. 2011; 2013; 2014). Despite these findings the synaptic hallmarks underlying PACAP-induced effects on VMN neurons has not been fully elucidated. In one study cell-attached recordings in VMN slices following exogenous application of PACAP (100 nM) in the presence of the glutamate receptor antagonist CNQX and the GABAAR antagonist picrotoxin, increased the frequency of spontaneous postsynaptic potentials in VMN neurons, a result consistent with PACAP induced alterations in VMN neuronal excitability (Hurley et al. 2016). No other studies have been conducted elucidating the cellular mechanisms underlying the PACAP- induced effects in the VMN. Given PACAPs link to appetite (Kocho-Schellenberg et al.

2014, Iemolo et al. 2015), such experiments are critical in elucidating the functional role of PACAP signaling in the VMN.

1.6.2 Hippocampus

The hippocampus is a “C-shaped” limbic structure localized to the caudal portion of the brain that mediates cognitive functions such as memory formation, storage and retrieval. The hippocampus is composed of three distinct subregions referred to as the dentate gyrus (DG), the hippocampus proper (CA1, CA2, and CA3 regions) and the subiculum. In traditional anatomical models, excitatory glutamatergic projections within the hippocampus are preserved in a polysynaptic loop or “tri-synaptic” pathway (van

Strien et al. 2009; Purves et al. 2012). Briefly, glutamatergic inputs arising from the entorhinal cortex (perforant pathway (PP)) innervate granule neurons in the dentate gyrus

(DG). The DG granule neurons project excitatory “mossy fiber” outputs to pyramidal neurons in the CA3 region which then project excitatory glutamatergic “schaffer collaterals” afferents to pyramidal neurons in the CA1 region. Excitatory projections in the hippocampus are not necessarily unilateral in that pyramidal neurons from the CA1 to and CA3 back project to neurons in the DG, the CA1, and other regions in the hippocampus (Reviewed in van Strien et al. 2009). The conservation of the “trisynaptic loop” has established the hippocampus as an integral model for synaptic plasticity and memory (Neves et al., 2008; Purves et al. 2012). Long-term potentiation (LTP) and long- term depression (LTD) represent two distinct forms of synaptic plasticity intricately studied in the hippocampus that are associated with learning and memory. LTP is physiologically characterized by a persistent (<60 min) increase in the size of evoked responses recorded from individual neuron following a high frequency stimulus (e.g. 5-

100 stimuli, 100 Hz) to the pathway of interest (Purves et al. 2012; Nicholls et al. 2012).

LTD is physiologically characterized by a persistent (<60 min) decrease of synaptic strength below the initial baseline following a low frequency stimulus (e.g. 900 stimuli, 1

Hz) (Purves et al. 2012; Nicholls et al. 2012).

PACAP exerts neuromodulatory effects on DG granule neurons. While direct release of PACAP has never been examined in the DG, neurons in the entorhinal cortex synthesize PACAP and PACAP-immunopositive fibers are localized to DG granule neurons (Köves et al. 1994; Skolglösa et al 1999; Hannibal 2002). DG neurons also express mRNAs encoding all PACAP receptor subtypes (Hashimoto et al.1996; Lutz et al., 1993; Sheward et al., 1996). Microelectrode recordings from DG neurons following exogenous application of PACAP to hippocampal slices (1 μM, 10 min) showed that

PACAP induced a long-term synaptic plasticity significantly elevating the slope and amplitude of the field EPSPs by ≈ 120% and ≈142% for 5 hours after PACAP washout

(Kondo et al. 1997). The PACAP induced enhancement in field EPSPs was concentration-dependent, independent of concomitant NMDAR activation, and did not require PKC, PKA or calmodulin-dependent protein kinase II (CaMKII) signaling.

Interestingly, despite inducing a long-term synaptic plasticity in the DG, LTP induction

in vivo was only slightly perturbed in PAC1R knock out and heterozygous PACAP deficient mice (Matsuyama et al. 2002). Consequently, while these studies suggest that

PACAP plays a minimal role in the induction of LTP in PPDG synapses, they demonstrate that PACAP has the capacity to induce long-term synaptic plasticity at excitatory PPDG synapses. Future studies are required to elucidate the specific mechanisms and intracellular effectors underlying the PACAP-induced long-term synaptic plasticity at excitatory PPDG synpases

LTP at mossy fiber synapses (DGCA3) is mediated directly by PACAP signaling. DG granule neurons synthesize PACAP and PAC1Rs are expressed in post- and presynaptic terminals localized to CA3 pyramidal neurons, however corelease of

PACAP has never been demonstrated from mossy fiber synapses (Köves et al., 1994;

Piggins et al., 1996; Skoglösa et al., 1999; Otto et al. 1999, Hannibal, 2002). High frequency stimulation of presynaptic mossy fiber inputs in brain slices from both PAC1R

-/- knock out and PACAP deficient mice (PAC1R KO and PACP ) fails to induce LTP on

CA3 pyramidal neurons. These effects were not accompanied by changes in either post- tetanic potentiation or paired-pulse facilitation (Otto et al. 2001). Interestingly, behavioral

-/- experiments from this same study demonstrated that both PAC1R KO and PACAP mice

18 exhibited impairments in associative memory without altering declarative memory or declarative learning. While no other studies have been conducted examining the mechanisms or signaling pathways mediating the effects of PACAP signaling at

DGCA3 synapses, these findings clearly establish a role for PACAP signaling in modulating LTP at DGCA3 synapses as well as associative memory.

PACAP induces a diverse array of neuromodulatory effects at CA3CA1 synapses. PACAP is synthesized by CA3 schaffer collateral neurons (Skoglösa et al.

1999), and CA1 pyramidal neurons express PAC1Rs, VPAC1Rs, and VPAC2Rs (Joo et al.

2004) but activity-dependent PACAP release has not been demonstrated. Interestingly, exogenous PACAP induces synaptic potentiation or depression depending on the concentration applied. At low concentrations, (<10 nM) PACAP enhances CA3→CA1 synaptic transmission, characterized by a prolonged increased NMDAR and AMPAR sensitivity (Costa et al. 2009; Macdonald et al. 2007). The PACAP-induced increase in

NMDAR sensitivity involved a PAC1R induced PLC- and PKC-dependent activation of non-receptor tyrosine kinase, Pyk2, and subsequent recruitment and phosphorylation of

NMDARs by Src (Macdonald et al. 2005; Ali et al. 2001). The PACAP-induced increase in AMPAR-sensitivity is activated through a PAC1R-induced AC and PKA signal transducing cascade (Costa et al. 2009) as is increased phosphorylation at S845 on the

GluA1 AMPAR subunit, while an accompanying decrease in phosphorylation of GluA1

T840 is mediated via protein phosphatases 1 and 2A (PP1/PP2A) (Toda et al. 2009). The effects of PACAP signaling on presynaptic glutamate release remains controversial as one study observed that low concentrations of PACAP does not alter the probability of presynaptic glutamate release (Roberto et al. 2000) while a second study does identify

19 that low concentrations of PACAP reduces the probability of presynaptic glutamate release (Pecoraro et al. 2017).

In addition to increasing NMDAR/AMPAR sensitivity the effects of low concentrations of PACAP require concomitant activation of NMDARs and muscarinic acetylcholine receptors (mAChRs) (Roberto et al. 2000; Roberto et al. 2001).

CA3CA1 synapses receive afferent cholinergic projections from the medial septal area and muscarinic acetylcholine receptors (mAChRs, M1-M4), α7 homopentameric nicotinic acetylcholine receptors (α7-nAChRs) and heteropentameric α4β2 as well as

α3β4 containing nAChRs exhibit mixed pre- and postsynaptic expression in CA3 and

CA1 synapses (Teles-Grilo Ruivo and Mellor, 2013). PACAP induces ACh release in

CA3CA1 synapses and inhibition of mAChRs with atropine blocked the PACAP- induced augmentation in neuronal activity at CA3→CA1 synapses (Masuo et al. 1993;

Roberto et al. 2001). These findings suggest a requirement for cholinergic signaling in mediating the potentiating effects of low concentrations of PACAP observed at

CA3→CA1 synapses (Roberto et al., 2001).

Contrasting with the low concentration, PACAP applied at ≥10 nM induces a sustained depression of CA3→CA1 synaptic transmission that is associated with an AC,

Epac, p38 MAPK-mediated reduction in AMPAR ion currents and presynaptic glutamate release (Kondo et al. 1997; Roberto et al. 2001; Ster et al. 2009; Costa et al. 2009;

Pecoraro et al. 2017). Surprisingly, the synaptic plasticity induced by high PACAP concentrations was independent of the PAC1R, PKA, PKC, and NMDAR activation but mimicked by activation of VPAC2Rs concluded by using the selective VPAC2R agonist

Bay 55-9837 (Kondo et al. 1997; Costa et al. 2009). These findings indicate that

VPAC2R signaling alone could be responsible for the PACAP mediated depression at

CA3→CA1 synapses. Like PACAP, VIP applied at low concentrations (1 nM) potentiates CA3→CA1 synaptic transmission to a similar extent as low concentrations of

PACAP. However, the potentiating effects of VIP were mediated by both VPAC1Rs and

VPAC2Rs and by subsequent activation of PKA mediated signal cascades (Cunha-Reis,

2005; Cunha-Reis, 2006), indicating that VPAC2R activation does not necessarily result in synaptic depression making it unlikely that the PACAP-induced potentiation or depression at CA3→CA1 synapses is mediated by the activation of distinct PACAP receptor subtypes.

Despite inducing synaptic depression, high concentrations of PACAP enhances

CA1 pyramidal neuronal excitability. Whole-cell patch clamp recordings from CA1 neurons following PACAP application (100 nM, 5 min) in hippocampal slices, induced a slow-onset depolarizing current (Di Mauro et al. 2003) and subsequent experiments conducted by Taylor et al. (2014), found that application of PACAP (100-500 nM) suppressed the slow afterhyperpolarizing current (sIAHP) in CA1 neurons resulting in decreased spike frequency adaptation. The suppression of sIAHP was mediated by both

VPACR and PAC1R-induced activation of PKA, and MAPK. Additional studies conducted by Gupte et al. (2015) also demonstrated that PACAP enhanced CA1 pyramidal neuron excitability via downregulation of the Kv 4.2 channel. PACAP treatment (100 nM, 20 min) increased phosphorylation of Kv4.2, an effect associated with a reduction in IA current density. The reduction in Kv4.2 was induced by a PAC1R- induced ERK1/2 mediated signaling cascade (Gupte et al. 2015). Taken together these findings indicate that PACAP applied at high concentrations alters CA1 hippocampal

21 neuronal excitability, an effect that appears counteractive and yet refractory to the depressing effects of PACAP.

The studies within the CA3CA1 synapses demonstrate that PACAP signaling exerts potentiation or depression in a concentration-dependent fashion at CA3CA1 synapses. While the outcomes of PACAP signaling appear inversely related to LTP and

LTD, it will be important to determine how different stimulation rates producing LTP versus LTD correlate with actual PACAP levels released by Schaffer collateral terminals.

In addition, while the PACAP-induced synaptic potentiation and depression effects are mediated by the activation of PAC1Rs and VPAC2Rs, respectively, the mechanisms whereby PACAP will potentiate or depress synaptic transmission remain unknown. A possible explanation is that the mechanisms regulating the differential effects of PACAP are associated with intracellular cAMP-induced signal transducing pathways. Because of the role for PKA signaling in the PACAP-induced potentiation and the role for ePAC in the PACAP-induced depression, it would be useful to examine possible connections between PACAP receptor surface localization, and intracellular cAMP dynamics in mediating the effects of PACAP on CA3→CA1 terminals. Additionally, since both low and high PACAP concentrations produced similar AMPAR GluA1 S845 phosphorylation and T840 dephosphorylation, further study of possible concentration dependent actions on AMPAR channel properties or insertion as proposed outcomes (Toda et al. 2015) seem warranted. Such studies could clarify how PACAP signaling can dynamically alter synaptic transmission at CA3→CA1 synapses and help elucidate relevant mechanisms.

1.6.3 Amygdala

The amygdala is a subcortical nucleus associated with motivational and emotional behaviors and processing anxiogenic stimuli, notably those producing innate and learned fear (LeDoux, 2003; Adhikari, 2014). Neurons in the basolateral amygdala (BLA) subnucleus functionally integrate anxiogenic sensory stimuli, and project glutamatergic outputs to neurons in the central amygdala (CeA) subnucleus that modify anxiogenic and fear associated behaviors (Adhikari, 2014). PACAP is expressed in inputs to the lateral

CeA (lCeA) (Cho et al. 2012) likely arising from PACAP-containing neurons in the vagus nerve (CN X) brainstem dorsal complex (Piggins et al. 1996; Das et al. 2007). In addition, CeA neurons express both PAC1Rs and VPACRs (Vaudry et al. 2009; Joo et al.

2004). Consistent with these observations, exogenous PACAP robustly alters activity at

BLA→lCeA synapses (Cho et al., 2012). Specifically, transient PACAP exposure (5 nM,

10 min) increased the number and amplitude of excitatory postsynaptic potentials

(EPSPs) in lCeA neurons evoked by stimulating presynaptic BLA neurons for up to 1 h.

Subsequent whole-cell recordings revealed that PACAP signaling enhanced AMPAR- mediated evoked EPSC amplitudes in lCeA neurons. Paired-pulse experiments revealed no significant differences between PACAP treated and control neuronal responses, making it unlikely that PACAP alters the probability of glutamate release from presynaptic BLA terminals, and supporting a postsynaptic mechanism. The effects of

PACAP were dependent on a VPAC1R induced, cAMP/PKA and CAMKII mediated signal transduction cascade. Interestingly, pretreatment with Pep1-TGL (200 μM), a synthetic peptide that inhibits GLU1R trafficking by disrupting the interaction of the C- terminus end of the GluA1 subunit with synapse associated protein SAP97, and critical

23 for intracellular AMPAR trafficking and surface expression (Rumbaugh et al. 2003) blocked the effects of PACAP. Taken together these results suggest that PACAP induces plasticity at BLA→lCeA synapses by increasing AMPAR density at postsynaptic sites through cAMP/PKA and CAMKII dependent trafficking of AMPARs (Cho et al. 2012).

Further experiments will be required to verify whether PACAP increases the number of

AMPARs expressed in postsynaptic densities. It would also be interesting to determine if higher PACAP concentrations induce synaptic depression in BLA→lCeA synapses as is observed at hippocampal CA3→CA1 synapses. Such studies might elucidate common signal transducing mechanisms underlying PACAP induced potentiation and depression at glutamatergic synapses throughout the central nervous system. Lastly, while the amygdala processes anxiogenic sensory cues linked to PTSD, and fear conditioning increases PAC1R mRNA in mouse amygdala (Mahan et al. 2012; Ressler et al. 2012) a direct relationship between amygdaloid synaptic plasticity induced by PACAP and fear consolidation has yet to be established.

As part of the “extended amygdala” the bed nucleus of the stria terminalis

(BNST) is a crucial integration center that regulates visceral homeostasis and behavioral responses to unpredictable and chronic stress (Hammack 2010; Crestani et al.

2013). BNST axons project to several nuclei in the hypothalamus and the brainstem that mediate homeostasis and autonomic circuitry. Among hypothalamic targets is the paraventricular nucleus, a region believed responsible for activating the hypothalamic– pituitary axis during responses to stress (Crestani et al. 2013). BNST neurons receive functional GABAergic and glutamatergic synaptic inputs. Additionally, presynaptic terminals to BNST neurons express several neuropeptides that regulate synaptic

24 transmission (Hammack et al. 2010; Crestani et al. 2013). While PACAP is present in presynaptic terminals localized to the lateral BNST (Kozicz et al. 1998; Kozicz et al.

2000) it is not known if PACAP is co-released by presynaptic activity with conventional neurotransmitters. Autoradiographic experiments have demonstrated that BNST neurons express VPAC1Rs and not VPAC2Rs (Vertongen et al. 1997). Several recent studies have supported a neuromodulatory role for PACAP signaling in the BNST. Of particular relevance to PTSD and stress-related disorders, rats exposed to chronic unpredictable stress developed concurrent increases in both PAC1R and PACAP mRNA synthesis in the

BNST. Subsequent experiments utilizing a single bilateral BNST infusion of PACAP identified that PACAP enhanced acoustic startle response both immediately and 7 days after PACAP infusion (Hammack et al. 2009) as well as caused significant weight loss in male and female rats (Kocho-Schellenberg et al. 2014). Furthermore, single infusions of

PACAP to the dorsal lateral BNST elevated blood plasma corticosterone (Lezak et al.

2014). Interestingly, these anxiogenic effects were found to be mediated by PAC1R activation (Roman et al. 2014) since infusions of the PAC1R agonist maxadilan mimicked the effects while infusion of the PAC1R antagonist PACAP(6-38) (40 μM) blocked them

(Roman et al. 2014). Taken together these findings identify that PACAP signaling in the

BNST can exert an anxiogenic influence on behavior. Additionally, these studies highlight critical questions regarding whether the behavioral effects of PACAP on the

BNST are associated with physiological alterations in the BNST. Specifically, it remains to be seen if PACAP exerts these anxiogenic effects by altering the excitable and/or synaptic properties of BNST neurons. Such studies could implicate a potential

25 endogenous role for PACAP signaling in regulating BNST neuronal activity underlying behavioral responses to stress and illuminate the basis of stress disorders such as PTSD.

1.7 PACAP Signaling at Autonomic Synapses

1.7.1 Ciliary Ganglion

The parasympathetic avian CG is a useful model for understanding the development and function of fast chemical synapses in the nervous system (reviewed in

Stanley, 1992; Dryer et al. 1994). Presynaptic accessory oculomotor cranial nerve (CN)

III (Edinger–Westphal, EW) nucleus inputs to postganglionic ciliary and choroid neuron populations within the CG release ACh, activating postsynaptic α3*- and perisynaptic α7- nAChR subtypes that underlie synaptic transmission through the ganglion (Martin and

Pilar, 1964; Landmesser and Pilar, 1972; Ullian et al. 1997; Sargent, 2009). At the giant, calyx-like presynaptic terminals of EW axons that innervate ciliary neurons, PACAP- and synapsin-I-immunolabeling co-localize, and KCl depolarization abolishes PACAP- without affecting synapsin-I-immunolabeling (Pugh et al. 2009). These results demonstrate that PACAP and ACh are present in the same presynaptic terminals within the CG, and that PACAP can be released by depolarizing stimuli. Neurotransmission from EW axons to ciliary neurons reliably follows stimuli delivered at frequencies up to

50 Hz (Dryer and Chiappinelli, 1985). Moreover 16–20 Hz stimulation is sufficient to trigger PACAP release from splanchnic nerve terminals and intercardiac nerve fibers

(Tornøe et al. 2000; Tompkins et al. 2007), making it likely that PACAP is released from

EW nerve terminals in vivo. Since CG neurons express abundant PAC1Rs (Margiotta and

Pardi, 1995; Pardi and Margiota, 1999) PACAP fulfills criteria as a neurotransmitter in

26 this system, and is also well positioned to modulate fast nAChR mediated transmission at

CG neuron synapses. Our past and recent findings support a role for PACAP as a potent agent of both short- and long-term synaptic plasticity.

PAC1Rs on CG neurons couple to AC and PLC effectors and efficiently mobilize cAMP and induce IP turnover (EC50 ≈ 1 nM for both) (Margiotta and Pardi, 1995; Pardi and Margiotta, 1999). Consistent with the ability of 8-Bromo-cAMP to increase the nAChR sensitivity of CG neurons (Margiotta et al. 1987) PACAP enhanced responses from both α7-nAChRs and α3, α5, 4, ±2-containing nAChRs (α3*-nAChRs) in a

PAC1R-, AC-, and PKA-dependent fashion (Margiotta and Pardi, 1995; Pardi and

Margiotta, 1999). As discussed above for NMDARs and AMPARs on hippocampal CA1 neurons, however, the relevant phosphorylation targets responsible for the PACAP- induced enhanced sensitivity of α3*- and α7-nAChRs have yet to be determined. Since nAChR-mediated synapses form between CG neurons in culture, and resemble those seen on the neurons in vivo, particularly regarding the differential expression of α3*- and α7- nAChRs at post- and peri/extra-synaptic sites, respectively (Margiotta and Berg, 1982;

Chen et al. 2001) we have used this accessible system to evaluate the impact of PACAP on nAChR-mediated synaptic transmission. Applying 100 nM PACAP for 5–15 min markedly enhanced synaptic activity in CG cultures, increasing the frequency and amplitude of spontaneous impulse-dependent nAChR-mediated EPSCs(sEPSCs) by 100–

300 % and 40–50 %, respectively (Pugh et al. 2009) and doing so without increasing neuronal excitability as with other autonomic neurons (See Below). The rapidly induced synaptic plasticity in CG neurons occurred by PAC1R activation and was AC- and PKA- dependent, and PLC-independent. Despite the ability of similar PACAP treatments to

27 enhance α3*- and α7-nAChR sensitivity, analysis of stimulus evoked nAChR-mediated

EPSCs revealed that PACAP enhanced synaptic function by increasing presynaptic ACh release (quantal content) without affecting the unitary post synaptic response (quantal size) assessed from the amplitude of mEPSCs acquired in the presence of tetrodotoxin

(TTX) (Pugh et al. 2009). The PACAP-induced presynaptic plasticity resulted from activation of Ca2+ /Calmodulin- and PKA-dependent neuronal nitric oxide synthase

(NOS1) (Bredt et al. 1992; Hurt et al. 2012) and subsequent increased production of NO

(Pugh et al. 2009) that was detected in CG neurons using the fluorescent NO indicator, 4- amino-5-methylamino-2′,7′-difluorofluorescein (Jayakar et al. 2014; Kojima et al. 1998).

Applying the NO scavenger 2-4-carboxyphenyl-4,4,5,5- tetramethylimidazoline-1-oxyl-3- oxide blocked the ability of PACAP to alter sEPSC frequency or amplitude, consistent with a mechanism where NO produced in postsynaptic neurons diffuses to presynaptic terminals thereby enhancing ACh release in a retrograde fashion (Etherington, 2004;

Bright and Brickley, 2008; Xue et al. 2011). Interestingly, co-immunoprecipitation experiments indicated that nAChRs and NOS1 associate in a molecular complex, and functional studies revealed that α7-nAChR activity was required for PACAP to induce

PKA-dependent synaptic plasticity by a process localized to the cell membrane by A- kinase anchoring proteins (reviewed by Wong and Scott, 2004). These results support a mechanism where PACAP enhances presynaptic ACh release via retrograde NO action, and where NO is produced by upregulating Ca2+ permeable NOS1 associated nAChRs, with the largest contribution from α7-nAChRs present at perisynaptic and extrasynaptic membranes of postsynaptic nyeurons (Jayakar et al. 2014). These results are more fully presented in Chapter 2 of this dissertation.

In accord with its neurotrophic actions, PACAP activates MAPK dependent pro- survival pathways (Pugh and Margiotta, 2006) and as mentioned above, induces nuclear translocation of cyclic AMP response element binding protein (CREB; Sumner and

Margiotta, 2008) indicating it can potently alter gene transcription, and suggesting it may have long-term transcription dependent effects on synaptic function and structure. Indeed,

PACAP enhanced synaptic function in CG cultures >48 h after a transient 15 min exposure (See Chapters 3 and 4; Pugh et al. 2009; Starr and Margiotta, 2014, 2016). This long-term (LT) plasticity was not simply a prolonged version of the short-term (ST) form seen immediately after PACAP exposure. While featuring similar increases in sEPSC frequency and quantal content, there was a more profound increase in sEPSC amplitude compared to similar measurements made immediately after PACAP exposure, and, consistent with a postsynaptic effect, a significant increase in postsynaptic quantal size.

Moreover, unlike the acute PACAP-induced plasticity, the sustained plasticity was independent of NOS1 and nAChR activities and required gene transcription (Starr and

Margiotta, 2014). In a gene array study we detected a number of transcripts relevant to

ACh processing that were upregulated 96 h after transient exposure to PACAP including the catalyzing enzyme, choline acetyltransferase (1.5-fold after 15 min and 2.2-fold after

24 h PACAP exposure), with nearly identical increases seen for the high-affinity choline transporter, responsible for rate-limiting active transport of choline into the nerve terminal (Sumner and Margiotta, 2008). Relevant to ACh storage, the vesicular acetylcholine transporter transcript increased 2.8-fold at 24 h. While the ability of

PACAP to upregulate gene transcripts associated with ACh release, synthesis, and storage is consistent with its persistent positive presynaptic influence on nicotinic

29 neurotransmission, the overall picture is still incomplete. Further studies are required to explore relevant cellular mechanisms, particularly to identify upregulated genes and, since nAChR transcripts were not altered by any of the PACAP treatments (Sumner and

Margiotta, 2008), to assess whether structural changes in synapse morphology or nAChR clustering may account for the postsynaptic changes associated with the sustained synaptic plasticity induced by PACAP (See section 1.8).

1.7.2 Cardiac Ganglion

The parasympathetic cardiac ganglion (CdG) controls heart rate and blood flow to the cardiac muscle. CdG neurons are innervated by CN X (vagus) axons and supply the sinoatrial node, the atrioventricular nodes, and the atrial and ventricular musculature

(Mitchell, 1953; Wallis et al. 1996; Singh et al. 1996; Purves et al. 2012). In guinea pig,

PACAP is co-expressed with choline acetyltransferase in CN X input terminals, released in an activity dependent fashion, and activates PAC1Rs on CdG neurons (Braas et al

1998; Parsons et al. 2000; Calupca et al. 2000; Dehaven and Cuevas, 2002; Parsons et al.

2006; Tompkins et al. 2007). Consistent with these neurotransmitter characteristics, application of PACAP via microperfusion (50 μM, 500 ms) caused a slow depolarization

(slow EPSP) in CdG neurons that elicited bursts of action potentials, like those seen following high frequency preganglionic stimulation. The results reveal an additional neuromodulatory action whereby the PACAP-induced depolarization reflects increased

CdG neuron excitability (Tompkins et al. 2007). Pharmacological studies indicate that both actions are mediated by a PAC1R-induced AC, PKA, MEK signal transduction pathway (Tompkins et al. 2007; Tompkins and Parsons 2008) requiring extracellular Ca2+

30 entry (Hardwick et al. 2006). In accord with the dependence of increased excitability on

AC activation, PACAP applied at a concentration of 100 nM induces a depolarizing shift in the activation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels

(Hardwick et al. 2006; Merriam et al. 2009). Treatment with the cAMP analog, 8-br- cAMP or AC agonist, forskolin only partially mimicked the effects of PACAP suggesting that other cellular mechanisms in addition to AC signaling contribute to the effects of

PACAP on CdG neuron excitability (Tompkins et al. 2008). Other studies have expanded upon these results showing that PACAP-induced increases in neuron excitability also depend on Ca2+ influx via T- and R-type VDCCs that are linked to slow depolarization and repetitive firing (Hardwick et al. 2006; Tompkins et al. 2015) as well as PAC1R internalization (Merriam et al. 2013). While the increases in membrane excitability elicited by the PACAP-induced depolarization would be expected to affect nAChR mediated transmission in the CdG, this possibility has not been tested. Performing such studies is supported by the finding that PACAP increases nAChR currents in CdG and submandibular ganglion (SMG) neurons (Liu et al. 2000) and could elucidate a dual role for PACAP as a transmitter in mediating slow EPSCs and as a modulator regulating the excitability of these neurons and nAChR- mediated ganglionic transmission.

1.7.3 Submandibular Ganglion

The parasympathetic SMG increases salivary secretion from and blood flow to the submandibular gland (Segal et al. 1996). The SMG receives preganglionic cholinergic fibers originating from the superior salivary nucleus of CN VII (Endoh, 2004). Several neuropeptides are expressed in these input terminals to the SMG (Endoh, 2004) but

31 neither the presence of PACAP in nor its release from these terminals has been demonstrated. Imaging and electrophysiological studies indicate that SMG neurons express nAChRs (Shida et al. 1993; McCann and Lichtman, 2008) as well as PAC1Rs and

VPACRs, but it is not known which VPACR subtypes are expressed (Hayashiet al. 2002;

Kamaishi et al. 2004). PACAP application typically reduces, and in fewer demonstrated cases enhances, VDCC currents. In hamster SMG neuronal cultures (Kamaishi et al.

2004) 100 nM PACAP inhibited P/Q-, N- and L-type VDCCs, with P/Q- and N-type inhibition mediated by PAC1Rs via G -protein βγ subunits, and L-type inhibition dependent on a VPACR-mediated, AC/PKA- and PLC/PKC- dependent signal transduction. Taken together, these studies indicate that PACAP can potently alter VDCC activation in SMG neurons via distinct pathways. Similar to the CdG, the superior cervical ganglion, the ciliary ganglion and the VMN, (SCG;See below) focal ejection of

10 μM PACAP (10 at 50 ms and 1 Hz) to dissociated SMG neurons induces a depolarizing current sufficient to generate bursts of action potentials (Hayashi et al. 2002) indicative of increased neuronal excitability. Inhibition of VDCCs in SMG neurons could lead to a depolarization and increased excitability by depressing activation of Ca2+ - activated K+ channels, particularly those responsible for the after-hyperpolarization

(Kamaishi et al. 2004) but this has yet to be demonstrated. It is not known if the PACAP- induced effects on VDCCs or excitability can alter the output of nAChR-mediated synapses on SMG neurons. Like the modulatory actions of PACAP in the CG and the

CdG, PACAP also enhances nAChR-mediated currents in SMG neurons. Following ectopic exposure to 10 μM PACAP nAChR mediated currents induced by focal perfusion of 100 μM ACh were enhanced, reflecting an increase in nAChR sensitivity (Liu et al.

2000). Finally, whole animal experiments support a role for PACAP as a potent neuromodulator of SMG transmission in vivo. Specifically, PACAP injections increase salivary secretion from and blood flow to the submandibular gland (Mirfendereski et al.

1997), consistent with enhanced parasympathetic innervation of the SMG. Additionally, intra-arterial PACAP injections alone did not induce submandibular salivary secretion in ferret while combined ACh and PACAP injections did, and PACAP injections during

“parasympathetic nerve evoked flow of saliva” also increased salivary secretion (Tobin et al. 1995). Taken together, these findings indicate that PACAP inhibits Ca2+ channels and increases excitability in SMG neurons. They further suggest that PACAP can alter nAChR-mediated synaptic activity in the SMG with concomitant regulation of submandibular salivary secretion in vivo.

1.7.4 Superior Cervical Ganglion

The SCG is a prototypical sympathetic paravertebral ganglion that innervates extensive targets in the head, neck and heart (Li and Horn, 2006; Lazarov et al. 2009).

Composed of multiple CAergic neuronal subpopulations SCG neurons express postsynaptic α3*-nAChRs (Mao et al. 2008) and α7-nAChRs (Skok et al. 1999) and principally receive preganglionic cholinergic inputs from neurons in the spinal cord intermediolateral (IML) cell column (Krishnaswamy and Cooper, 2009; Rando et al.

1981; Reuss et al. 1989; Strack et al. 1988). IML neurons synthesize and store PACAP in cholinergic preganglionic terminals within the SCG (Beaudet et al. 1998; May et al.

1998). While PACAP release from IML terminals has not been verified, SCG neurons do express PAC1Rs (May et al. 1998; Nogi et al. 1997; Braas et al. 1999; Beaudet et al.

2000). PACAP has a significant neuromodulatory impact on SCG neuron excitability like that in the CdG. Focal PACAP perfusion (50 μM, 1 s) produced a prolonged depolarizing inward current in SCG neurons in culture that was insensitive to TTX indicative of activity-independence. Pharmacological studies indicated that the PACAP-induced inward current was mediated by a PAC1R induced PLC-IP3-IP3R signal transduction pathway involving sodium and potassium dependent components (Beaudet et al. 2000).

In Na+ deficient solution, the PACAP induced inward currents were reduced by 70%, while increasing extracellular K+ concentration reduced PACAP induced currents by

35%. When extracellular Ca2+ and Na+ were reduced, the PACAP-induced reduction in inward current was not significantly different from the sodium deficient condition alone.

These findings indicate that the PACAP-induced depolarization of SCG neurons is mediated by extracellular Na+ entry and inhibition of a K+ outward conductance.

However, other possible options are cited such that the specific ion channels underlying these PACAP-induced effects remain unknown. The ability of PACAP to elicit a sustained depolarization in SCG neurons suggests it can increase SCG neuron excitability and output. Indeed, when total medium CA metabolites (DOPAC and HVA) were measured in dissociated SCG neuronal cultures, ectopic exposure to concentrations of

PACAP (≥10 nM, 48 h) resulted in a threefold increase in extracellular CA metabolite concentrations (May and Braas, 1995) suggesting that PACAP enhanced CA release from

SCG neuronal cultures. Although more detailed studies remain to be conducted, these findings suggest that PACAP can induce changes in postganglionic SCG neuronal output, consistent with enhanced SCG neuronal excitability. Alternatively, PACAP could act as a secretagogue in the SCG as it does in the closely related sympathetic-like adrenal gland.

Unfortunately, no experiments have been conducted to determine whether PACAP also alters nAChR-mediated synaptic properties of SCG neurons or ACh release from their cholinergic IML inputs. Such findings would complement those conducted in parasympathetic ganglia and might reveal multiple roles for PACAP in modulating excitability, secretion and synaptic transmission in sympathetic ganglia.

1.8 Conclusions and Dissertation Hypothesis

Despite its ubiquitous distribution and diverse functions, PACAP is well positioned to mediate and modulate synaptic transmission via its cognate GPCRs and their associated intracellular signaling effectors. Sections 1.6, 1.7 indicates that in most cases PACAP satisfies two of the three criteria to qualify it as an endogenous neurotransmitter. Furthermore, PACAP is also a potent neuromodulator that triggers acute and persistent alterations of membrane components underlying neuronal synaptic transmission. While studies using knockout mice lacking PACAP support its role as a synaptic neurotransmitter and/or neuromodulator in vivo, a critical caveat to this conclusion is that the ability of endogenous PACAP to modulate synaptic properties has not been demonstrated directly.

Two themes that emerge from PACAP’s actions are its ability to rapidly alter membrane excitability and specific synaptic components. The first theme has been demonstrated in neurons in the autonomic and central nervous system and appears entwined with the activation or inhibition of VDCCs. Here, the resulting depolarization or indirect action of Ca2+ influx on K+ or other channels enhances excitability by increasing the capacity for repetitive firing (Fig. 1.1b). Interestingly the coupling of

35 increased excitability to VDCCs and depolarization exemplifies the cell-specific nature of

PACAP signaling since, while VDCC modulation is a common outcome, resultant increased excitability is seen in some autonomic neurons but not in CG neurons where

PACAP induces a sustained ≈5 mV depolarization (Pugh et al. 2009) and VDCC current inhibition but these effects are associated with reduced action potential firing (Pugh et al.

2009). In accord with other PACAP-signaling outcomes, the cellular mechanisms integrating PACAPs actions on specific classes of ion channels to produce increased excitability in some autonomic neurons but not others remain to be determined, as does the possible extension of these effects to central neurons.

The second theme involves the ability of applied PACAP to rapidly induce synaptic plasticity in both the central and autonomic nervous system by altering the postsynaptic response to conventional neurotransmitters and/or the release of conventional neurotransmitters from presynaptic nerve terminals (Fig. 1.2C). A common outcome is the concentration dependent regulation of postsynaptic AMPAR and NMDAR responses in the amygdala, the SCN, and CA1 pyramidal neurons and the upregulation of nAChR responses in autonomic CdG, SMG, and CG neurons, possibly driven by their differential phosphorylation and/or insertion into postsynaptic sites as has been demonstrated for CA1 hippocampal neurons. Less common is the ability of PACAP to increase the release of conventional neurotransmitter from presynaptic terminals as seen with ACh release in CA3CA1 (Masuo et al. 1993; Roberto et al 2000, 2001) synapses and CG neuronal cultures. Our findings in the CG indicate one potential mechanism whereby PACAP signaling via PKA integrates AKAP-targeted nAChR upregulation, and

NOS1 activation to focally release NO and thereby enhance ACh release from juxtaposed presynaptic terminals in a retrograde fashion (Pugh et al. 2009; Jayakar et al. 2014).

PACAPs well-established neuromodulatory role at excitatory synapses suggests that its capacity to induce synaptic plasticity is critical in mediating visceral, cognitive and behavioral function. Within the hippocampus as well as in the ciliary ganglion (CG)

PACAP has been demonstrated to induce long-term, persistent forms of synaptic plasticity. This is particularly true in the CG, where the synaptic effects of PACAP persist

48 h after PACAP washout (Pugh et al. 2009). However, no research has examined the mechanisms underlying long-term (LT) PACAP-induced synaptic plasticity at excitatory synapses. Therefore, the central goal of this dissertation is to characterize as well a distinguish the mechanisms underlying long-term PACAP-induced synaptic plasticity relative to the short-term synaptic plasticity at CG cholinergic synapses. Using CG neuronal cultures, we hypothesized that PACAP does induce a novel long-term synaptic plasticity at cholinergic synapses that exhibit distinguishable biochemical, electrophysiological, and synaptic hallmarks from the short-term PACAP-induced synaptic plasticity.

Fig. 1.3: Simplified summary of PACAP actions at synapses where PACAP is detected in presynaptic terminals and its receptor isoforms are found on postsynaptic cells. A. In the adrenal gland, PACAP is an endogenous neurotransmitter that acts via chromaffin cell PAC1Rs to stimulate CA release. B. Notably in autonomic ganglion neurons, applied PACAP acts via PAC1Rs and/or VPACRs to alter VDCC function that causes membrane depolarization and, in most cases, enhances excitability (see however C). C. Notably in central and parasympathetic neurons, PACAP acts via PAC1Rs and VPAC2Rs to induce synaptic plasticity in a time-dependent fashion. Acute effects are mediated largely via PAC1Rs and AC and can be postsynaptic, featuring enhanced ionotropic receptor currents, and/or presynaptic featuring enhanced transmitter release, mediated as exemplified by CG neurons via nAChR dependent activation of NOS1. Persistent synaptic plasticity, requiring gene transcription and exemplifi ed by CG neurons, features increases in both conventional presynaptic transmitter (ACh) release and postsynaptic unitary nAChR-mediated mEPSC amplitudes. In CG neurons acute PACAP application also reduces VDCC currents and depresses membrane excitability. Filled dark circles depict PACAP-, conventional transmitter- or CA-containing vesicles (black , blue , or red , respectively) with their respective cargos depicted in the same colors. ΔVm refers to a change in resting potential. Positive and negative actions are indicated by encircled + and – symbols 38

Chapter 2

PACAP induces plasticity at autonomic synapses by nAChR-dependent NOS1 activation and AKAP- mediated PKA targeting

(This Chapter appeared as a published research article in Molecular and Cellular

Neuroscience. I am a participating author on the article.)

Jayakar S. S., Pugh, P. C, Dale, Z., Starr, E. R., Cole, S., and Margiotta, J. F. (2014).

PACAP induces plasticity at autonomic synapses by nAChR-dependent NOS1 activation and AKAP-mediated PKA targeting. Mol. Cell Neurosci, 63: 1-12

2.1 Abstract

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleitropic neuropeptide found at synapses throughout the central and autonomic nervous system.

We previously found that PACAP engages a selective G-protein coupled receptor

(PAC1R) on ciliary ganglion neurons to rapidly enhance quantal acetylcholine (ACh) release from presynaptic terminals via neuronal nitric oxide synthase (NOS1) and cyclic

AMP/protein kinase A (PKA) dependent processes. Here, we examined how PACAP

39 stimulates NO production and targets resultant outcomes to synapses. Scavenging extracellular NO blocked PACAP-induced plasticity supporting a retrograde (post- to presynaptic) NO action on ACh release. Live-cell imaging revealed that PACAP stimulates NO production by mechanisms requiring NOS1, PKA and Ca2+ influx. Ca2+- permeable nicotinic ACh receptors composed of α7 subunits (α7-nAChRs) are potentiated by PKA-dependent PACAP/PAC1R signaling and were required for PACAP- induced NO production and synaptic plasticity since both outcomes were blocked following their selective inhibition. Co-precipitation experiments showed that NOS1 associates with α7-nAChRs, many of which are perisynaptic, as well as with heteromeric

α3*-nAChRs that generate the bulk of synaptic activity. NOS1-nAChR physical association would facilitate NO production at perisynaptic and adjacent postsynaptic sites to enhance focal ACh release from juxtaposed presynaptic terminals. The synaptic outcomes of PACAP/PAC1R signaling are localized by PKA anchoring proteins

(AKAPs). PKA regulatory-subunit overlay assays identified five AKAPs in ganglion lysates, including a prominent neuronal subtype. Moreover, PACAP-induced synaptic plasticity was selectively blocked when PKA regulatory-subunit binding to AKAPs was inhibited. Taken together, our findings indicate that PACAP/PAC1R signaling coordinates nAChR, NOS1 and AKAP activities to induce targeted, retrograde plasticity at autonomic synapses. Such coordination has broad relevance for understanding the control of autonomic synapses and consequent visceral functions.

2.2 Introduction

PACAP is a neurotrophic, neuromodulatory, and anxiogenic neuropeptide found throughout the nervous system (Arimura, 1998; Sherwood et al., 2000; Hannibal, 2002).

PACAP and a PAC1R gene mutation have been proposed as biomarkers for posttraumatic stress disorder because they correlate with human and animal models of trauma, stress and fear (Hammack et al., 2009a; Vaudry et al., 2009; Ressler et al.,

2011a). Consistent with its neuromodulatory and anxiogenic actions, PACAP potently influences synaptic function and plasticity. In the amygdala, where PACAP is present in presynaptic terminals, fear conditioning increases PAC1R mRNA levels (Ressler et al.,

2011b) while exogenous PACAP enhances anxiogenic responses (Legradi et al., 2007) and intrinsic synaptic transmission (Cho et al., 2012). The autonomic nervous system

(ANS) regulates cardiovascular, respiratory, and sexual functions that are imbalanced in stress and anxiety disorders (Cohen et al., 2000; Kotler et al., 2000; Blechert et al., 2007;

Chang et al., 2013; Shah et al., 2013; Williamson et al., 2013) and PACAP/PAC1R signaling potently impacts autonomic synapses, regulating output to visceral targets

(Tompkins et al., 2007; Pugh et al., 2010; Stroth et al., 2012). PACAP/PAC1R signaling may therefore contribute to stress disorders by regulating central and autonomic synapses.

In parasympathetic ciliary ganglion (CG) neurons, PAC1Rs couple via Gαs to adenylate cyclase (AC) signaling, potentiating the function of α3*- and α7-nAChRs

(Margiotta and Pardi, 1995; Pardi and Margiotta, 1999; Pugh and Margiotta, 2006) that underlie synaptic transmission (Ullian et al., 1997; Chen et al., 2001; Sargent, 2009).

PACAP is present in cholinergic presynaptic terminals on CG neurons and released by

41 depolarization, supporting a functional role during activity driven ganglionic transmission

(Pugh et al., 2010). Functional synapses established between CG neurons in cell culture are mediated exclusively by nAChRs clustered in the postsynaptic membrane adjacent to juxtaposed presynaptic terminals, and thereby provide a useful model for synapses formed on CG neurons in vivo (Margiotta and Berg, 1982; Chen et al., 2001; Zhou et al.,

2004; Pugh et al., 2010; Jayakar and Margiotta, 2011). Consistent with its actions in the amygdala, exogenous PACAP enhances synaptic transmission in CG cultures within minutes, increasing the frequency and amplitude of impulse-driven, spontaneous nAChR- mediated excitatory postsynaptic currents (sEPSCs) by enhancing vesicular ACh release from presynaptic terminals (quantal content) without affecting the unitary postsynaptic response (miniature EPSC amplitude; quantal size) (Pugh et al., 2010). The rapid

PACAP-induced plasticity requires PAC1Rs, AC, and subsequent activation of PKA to drive protein phosphorylation, as well as NOS1 to synthesize NO (Pugh et al., 2010).

NOS1 is activated by calmodulin binding in response to Ca2+ elevation (Bredt and

Snyder, 1990; Abu-Soud et al., 1994). In central nervous system (CNS) neurons, Ca2+ influx via N-methyl-D-aspartate (NMDA)-type glutamate receptors (NMDARs) likely triggers NOS1 activation (Garthwaite et al., 1988; Garthwaite, 2008) and NO produced in this manner acts as a retrograde messenger to regulate presynaptic function (Regehr et al.,

2009). In the ANS, elevated NO production has been similarly linked to LT presynaptic potentiation resulting from increased quantal content (Lin and Bennett, 1994).

Interestingly PACAP stimulates NOS1 in pituitary gonadotrophs (Garrel et al., 2002), and in sensory neurons NOS1 is activated in both a Ca2+/calmodulin- and PKA- dependent manner (Bredt and Snyder, 1990; Bredt et al., 1992; Hurt et al., 2012). Given

42 these precedents we examined the interplay between NOS1- and PKA-dependent mechanisms in transducing the actions of PACAP at synapses on CG neurons where transmission is mediated by Ca2+-permeable nAChRs. A NO scavenger (C-PTIO) was first used to show that NO likely acts in a retrograde manner to induce synaptic plasticity.

We then examined mechanisms whereby PACAP/PAC1R signaling activates NOS1 to increase NO levels in CG neurons, and localizes these effects to synaptic components via

PKA-anchoring proteins (AKAPs) to enhance synaptic function. The results reveal that

PACAP/PAC1R signaling tightly coordinates α3*- and α7-nAChR with NOS1 and AKAP activities to induce targeted, retrograde synaptic plasticity. Such coordination has broad relevance for understanding the control of autonomic synapses and consequent visceral functions, possibly including those impacted by stress disorders.

2.3 Methods

2.3.1 Neuronal Preparation

Neuronal cultures were prepared from embryonic day 8 (E8, Stage 34 - 35) chick ciliary ganglia using approaches similar to those previously described (Chen et al., 2001;

Zhou et al., 2004; Pugh et al., 2010; Jayakar and Margiotta, 2011). Dissected ganglia were treated with trypsin (0.025%, 15 min, 37°C) and the neurons dispersed by mechanical trituration and grown on glass bottom 35 mm dishes (WillCo-dish, BioSoft

International) pre-coated with 0.2 mg/ml poly-D-ornithine and 12.5 µg/ml mouse laminin

(a gift from Dr Salvatore Carbonetto, McGill University). The growth medium consisted of Eagle’s minimum essential medium (MEM) supplemented with 2 mM glutamine, 100

U/ml penicillin, 100 µg/ml streptomycin, 10% heat-inactivated horse serum (all components from Invitrogen, Rockville, MD) and with freshly-prepared E17 chick eye

eye extract (3% v/v, MEM ). Cultures were maintained at 37°C in 95% air/5% CO2, and

MEMeye replenished every 2-3 d. For some experiments freshly-dissociated neurons were isolated from E14 (Stage 40) ciliary ganglia by combined collagenase-A (1 mg/ml,

30 min, 37°C) and trituration treatment and plated on 12 mm glass coverslips coated with

1-2 mg/ml poly-D-lysine (Sigma Aldrich, St. Louis, MO) as previously described (Nai et al., 2003; Jayakar and Margiotta, 2011).

2.3.2 Electrophyiological Recording

After 5-7 d in culture, whole-cell recordings were obtained from CG neurons as previously described (Chen et al., 2001; Pugh et al., 2010). A physiological solution (RS; pH 7.4) containing (in mM): 145 NaCl, 5.3 KCl, 5 HEPES, 5.4 Glucose, 0.8 MgSO4, 2

hs CaCl2 was supplemented with 10% heat inactivated horse-serum for recordings (RS ).

Patch pipettes (2-5 MΩ) were fabricated from Corning 8161 glass tubing and filled with an internal solution containing (in mM) 145.6 CsCl, 0.6 CaCl2, 2.0 EGTA, 5.4 glucose, and 5.0 Na-HEPES (pH 7.3). Synaptic function was assessed by acquiring nAChR- mediated sEPSCs at -70 mV in whole-cell recording mode. Specific reagents tested for effects on synaptic transmission were added to MEMeye or RShs from frozen stocks as described in RESULTS. Synaptic currents were examined off-line using Mini Analysis

(6.0.3, Synaptsoft, Fort Lee, NJ) as previously described (Chen et al., 2001; Zhou et al.,

2004; Pugh et al., 2010). Only those events that activated abruptly and had peak amplitudes 2-3X baseline RMS current noise (typically 1.5 - 2.5 pA) were selected for

44 analysis. For each neuron, overall sEPSC frequency (Fs, Hz) was determined from the total number of events (130 ± 14 from 100 control neurons) divided by the recording epoch duration (usually 120 s) while sEPSC amplitudes were determined from the average of individual event amplitudes (As, pA). In each experiment Fs and As values from every control and treated neuron (e.g. PACAP) were routinely normalized to their average values from control neurons tested in parallel. This procedure allowed comparisons to be made from experiments conducted on different cultures and did not change overall conclusions. For example, in 20 experiments normalized sEPSC Fs and As values from PACAP treated neurons (n = 109) were 3.72 ± 0.33 and 1.35 ± 0.06 times higher, respectively, than those from control neurons (1.00 ± 0.05 and 1.00 ± 0.03, n =

100) tested in the same experiments (p < 0.001 for both). These differences compare well to those seen when “raw” Fs and As averages from PACAP-treated neurons (2.9 ±

0.3 Hz and 19.8 ± 0.8 pA) and control neurons (1.1 ± 0.1 Hz and 15.7 ± 0.7 pA) were compared (p<0.001 for both). For some studies the soma membrane nAChR population was sampled by focally applying nicotine (20 µM in RS) by pressure microperfusion (5 -

10 psi). Peak nAChR current values were measured using Clampfit (pClamp 6.0 or 8.0,

Axon Instruments, Burlingame, CA) as previously described (Nai et al., 2003; Zhou et al., 2004).

2.3.3 Nitric Oxide Imaging

A live-cell imaging approach based on the fluorescent NO indicator dye 4-amino-

5-methylamino-2’,7’-difluorofluorescein (DAF-FM) (Kojima et al., 1998) was used to assess changes in NO levels in CG neurons. Most experiments were conducted using E8

CG neurons grown for 5-7 d in culture. Cases where acutely dissociated E14 CG neurons were used are specified in the text. Neurons were loaded with membrane-permeable

DAF-FM diacetate (Molecular Probes, 2 µM) for 20 min, with this and all subsequent steps conducted at room temperature (RT). In most cases, the loading, wash and incubation solution was RS supplemented with 1 mM L-arginine (RSarg). After washing

(2X, 5 min) and recovery (20 min) to allow time for deacetylation of the dye, neurons were washed (1X, 5 min) and then incubated with RSarg alone (control), RSarg plus the

NO donor (Z)-1-(N-Methyl-N-[6-(N-methylammoniohexyl)amino])-diazen-1-ium1,2,- diolate] (MAHMA-NANOate, Enzo Life Sciences, 100 µM, positive test), or RSarg plus

PACAP (PACAP-38, Bachem) and/or other test reagents (see Results) all for 20-40 min.

Since MAHMA-NANOate is stable at pH 9 and releases NO with a half-time of 2.7 min at pH 7.4, stock solutions (100 mM) were prepared in 10 mM NaOH (pH 9.5) and stored at -20°C. MAHMA-NANOate stocks were diluted to 100 µM in RSarg (pH 7.4) and immediately applied to cells. A Zeiss 200M inverted microscope equipped with 10X

(0.75 N.A.) objective, 495 nm excitation, and 517 nm emission filters was used for imaging. Two to three bright field and fluorescent image pairs were acquired from each coverslip or culture dish condition (12 bit, 1X1 bins) using a cooled digital CCD camera

(Q-Imaging Retiga 1434) under the control of IP Lab software (Version 4 Scanalytics;

Reading, PA). The time between acquiring control and test images was ≤ 5 min.

Fluorescence exposure times were usually 2.0 s (range = 0.5 - 4.0 s) and kept constant within a given experiment. DAF-FM fluorescence (FLs) was quantified off-line from microscope fields (each containing 10-50 neurons) using NIH ImageJ 1.45s software. To assess neuronal FLS, the fluorescence intensity within a region of interest superimposed

46 over the soma minus the background fluorescence intensity (FLT - FLB = ΔF) was determined for each neuron in a given field relative to FLB in the same field (FLS =

ΔF/FLB). Changes in DAF-FM fluorescence (FLS*) in response to treatments are expressed as the ratio of FLS values for treated to untreated (control) neurons (FLS* =

FLS,t/FLS,c). The same procedure was used to assess FLS* for glial support cells. In a few experiments, neurons were fixed in paraformaldehyde (2-4% in PBS with 4% sucrose, 15 min), and then washed in PBS (2X, 5 min) prior to imaging. Changes in DAF-FM levels assessed in live and fixed cells were qualitatively similar, but all assessments presented here are from live cells.

2.3.4 Detection of Endogenous NOS1

Endogenous NOS1 was detected using an anti-NOS1 polyclonal antibody (BML-

SA227, Enzo Life Sciences) raised against a synthetic peptide corresponding to C- terminal amino acids 1414-1434 of human NOS1 having 100% homology with the predicted chicken sequence. NOS1 localization was assessed by fluorescence immunolabeling, image acquisition and analysis using CG neurons, either grown in culture for 5-7 d or acutely dissociated at E14. Neurons were brought to RT washed in

0.1 M phosphate-buffered saline (PBS, pH 7.4; 2 min) and fixed (4% paraformaldehyde in PBS, 20 min). Fixed neurons were washed with PBS (3X, 5 min), blocked and permeabilized in a block solution containing PBS with 5% Normal Goat Serum and 0.1%

TX-100 (BS1) for 1 h, and incubated in BS1 containing anti-NOS1 (1:2000, 16 h, 4°C).

Neurons were then brought to RT, washed in PBS containing 0.1% Triton X-100 (WS,

3X, 5 min) and incubated in BS containing Alexa Fluor 488 conjugated goat anti-rabbit

47 secondary antibody (1:400, Jackson Immunoresearch Laboratories; 1 h). After washing in WS (3X, 5 min) and PBS (1X, 5 min) coverslips were mounted in Vectashield anti- fade mounting medium (Vector Laboratories, Burlingame, CA) and viewed.

For biochemical detection, 20 frozen E17 ciliary ganglia (-80°C) were thawed on ice and homogenized using a 100 µl tissue homogenizer (Wheaton, Millville, NJ) in Tris-

Triton buffer (TB; 300 µl final volume), containing 50 mM Tris (pH 7.4), 0.5% Triton X-

100 and protease inhibitor cocktail (e.g. Sigma-Aldrich, St.Louis, MO), and solubilized for 1 h at 4°C with gentle rocking. Insoluble material was removed by centrifugation

(20,000 x g, 15 min, 4°C). Lysate supernatants were boiled and vortexed (5X, 1 min) loaded on 6% polyacrylamide/0.1% SDS (PAGE/SDS) gels in sample buffer (SB) containing 32 mM Tris (pH 6.8), 13% v/v Glycerol, 1% SDS, and 0.005% Bromphenol

Blue, subjected to electrophoresis (50 V, 30 min then 150V, 1 h) and electro-blotted to nitrocellulose filters (30 V, 16 h, 0°C). Filters were blocked in PBS containing 0.1%

Tween and 5% (w/v) dried milk powder (BS2), and probed in BS2 with anti-NOS1 antibody (1:3000) followed by HRP-conjugated anti-rabbit secondary antibody (1:5000,

Jackson Immunoresearch, West Grove, PA). Signals were visualized by enhanced chemiluminescence (Immun-StarTM HRP Substrate kit; Bio-Rad, Hercules, CA) and captured on autoradiography film.

2.3.5 nAChR-NOS1 Co-Precipitation

A modified pull-down assay (Conroy and Berg, 1998; Conroy et al., 2003) was used to determine if α3*- and/or α7-nAChRs exist in a complex with NOS1. CG lysates

48 were prepared by homogenizing 40-75 frozen E17 ganglia in 1-2 ml TB (0°C) followed by centrifugation (20,000 x g, 15 min, 4°C). To isolate α3*-nAChRs and associated proteins, half of the lysate was incubated with the α3/α5 selective rat monoclonal antibody (mAb35; 1:250, pull-down positive (PD+)) (Vernallis et al., 1993), and half incubated with an equal volume of non-immune rat serum (PD-), at 4°C for 1 h with gentle rocking. Protein AG UltraLink resin (AG, 20 l/ml, Pierce/Thermo Scientific) was added to both lysates and the samples incubated at 4°C for 1 h with gentle rocking.

To isolate α7-nAChRs and associated proteins, half of the CG lysate was incubated (4°C,

1 h, gentle rocking) with 20-50 µl αBgt conjugated to Actigel ALD resin (αBgt/Actigel,

Sterogene Bioseparation) according to the manufacturer’s specifications (PD+), and half with αBgt/Actigel plus 50 nM free αBgt (PD-) (Blumenthal et al., 1997; Gomez-Varela et al., 2012). The AG or Actigel resins were allowed to settle (5 min, 0°C) and the resins washed in TB (1 ml, 2-6 X). Proteins bound to the resins were eluted by boiling and vortexing the samples in 50 µl SB (5X, 1 min each). Eluent (25 µl) was collected from each sample (PD+, PD-), boiled as above, and the samples loaded on 6% and 8%

PAGE/SDS gels to detect NOS1 or nAChR, respectively. Samples were subjected to electrophoresis (50V for 30 min, 150V 60 min) and the gels electro-blotted to nitrocellulose filters (30 V, 16 h, 0°C). The blots were blocked, probed in BS2 with

NOS1 polyclonal antibody (1:1000) or subtype-specific nAChR antibodies (mAbA3-1

(1:1000) for α3-, or mAb319 (1:500) for α7-nAChR subunit; 2 h, RT), incubated with appropriate HRP-conjugated secondary antibodies (1:5000, Jackson Immunoresearch), and developed and exposed as above. The nAChR antibodies were gifts from Darwin

Berg (mAbA3-1, University of California San Diego) and Jon Lindstrom (mAb319,

University of Pennsylvania).

2.3.6 AKAP5 Detection

Endogenous AKAPs were identified by their ability to bind PKA regulatory subunit II (RII) using a modified overlay method (Hausken et al., 1994). Frozen E14 ciliary ganglia were homogenized in 1 ml TB (0°C). Solubilized CG lysates were centrifuged and 10 μl diluted in SB, boiled for 5 min, subjected to electrophoresis on 8%

PAGE/SDS gels (120V, 2h) and electro-blotted onto nitrocellulose membranes. RII probe solutions contained 200 ng of recombinant PKA regulatory subunit type II β (RIIβ;

Biaffin GmbH & Co, Kassel Niederzwehren, Germany) alone or RIIβ together with a synthetic stearated peptide that mimics the AKAP RIIβ binding site and blocks its binding (Ht31) or a proline derivative that does not block binding (Ht31P) (5 µM for both, Promega, Madison, WI). Probe solutions were incubated in 1 ml BS2 (1h, 4°C) and then diluted 3-fold. Individual nitrocellulose membranes either exposed to the different

RIIβ probe solutions, or containing 50 ng transferred RIIβ were washed in PBS containing 0.1% Tween, blocked in BS2 (both for 1 h, 22°C), and probed with anti-RII mAb (BD Biosciences, San Jose, CA; 1:2000, 16h, 4°C). Lastly, a lysate-transferred membrane not exposed to RII was probed with anti-AKAP5 mAb (BD Biosciences,

1:250, 16h, 4°C). All membranes were washed and incubated (1 h, RT) in HRP- conjugated anti-Mouse IgG (1 h, 22°C, 1:5000, Jackson) and reactive proteins visualized using HRP chemiluminesence on autoradiography film. AKAP5 was identified in CG neurons by labeling permeablized ganglion sections or CG cultures with a rabbit AKAP5

50 antiserum (H-105, Santa Cruz) followed by Alexa Fluor 488-conjugated goat anti-rabbit

IgG (Molecular Probes/Invitrogen).

2.3.7 Statistics

Data values are presented as mean ± SEM. Mean values were compared using

Students unpaired, two-tailed t-test with Welch’s correction for unequal variance, or with one-way ANOVA followed by the Bonferroni post-hoc test. Group mean values with p <

0.05 were considered significantly different. All statistical tests were performed using

Prism (Graph Pad Version 5.0d, La Jolla, CA) software.

2.4 Results

2.4.1 PACAP stimulates NOS1 to increase NO levels.

We previously reported that PACAP signaling via PAC1Rs rapidly and potently increases the frequency and amplitude of sEPSCs on CG neurons in culture by increasing presynaptic ACh release (quantal content) (Pugh et al., 2010). We also found that this plasticity required NOS1 activity and could be mimicked by an NO donor (Pugh et al.,

2010). The latter finding predicts that PACAP/PAC1R signaling leads to NOS1 activation and NO production responsible for the subsequent presynaptic plasticity. We therefore used an NO imaging approach based on the fluorescent indicator dye DAF-FM to assess the ability of PACAP to increase NO production and reveal underlying mechanisms. Throughout this study, PACAP was applied at 100 nM, a concentration we

51 previously found maximally enhances PAC1R signaling and subsequent synaptic function

(Margiotta and Pardi, 1995; Pardi and Margiotta, 1999; Pugh et al., 2010). PACAP increased the fluorescence intensity of DAF-FM-loaded CG neuron somas compared to sham-treated control neurons from the same cultures (Fig. 2.1A, B). The higher fluorescence intensity associated with PACAP application was indicative of increased cellular NO since the NO donor MAHMA-NANOate similarly increased DAF-FM soma fluorescence. Subsequent quantification revealed that PACAP and MAHMA-NANOate significantly increased specific DAF-FM fluorescence (FLS*) in neurons from CG cultures to levels that were on average 2.3 and 5.0 times higher, respectively, then those of untreated controls tested in parallel. No detectable increase in FLS* was observed when the neurons were co-treated with PACAP and NOS inhibitor (L-NAME) indicating that the PACAP-stimulated increase required endogenous NOS1 activity. Functional

NOS expression has been reported previously in CG neurons (Nichol et al., 1995) and consequent NO production linked to increased acetylcholine (ACh) release from preganglionic terminals (Lin and Bennett, 1994). Further evidence for the presence of endogenous NOS1 was obtained using an anti-NOS1 antibody that detected a ≈160 kDa protein in E14 CG extracts consistent with the predicted size of chicken NOS1, and revealed specific immunofluorescence localized to CG neurons (Fig. 2.1C). In accord with our previous findings indicating that NOS1 signaling is required for PACAP to induce synaptic plasticity, these results indicate that PACAP triggered signals converge on endogenous NOS1 to increase NO production in CG neurons.

Figure 2.1: PACAP increases NO production in CG neurons by activating endogenous NOS1. A. NO production assessed by DAF-FM fluorescence. Left and right pairs show bright field (Top) and DAF-FM fluorescence (Bottom) images from live CG neuron cultures treated with RSarg incubation medium alone (Control, Left) or with RSarg containing PACAP (Right, 30 min). Calibration: 25 μm. B. Specific neuronal DAF-FM fluorescence levels (FLS*) associated with PACAP, L-NAME, or L-NAME + PACAP treatments are expressed here and subsequently relative to control levels from 204 to 739 neurons as described in Experimental Methods. Inset shows FLS* quantification for neurons treated with MAHMA-NANOate (MAHMA) relative to untreated controls from90 and 112 neurons, respectively. Here and subsequently, an asterisk above the treatment group value indicates a significant difference compared to the control group (p b 0.05). Concentrations of PACAP, MAHMA-NANOate, and L-NAME were 100 nM, 100 μM, and 1mM, respectively. C. Endogenous NOS1 expression in CG and neurons. Left: E14 CG homogenates probed with NOS1 pAb (WB, +) revealed a major immunoreactive protein having the size predicted for chicken NOS1 (160 kDa, arrow) that was specific since it was absent when the primary antibody was omitted (WB, −). Right: Somas of acutely dissociated E14 CG neurons (Left) and E8 CG neurons in culture (Right) displayed NOS1-specific immunofluorescence. Calibration: 10 μm.

2.4.2 NO is a retrograde messenger

To determine whether NO generated by PACAP signaling acts as a retrograde messenger to impact presynaptic function, we examined the effect of the NO scavenger

2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO). In accord with our previous findings, the frequency (Fs) and amplitude (As) of spontaneous EPSCs recorded from CG neurons treated with PACAP (100 nM, 15 min) were, respectively, 3.5 and 1.5 times higher than those obtained from untreated neurons tested in parallel (Fig.

2.2 A-C). By contrast, when neurons from the same cultures were co-treated with

PACAP and cPTIO (200 µM) there was no detectable increase in either Fs or As. The effect was specific in the sense that Fs and As values from untreated neurons and neurons treated with cPTIO alone were statistically indistinguishable. Since cPTIO scavenges extracellular NO and is membrane impermeable, these results indicate that NO synthesized by NOS1 as a consequence of PACAP/PAC1R signaling crossed a cell membrane to exert its presynaptic effects (Etherington, 2004; Bright and Brickley, 2008;

Xue et al., 2011). This finding supports the idea that NO acts by a retrograde mechanism since it indicates the NO originated from a source outside of presynaptic terminals such as postsynaptic neurons or non-neuronal support (i.e. glial) cells. Any contribution from glial support cells is unlikely, however, because the culture conditions restrict their growth (Nishi and Berg, 1981) and because PACAP failed to detectably increase FLS* in the few glial cells that were present (Fig. 2D).

Figure 2.2: NO generated by PACAP signaling in CG neurons acts as a retrograde messenger to induce synaptic plasticity. A. Exemplar sEPSC recordings showing that scavenging extracellular NO with cPTIO blocks PACAP-induced synaptic plasticity. Calibrations: 50 pA vertical, 500ms horizontal. Panels B and C show quantification of the cPTIO and PACAP treatment effects on sEPSC frequency and amplitude values normalized to those obtained from untreated controls (Fs* and As*, respectively). In panels A–C CG neuron cultures were pre-treated with culture medium (MEMeye) alone (Control, 20min) or MEMeye containing cPTIO (200 μM, 20min) and then treated with MEMeye alone or MEMeye containing PACAP (100 nM, 15min) ± cPTIO (200 μM,15 min). Plus (+) and minus (−) signs denote the presence or absence of PACAP or cPTIO in the treatment condition. Cultures treated with MEMeye alone or MEMeye containing PACAP were washed 1× in RShs and recording smade in RShs. Cultures treated with MEMeye containing cPTIO± PACAP were washed 1× in RShs containing 200 μM cPTIO, and recordings made in RShs containing 200 μM cPTIO. Data in panels B and C were compiled from 8 to 14 neurons for each of the four treatments. D. Glial support cell NO levels are unaffected by PACAP. The specific DAF-FM fluorescence increase (FLS*) was quantified as described in Experimental Methods, for control and PACAP-treated neuron somas (n = 92 and 113, respectively) and glial support cells (n= 23 and 25, respectively).

2.4.3 PACAP-Induced PKA Dependent Up-regulation of α7-nAChRs Underlies NO

Elevation

CG neurons express abundant, high-affinity, PACAP-specific PAC1Rs that couple via Gαs and Gαq to engage AC and phospholipase-C (PLC) signaling pathways, respectively (Deutsch and Sun, 1992; Spengler et al., 1993; Pisegna and Wank, 1996).

We previously showed that PAC1R signaling via PLC causes inositol phosphate turnover and Ca2+ release from intracellular stores (Ca2+in) while AC signaling elevates intracellular cyclic AMP (cAMP) (Margiotta and Pardi, 1995; Pardi and Margiotta, 1999;

Woo and Margiotta, 2007) causing subsequent activation of cAMP dependent protein kinase (PKA). Since NOS1 activity initiated by Ca2+/Calmodulin is sustained by PKA- dependent phosphorylation (Bredt et al., 1992; Hurt et al., 2012) and PKA is required for

NO-dependent LTP in the cerebellum (Jacoby et al., 2001) we next tested the relative contributions of PLC and AC signaling pathways to PACAP-induced NO production in

CG neuronal cultures (Fig. 2.3A). Consistent with previous results showing that PLC is dispensable for PACAP to enhance nAChR-mediated synaptic function (Pugh et al.,

2010) PLC inhibition with U73211 failed to block the PACAP-induced increase in FLS*.

By contrast, PKA was required for PACAP to induce synaptic plasticity (Pugh et al.,

2010) and is similarly required for PACAP to increase FLS* since co-treatment with the highly specific PKA inhibitor PKI(14-22) blocked the increase. These findings indicate that PACAP signaling via PKA is necessary for NOS1 activation and increased NO,

2+ while concomitant signaling via PLC and Ca in release is dispensable.

Although elevation of Ca2+ derived from PLC stimulated-intracellular stores is dispensable for NOS1 activation, localized Ca2+ influx via NMDA or α-Amino-3-

56 hydroxy-5-methyl-4-isoxzolepropionic Acid (AMPA) type glutamate receptors does activate NOS1 and lead to NO production (Garthwaite et al., 1988; Garthwaite, 2008;

Socodato et al., 2012). Like NMDARs, neuronal nAChRs display considerable Ca2+

2+ + permeability. The Ca relative to Na permeability (PCa/PNa) for α3β4-containing nAChRs is ≈1 and 10-20 for α7-nAChRs (Séguéla et al., 1993; Vernino et al., 1994; Role and Berg, 1996), the latter equaling or surpassing that reported for NMDARs (PCa/PNa =

10) (Mayer and Westbrook, 1987). Moreover, unlike NMDARs, α7-nAChRs are not susceptible to Mg2+ block at resting or hyperpolarizing voltages thereby providing a route for Ca2+ influx at physiological membrane potentials (Role and Berg, 1996). We therefore next examined the Ca2+ and nAChR dependence of NO production (Fig. 3B).

Stimulating Ca2+ influx via voltage-dependent Ca2+ channels (VDCCs) by treating the neurons with depolarizing KCl (50 mM) elevated neuronal FLS*, and this effect was blocked when external Ca2+ was reduced to ≈5 µM with EGTA (5 mM). Similarly, activating Ca2+-permeable α3*- and α7-nAChRs by treating E14 neurons with carbamylcholine (CCh, 1 mM) also increased FLS* (Fig. 2.3B. In addition to nAChRs,

CCh will stimulate muscarinic AChRs (mAChRs) present on CG neurons that couple via

2+ PLC to induce Ca in release from intracellular stores (Rathouz et al., 1995; 1996). Any contribution from mAChRs stimulated by CCh was minimal, however, because the increase in FLS* was blocked by external EGTA and therefore dependent on external

Ca2+ influx, and because co-treatment with the reversible, pan-selective nAChR antagonist d-tubocurarine (dTC, 100 µM) completely blocked CCh-stimulated FLS*.

These results are consistent with the NOS1-, nAChR-, and VDCC-dependent NO increases produced by nicotine in dorsal root ganglion neurons (Haberberger et al., 2004)

57 and indicate that activation of Ca2+-permeable nAChRs stimulates neuronal NO production in CG neurons. nAChR activation would induce Ca2+ entry and depolarization to activate VDCCs thereby increasing Ca2+ influx, and possibly also triggering Ca2+-induced Ca2+ release (CICR) from intracellular stores.

Since PACAP increases α3*- and α7-nAChR agonist-induced currents by >2-fold via AC/PKA-dependent up-regulation (Margiotta and Pardi, 1995; Pardi and Margiotta,

1999) we tested the hypothesis that nAChRs are necessary intermediates in the PACAP- induced elevation of neuronal NO production. In CG cultures, ongoing impulse- dependent ACh release engages postsynaptic nAChRs leading to sEPSCs (Chen et al.,

2001; Pugh et al., 2010) and is also expected to spread to peri- and extra-synaptic nAChRs. In this context, inhibiting all endogenous nAChR activity with dTC (100 µM) blocked the PACAP-induced increase in NO (Fig. 2.3C) supporting a mechanism whereby PACAP enhances synaptic function by an obligatory AC/PKA-dependent participation of nAChRs that increases Ca2+ influx to activate NOS1 and induce NO production. To determine relative contributions of nAChR subtypes in PACAP-induced

NO production, α3*- or α7-nAChR mediated currents were selectively antagonized prior to and during PACAP treatment. We previously found that 10 µM alpha-conotoxin AuIB

(αCTx-AuIB) inhibited α3*-nAChR-mediated currents by >90% without affecting α7- nAChR currents, while 50 nM αBgt blocked α7-nAChR-mediated currents without affecting α3*-nAChR-mediated currents (Nai et al., 2000). Interestingly, the ability of

PACAP to increase FLS* in CG neuron cultures was primarily α7-nAChR dependent

Fig. 2.3: PACAP-induced NO elevation assessed by increased DAF-FM FLS* requires PKA, Ca2+ influx and α7-nAChR activation. A. PACAP-induced increases in FLS* are blocked by prethen co-treatment with PKI(14–22) to inhibit PKA (Left) but unaffected by similar treatments with U7-3122 to inhibit PLC (Right). B. Activation of voltage- dependent Ca2+ channels with 50 mM KCl (Left) or nAChRs with 1 mM CCh (Right) increase FLS*, effects indicative of a requirement for Ca2+ influx since they are reversed by chelating extracellular Ca2+ with EGTA or by blocking nAChRs with dTC. C. PACAP-induced increases in FLS* are abolished by co-treatment with dTC to block both α3*- and α7-nAChRs (Left). PACAP-induced increases in FLS* are nominally reduced by cotreatment with αCTx-AuIB to selectively block α3*-nAChRs (Middle), but are abolished by co-treatment with αBgt to block highly Ca2+-permeable α7-nAChRs (Right). DAFFM FLS* was quantified as described in Experimental Methods. Cultures were pretreated with incubation medium (RSarg) alone (15 min) or RSarg containing the indicated reagents PKI(14–22) (1 μM, 15 min), U7-3122 (1 μM, 15 min), EGTA (5 mM, 15 min), dTC (100 μM, 10–20 min), αCTx-AuIB (10 μM, 10 min) or αBgt (50 nM, 30 min). Cultures were then treated for 30 min with RSarg alone or RSarg containing PACAP (100 nM) ± PKI(14–22) (1 μM), dTC (100 μM), αCTx-AuIB (10 μM) or αBgt (50 nM), with RSarg containing KCl (50 mM) ± EGTA (5 mM), or with RSarg containing CCh (1 mM) ± dTC or EGTA (5 mM). Plus (+) and minus (−) signs denote the presence or absence of the indicated reagent in the treatment condition. Each bar represents FLS* measurements from 25–370 neurons.

60 since it was only nominally diminished by co-treatment with 10 µM αCTx-AuIB (a gift from J Michael McIntosh Department of Biology, University of Utah, Salt Lake City,

Utah) but abolished by 50 nM αBgt. In fact, αBgt applied with PACAP drove FLS* significantly below control levels. Since somatic α7-nAChRs are up-regulated by

PACAP/PAC1R signaling (Margiotta and Pardi, 1995; Pardi and Margiotta, 1999) the latter finding indicates that ongoing α7-nAChR activity exerts a powerful influence on

NO production. Taken together, these results indicate that highly Ca2+-permeable α7- nAChRs are necessary intermediates in PACAP-induced NO production, whereas α3*- nAChRs are minor or dispensable participants in this process despite their central role in mediating the bulk of transmission at synapses on CG neurons (Chen et al., 2001; Pugh et al., 2010).

2.4.4 Concommitant nAChR Activation Underlies PACAP/PAC1R-Induced

Synaptic Plasticity

Our findings thus far indicate that PACAP/PAC1R signaling induces synaptic plasticity via NOS1-mediated increases in NO production, and that such increases require recruitment of α7- but not α3*-nAChRs. Since both α3*- and α7-nAChR populations are up-regulated by PACAP (Margiotta and Pardi, 1995; Pardi and Margiotta, 1999) we next tested whether either or both are required for PACAP/PAC1R induced synaptic plasticity by using dTC to block both subtypes and αCTx-AuIB or αBgt to respectively block α3*- or α7-nAChRs (Fig. 2.4). As with NO mobilization, concomitant nAChR activity is required for synaptic plasticity since co-application of the reversible, pan-selective nAChR antagonist dTC (100 µM) followed by washing to remove the drug, blocked the

61 increase in Fs and As normally produced by PACAP (Fig. 2.4A). To assess contributions from α3*- or α7-nAChRs we relied on previous observations that while αCTx-AuIB is specific for α3*-nAChRs, that mediate virtually all sEPSCs in the cultures, the toxin has relatively low potency (IC50 ≈ 350 nM) and is readily reversible (Chen et al., 2001; Nai et al., 2003). Conversely while αBgt has very high potency for blocking α7-nAChRs (IC50

≈ 10 nM) with high affinity (KD ≈ 1 nM) and displays slow reversal (McNerney et al.,

2000) α7-nAChRs mediate a tiny fraction of sEPSCs in CG cultures (Chen et al., 2001).

We first confirmed the degree and reversibility of αCTx-AuIB block by testing the toxin on neurons displaying only α3*-nAChR currents (achieved by blocking α7-nAChRs with

50 nM αBgt). In these experiments, peak whole-cell α3*-nAChR currents induced by microperfusion with 20 µM nicotine were reduced by >90% in neurons co-treated with

αBgt and 10 µM αCTx-AuIB compared with responses from control neurons treated with

αBgt alone. After 2 washes (5 min each) peak α3*-nAChR currents from αCTx-AuIB treated neurons recovered and were indistinguishable from those obtained from controls

(ER Starr and JF Margiotta, unpublished findings) indicating rapid and complete toxin reversibility. Consistent with these actions, Fs and As sEPSC values are drastically reduced by αCTx-AuIB treatment (Chen et al., 2001) and were restored to control levels after toxin washout (Fig. 2.4B). In accord with their nominal effect on NO production, blocking α3*-nAChRs with αCTx-AuIB during PACAP treatment only partially impacted PACAP-induced synaptic plasticity. Specifically, while prethen co-treatment with αCTx-AuIB followed by washing eliminated the PACAP-induced increase in As, the increase in Fs was only nominally affected. As with NO production, results with αBgt were more extreme since blocking α7-nAChRs with αBgt before and then during PACAP

62 treatment abolished both aspects of PACAP-induced synaptic plasticity. Specifically, when neurons were co-incubated with αBgt and recordings made with the toxin present, the PACAP-induced increase in sEPSC Fs was greatly reduced and the increase As abolished (Fig. 2.4C). The failure was not due to an anomalously high fraction of α7- nAChR mediated sEPSCs in these experiments since sEPSCs acquired in the presence of

αBgt alone displayed Fs and As values that were indistinguishable from those of naïve controls tested in parallel. These experiments indicate that, although both α7- and α3*- nAChRs are up-regulated by PACAP/PAC1R signaling (Margiotta and Pardi, 1995; Pardi and Margiotta, 1999) Ca2+-permeable α7-nAChRs are necessary intermediates for both

PACAP-induced NO production and consequent synaptic plasticity, whereas α3*- nAChRs, that mediate the bulk of sEPSCs, play a lesser role in these processes. In order to assess whether presynaptic nAChRs contribute to synaptic plasticity, the selective agonist GTS-21 was focally applied at low concentration to activate α7-nAChRs on presynaptic inputs (McGehee et al., 1995; McGehee and Role, 1996). Interestingly sEPSC Fs and As values recorded before and after 3 µM GTS-21 application were indistinguishable (p>0.05 for both, Starr and Margiotta, unpublished findings). The inability of GTS-21 to alter these synaptic parameters indicates that if α7-nAChRs are present on presynaptic terminals, they are unable to increase ACh release and therefore unlikely responsible for triggering the observed plasticity. Instead, α7-nAChRs on the postsynaptic cell soma seem more likely to play this role, a conclusion that is consistent with our findings that NO acts in a retrograde fashion to impact synaptic plasticity (Fig.

2.2B and C) and that α7-nAChR activation generates NO in CG neuron somas (Fig.

2.3C).

Fig. 2.4: nAChR participation in PACAP-induced synaptic plasticity. A. PACAP- induced increases in sEPSC frequency (Fs*, Left) and amplitude (As*, Right) are blocked after prethen co-treatment with dTC (to reversibly inhibit both α3*- and α7-nAChRs). B. Prethen co-treatment with αCTx-AuIB to reversibly inhibit α3*-nAChRs blocked PACAPinduced increases in sEPSC amplitude (Right) but only nominally reduced the increases in frequency (Left). C. Pre- then co-treatment with αBgt to block α7-nAChRs, attenuated the PACAP-induced increase in sEPSC frequency (Left) and abolished the increase in amplitude (Right). Cultures were pre-treated with MEMeye alone (15 min) or MEMeye containing the indicated nAChR blockers dTC (100 μM, 10–20 min), αCTx- AuIB (10 μM,10 min) or αBgt (50 nM, 30 min). Cultures were then treated for 30 min with MEMeye alone or MEMeye containing PACAP (100 nM) ± dTC (100 μM), αCTx- AuIB (10 μM) or αBgt (50 nM). Plus (+) and minus (−) signs denote the presence or absence of the indicated reagent in the treatment condition. Cultures treated with dTC or αCTx-AuIB were washed (3×, 5 min each) in RShs and sEPSC recordings made in RShs whereas those treated with αBgt were washed 1× in RShs containing αBgt (50 nM) and sEPSC recordings made in RShs containing αBgt (50 nM). The ^ symbol indicates a significant difference (p b 0.05) in the indicated parameter relative to PACAP treatment. Each bar represents measurements from 9 to 25 neurons. that is consistent with our findings that NO acts in a retrograde fashion to impact synaptic plasticity (Fig. 2B and C) and that α7-nAChR activation generates NO in CG neuron somas (Fig. 3C).

3.4.5 nAChRs Associate with NOS1.

The requirement for α7-nAChRs in PACAP-induced NO production and consequent synaptic plasticity, and the partial involvement of α3*-nAChRs in NO- dependent synaptic plasticity could indicate that nAChRs and NOS1 are physically associated. To test this idea, co-precipitation experiments were conducted using either

αBgt conjugated with Actigel resin (αBgt/Actigel) or an α3/α5-subunit specific antibody

(mAb35) conjugated with Protein AG resin (mAb35/AG) to respectively purify α7- or

α3*-AChRs and interacting proteins from CG homogenates (Fig. 2.5). α Bgt/Actigel specifically isolated α 7-AChRs since an α 7 subunit-specific mAb (mAb319) detected a protein having appropriate mass (≈55 kDa) that was absent when free αBgt was added

(Fig. 2.5A). Interestingly, αBgt/Actigel co-precipitated a protein recognized by anti-

NOS1 having a molecular mass expected for NOS1 (≈160 kDa). A similar pattern was observed for α3*-nAChRs (Fig. 2.5B). Here mAb35/AG specifically immuno-purified

α3*-nAChRs since an α3 subunit-specific mAb (mAbA3-1) detected a protein of appropriate mass (≈50 kDa) that was not seen when non-immune serum was substituted for mAb35. mAb35/AG co-immunoprecipitated a protein recognized by anti-NOS1 having the expected ≈160 kDa size and this interaction was specific since none was detected when non-immune IgG was substituted for mAb35. The specificity of mAb319 and mAbA3-1 for their respective α7- and α3-nAChR subunit gene products over other nAChR subunits has been previously tested and validated (Vernallis et al., 1993). Thus, these results are consistent with the requirement for α7-nAChRs and nominal / partial involvement of α3*-nAChRs in NO production and synaptic plasticity (Figs. 2.3 and 2.4) and suggest that these processes are facilitated by nAChR/NOS1 interaction.

Fig. 2.5: α7- and α3*-nAChRs interact with NOS1. A, top. Precipitation with αBgt/Actigel resin (αBgt PD) isolated α7-nAChRs from CG lysates as indicated by detection of an ≈55 kDa eluted protein after probing with mAb319, an α7- subunit specific mAb (+). The pull down was specific since α7-like immunoreactivity was absent in negative controls where excess free αBgt (50 nM) was added to the αBgt/Actigel resin and lysate (−). A, bottom. αBgt PD specifically co-precipitated NOS1 as indicated by the detection of an ≈160 kDa protein when probed with anti-NOS1 (+) that was absent in negative controls (−). B, top. Immuno-precipitation with mAb35/AG resin (mAb35 PD) isolated α3*-nAChRs from CG lysates as indicated by detection of an ≈50 kDa protein when probed with A3-1, an α3-subunit specific mAb (+) that was absent in negative controls where non-immune rat serum was substituted formAb35 (−). B, bottom.mAb35 PD specifically co-immunoprecipitated NOS1 as indicated by an ≈160 kDa protein detected when probed with anti-NOS1 (+) that was absent in negative controls (−). Molecular mass markers indicate 50 kDa (top) and 150 kDa (bottom). Similar results were obtained in 2 other experiments for both nAChR subtypes. with the same antibody revealed significant AKAP5 immunoreactivity confined to CG neurons both in vivo and in culture (Fig. 6B).

3.4.6 AKAP5 is Present in CG Neurons and Targets PACAP/PAC1R Signals to

Synapses.

The preceding results indicate that PACAP/PAC1R signaling induces synaptic plasticity by elevating NO through α7-nAChR-dependent stimulation of NOS1 that may be facilitated by nAChR/NOS1 interaction. Since PKA is required for PACAP to upregulate α7-nAChRs (Pardi and Margiotta, 1999), sustain NO production (Fig. 2) and induce synaptic plasticity (Pugh et al., 2010) we next explored the possibility that these processes are coordinated by A-kinase anchoring proteins (AKAPs) that are known to act as platforms for delivering PKA to appropriate target substrates (Wong and Scott, 2004).

AKAP5 (Gene Nomenclature Committee name for proteins previously known as

AKAP79/150) is a prominent neuronal subtype known to be involved in synaptic plasticity (Wong and Scott, 2004; Sanderson and Dell'Acqua, 2011). We found evidence for a family of AKAPs including AKAP5 in CG extracts and neurons (Fig. 2.6). Filter overlays using PKA regulatory subunit II isoform α (RIIα) probed with anti-RII mAb revealed the presence of a family of interacting proteins in CG extracts. Proteins at 150,

127, 98, 66, and 56 kDa were identified as AKAPs because their binding was eliminated or greatly reduced when RIIα was co-incubated with an AKAP fragment that encompasses its RII-binding domain (Ht31) but not by an inactive proline analog

(Ht31P). Of the five AKAPs present, the 66 kDa protein likely represents AKAP5 since an anti-AKAP5 mAb recognized a protein of identical size in CG homogenates (Fig.

2.6A). Moreover, probing with the same antibody revealed significant AKAP5, immunoreactivity confined to CG neurons both in vivo and in culture (Fig. 2.6B).

Having established that AKAPs including AKAP5 are expressed in the CG system, we next examined their role in localizing PACAP/PAC1R signaling to synapses.

To do so, the effects of PACAP on synaptic function were assessed after blocking

AKAP-RIIα interactions with Ht31 to prevent subsequent PKA-dependent phosphorylation, a previously established requirement for synaptic plasticity (Pugh et al.,

2010). Interestingly, the ability of PACAP to enhance nAChR mediated sEPSC parameters (Fs and As) was abolished in Ht31-treated CG neurons (Fig. 2.6C).

Supporting the specificity of these effects, neurons not challenged with PACAP but treated with Ht31 alone displayed Fs and As levels that were not significantly different in neurons from control cultures tested in parallel. Based on ultrastructural and autoradiographic studies that distinguish nAChR abundance at postsynaptic versus peri- and extra-synaptic sites (Jacob et al., 1986; Loring and Zigmond, 1987; et al., 2001) we previously calculated that less than 5% of all surface α3*-nAChRs on CG neurons in culture are synaptic (Pugh et al., 2010). If AKAP actions were confined to synapses, blocking AKAP-RIIα interactions with Ht31 would be expected to have little effect on the ability of PACAP to up-regulate the total surface α3*-nAChR population (Margiotta and Pardi, 1995; Pardi and Margiotta, 1999). Consistent with this expectation, the 2X increase in α3*-nAChR currents typically induced by nicotine application seen after

PACAP treatment was maintained after pre-incubation and co-treatment with Ht31 (Fig.

2.6D). These results implicate AKAPs, possibly AKAP5, in confining PKA-dependent consequences of PACAP/PAC1R signaling to synapses as a co-requirement with NOS1 for triggering synaptic plasticity. Interestingly MAGUK scaffolding proteins such as

PSD-95 provide a molecular framework that, in addition to interacting with

Fig. 2.6: AKAP expression and PKA targeting to nAChR-mediated synapses by PACAP/PAC1R signaling. A. Detection of multiple AKAPs. Proteins in E14 CG homogenates were separated on PAGE/SDS gels and transferred to nitrocellulose membranes (Lanes 1–3, 5). In lanes 1–3, the membranes were overlaid with PKA regulatory subunit type II β (RIIβ) and then probed with anti-RII mAb (α-RII). Five RIIβ binding proteins were detected (Lane1) that represent AKAPs (arrows at indicated kDa) since their binding was blocked or drastically reduced when RIIβwas incubated with excess Ht31 inhibitor peptide (Ht31, Lane 2) but unaffected by incubation with control Ht31P peptide (Ht31P, Lane 3). Recombinant RIIβ migrates at the same apparent size (51 kDa) as the Ht31-insensitive protein (Lane 4; open triangles). An anti-AKAP5 mAb recognizes a 66 kDa protein in CG homogenates (Lane 5) corresponding to a prominent AKAP of identical size in RII overlays (Lanes 1, 3). Molecular weight markers indicate 250, 150, 100, 75, and 50 kDa. B. Detection of AKAP5 in CG neurons. Neurons in CG cultures (Left) and E14 ganglion sections (Right) are strongly immunoreactive after labeling with AKAP5 antiserum. Immunoreactivity was undetectable when the primary antiserum was omitted (data not shown). Accompanying bright field images are included below for reference. Scale bar, 15 μm. C. PACAP/PAC1R induced synaptic plasticity requires AKAP signaling. PACAP (100 nM, 15 min) increased sEPSC frequency (Fs, Left) and amplitude (As, Right) to levels that were, respectively, 5.8 and 1.5 times higher (black bars) than those of untreated controls (white bars). When combined with Ht31 pretreatment (5 μM, 1 h), however, co-treatment with PACAP failed to significantly increase either Fs or As (checked bars). Pretreating CG cultures with AKAP Ht31 inhibitor peptide alone had no effect on Fs or As (gray bars) compared to sham treated controls tested in parallel. Results are from 12–17 neurons for each condition in 3 separate experiments. D. AKAP signaling effects on α3*-nAChR mediated sEPSCs are localized to synapses. Records (at left) show averaged whole-cell α3*-nAChR currents induced by rapid microperfusion with 20 μM nicotine (bar) from neurons after sham treatment (Control, Black) after PACAP treatment (100 nM, 15 min, Blue) or after AKAP inhibition with Ht-31 (5 μM, 1 h) followed by PACAP and Ht31 co-treatment (15

71 min, Red). Cultures were preincubated in 100 nM, 30 min α-bungarotoxin to block α7- nAChRs. Calibration bars indicate 100 pA and 500 ms. Bar graph (at Right) shows corresponding mean peak current values normalized to cell membrane capacitance (Cm). Results depicted are from 8–12 neurons in two experiments.

NMDARs, AMPARs and nAChRs, also bind AKAPs and NOS1 (Christopherson et al.,

1999; Colledge et al., 2000; Wong and Scott, 2004; Funke et al., 2005; Sanderson and

Dell'Acqua, 2011). In a similar fashion, PSD scaffolds could orient the actions of PKA and NO via AKAP5 and NOS1, respectively, to appropriate nAChRs and synaptic components.

2.5 Discussion

Our results reveal that PACAP/PAC1R signaling rapidly confers plasticity at autonomic synapses by coordinating NOS1, nAChR, and AKAP activities. PACAP elevated NO production (Fig. 1) required to increase sEPSC frequency and amplitude in

CG neurons (Pugh et al., 2010) and such increases were blocked by scavenging extracellular NO with cPTIO (Fig. 2). NOS1 and PKA activities were both necessary for

PACAP to elevate NO (Figs. 1, 3A, B). The PKA dependence is consistent with previous results indicating that PKA phosphorylation “sensitizes” NOS1 thereby sustaining NO elevation (Bredt et al., 1992; Hurt et al., 2012). Finding that K+ depolarization-induced

Ca2+ influx via VDCCs elevated NO is in accord with the Ca2+/Calmodulin dependence of NOS1. The additional observation that Ca2+ influx rather than PLC-dependent intracellular Ca2+ release was required for stimulating NOS1 is consistent with results indicating that NOS1 can be activated via VDCCs and Ca2+-permeable NMDARs and

AMPARs (Garthwaite et al., 1988; Garthwaite, 2008; Socodato et al., 2012). This finding may reflect localized, possibly Ca2+/Calmodulin sensor-dependent signaling

(Burgoyne, 2007) relevant to PACAP-induced plasticity, but we cannot exclude the possibility that Ca2+ influx and subsequent CICR-driven cytoplasmic Ca2+ elevation may both be required. Since cPTIO is membrane impermeable and PACAP did not elevate

NO in glia, the scavenging experiments indicate that NO generated in postsynaptic neurons enters the extracellular space to increase presynaptic ACh release (Etherington,

2004; Bright and Brickley, 2008; Xue et al., 2011) supporting its role as a retrograde messenger (Hölscher, 1997; Garthwaite, 2008; Hardingham et al., 2013).

nAChR activity was required for PACAP-induced NO elevation and consequent synaptic plasticity. Stimulating nAChRs with CCh elevated neuronal NO, and this and the NO elevation produced by PACAP were both blocked by inhibiting nAChR function with dTC (Fig. 3). Experiments with αCTx-AuIB and αBgt revealed nominal and major roles for α3*-nAChRs and α7-nAChRs, respectively, in PACAP-induced NO elevation.

These findings are consistent with spontaneous nAChR-mediated EPSCs evident in CG neuron cultures (Margiotta and Berg, 1982; Chen et al., 2001), and with previous results indicating that α3*- and α7-nAChR subtypes are both up-regulated by PACAP/PAC1R signaling via PKA (Margiotta and Pardi, 1995; Pardi and Margiotta, 1999), and that α3*- and α7-nAChRs, respectively, display moderate and high Ca2+ permeability (Séguéla et al., 1993; Vernino et al., 1994; Role and Berg, 1996). The α7-nAChRs responsible for triggering NOS1 activation are likely on the soma rather than presynaptic terminals

(McGehee et al., 1995; McGehee and Role, 1996) because the bulk of NO fluorescence is localized to the soma (Fig. 1) and because we were unable to detect an influence of

73 presynaptic α7-nAChRs on transmitter release in CG cultures (Starr and Margiotta, unpublished findings). nAChRs were also required for PACAP-induced synaptic plasticity (Fig. 4). As with NO elevation, reversible nAChR inhibition with dTC blocked the ability of PACAP to increase sEPSC parameters. Similarly, reversible selective inhibition of α3*-nAChRs with αCTx-AuIB during exposure to PACAP had a modest effect, significantly reducing sEPSC amplitude, but only nominally reducing the PACAP- induced increase in frequency. In contrast, α7-nAChRs were essential since blocking their function with αBgt abrogated the increase amplitude and greatly attenuated the increase in frequency.

The importance of α7-nAChRs may appear surprising since the vast majority of sEPSCs in the cultures are mediated by α3*-nAChRs, compared with only ≈10% mediated by α7-nAChRs (Chen et al., 2001). Nevertheless, the central role of α7- nAChRs in mediating the PACAP-induced increase in NO production and synaptic plasticity is consistent with their emerging signaling functions. Examples include an α7- nAChR dependence in supporting CG neuron survival (Pugh and Margiotta, 2000), controlling the inhibitory phenotype of GABAergic inputs (Liu et al., 2006), determining the fate of adult born neurons (Campbell et al., 2010) and promoting glutamatergic synapse formation (Lozada et al., 2012). While Ca2+ influx via α7-nAChRs was suggested as the critical trigger for all of these functions, the present work is the first to indicate a mechanistic role for Ca2+ permeable α7-nAChRs. It remains to be seen whether Ca2+ influx via α7-nAChRs is sufficient trigger NO production and synaptic plasticity, or whether synaptic co-activation of VDCCs and CICR are also required. The importance of α7-nAChRs in PACAP-induced synaptic plasticity may also seem at odds

74 with the low expression of α7-nAChR mRNA in CG cultures (Corriveau and Berg,

1994). Nevertheless CG neurons do express functional α7-nAChRs in culture since α7- nAChR mediated sEPSCs can be identified (Chen et al., 2001), and nicotine induces α7- nAChR currents (Zhou et al., 2004) and supports α7-nAChR-mediated cell survival

(Pugh and Margiotta, 2000). Interestingly, previous studies indicated that α7-nAChRs are predominantly found at perisynaptic and extrasynaptic sites in culture (McNerney et al., 2000; Chen et al., 2001) an arrangement similar to that seen in the ganglion (Horch and Sargent, 1995; Sargent, 2009; Stanchev and Sargent, 2011). Since α7-nAChR mediated sEPSCs are rare (Chen et al., 2001) perisynaptic and extrasynaptic α7-nAChRs up-regulated by PACAP signaling may contribute significantly to the plasticity observed here. In preliminary studies, kinetically identified α7-nAChR mediated sEPSCs displayed increases in frequency and amplitude after PACAP treatment that were similar to those for total sEPSCs from the same neurons (data not shown). Such α7-nAChR mediated sEPSCs represented <10% of total events in control and PACAP-treated neurons, however, a factor that could limit their causal contribution to PACAP-induced plasticity.

Consistent with their functional coupling, α7- and α3*-nAChRs exist in a protein complex with NOS1 (Fig. 5). Scaffolding proteins could link nAChRs with NOS1 as at glutamatergic synapses where NMDARs are linked to NOS1 via PSD-95 or PSD-93, forming a ternary molecular complex required to efficiently couple Ca2+ influx to NOS1 activation for on-demand NO synthesis (Garthwaite et al., 1988; Bredt and Snyder, 1990;

Christopherson et al., 1999; Cui et al., 2007). Previous studies indicate that α3*- and α7- nAChRs do bind PSD scaffolding proteins, thereby optimizing synaptic function (Conroy

75 et al., 2003; Parker et al., 2004). In CG homogenates, specific interactions were found between α3*-nAChRs and PSD-95, and between α7-nAChRs and an unidentified PDZ containing protein, possibly a variant of PSD-93 (PSDx) (Conroy et al., 2003). The observed association of α7- and α3*-nAChRs with NOS1 are consistent with their respective peri- and postsynaptic localizations (Horch and Sargent, 1995; Chen et al.,

2001; Sargent, 2009). By analogy with NMDARs, PSDx and PSD95, could provide the structural scaffold for α7- and α3*-nAChR/NOS1 interactions at peri- and postsynaptic sites, respectively. In such an arrangement, Ca2+ entry from perisynaptic α7-nAChRs and

VDCCs could spread to activate NOS1 at both perisynaptic α7-nAChR/PSDx/NOS1 and adjacent postsynaptic α3*-nAChR/PSD95/NOS1 complexes thereby targeting the resulting NO production to juxtaposed presynaptic sites (See below).

AKAPs are required for targeting the PKA-dependent outcomes of

PACAP/PAC1R signaling to influence synaptic function (Fig. 6). A family of AKAPs including neuron-associated AKAP5 were identified in CG extracts, and inclusion of the

Ht31 AKAP inhibitor peptide blocked the ability of PACAP to increase sEPSC frequency and amplitude. Since AKAPs localize PKA actions, and both PACAP-induced synaptic plasticity and α7-nAChR up-regulation are dependent on PKA phosphorylation (Pardi and Margiotta, 1999; Pugh et al., 2010), perisynaptic α7-nAChRs may be targets of, or influenced by the AKAP mediated PKA phosphorylation that results in their upregulation. In support of a local, AKAP-dependent up-regulation arrangement, we found that Ht31 was unable to affect the ability of PACAP to enhance global nAChR-mediated responses to nicotine. Since extrasynaptic nAChRs vastly outnumber those associated with synapses (Jacob et al., 1986; Loring and Zigmond, 1987; Shoop et al., 2001; Pugh et

76 al., 2010) they are likely responsible for such global responses, and unlike synaptic nAChRs are apparently regulated by an AKAP-independent mechanism.

MAGUKs bind NOS1, AKAP5, and nAChRs allowing their activities to coordinately and specifically impact synaptic plasticity. For example, PSD-95 is known to bind NMDA and AMPA receptors and NOS1 through its first and second PDZ modules, respectively, allowing it to act as a platform for coordinating synaptic function

(Christopherson et al., 1999; Funke et al., 2005). In addition PSD-95 and SAP97 are known to interact with AKAP5 via their C-terminal SH3/GK domains, an arrangement consistent with focal AMPAR phosphorylation and synaptic plasticity (Christopherson et al., 1999; Colledge and Scott, 1999). The hypothetical model in Fig. 2.7 incorporates similar interactions for nAChR-mediated synapses. Here AKAP5 and perisynaptic α7- nAChR are placed in close proximity by interacting with a scaffold protein, possibly

PSDx (Conroy and Berg, 1995). PACAP, applied exogenously or released during periods of high-frequency activity in vivo, stimulates PAC1Rs activating AC, increasing cAMP production, and causing PKA-dependent up-regulation of AKAP-associated α7- nAChRs and NOS1 sensitization. ACh spillover onto these functionally enhanced α7- nAChRs results in increased Ca2+ influx (and increased depolarization of adjacent

VDCCs (not shown)). The resultant perisynaptic Ca2+ influx also spreads to postsynaptic sites thereby stimulating NOS1 activity in α7-nAChR/PSDx/NOS1 and α3*- nAChR/PSD-95/NOS1 complexes and causing focal NO elevation. Finally, retrograde diffusion of NO across to juxtaposed presynaptic terminals enhances quantal ACh release thereby increasing the frequency and amplitude of EPSCs mediated largely by postsynaptic α3*-nAChRs.

While autonomic synapses have been characterized as simple relays, they display plasticity triggered by factors such as PACAP and BDNF that may facilitate unanticipated functions. We previously showed that BDNF/TrkB signaling also enhances nAChR-mediated autonomic transmission (Zhou et al., 2004). Consistent with their ability to modify synaptic function, both PACAP and BDNF are anxiogenic factors that gate responses to stress and fear (Martinowich et al., 2007; Hashimoto et al., 2011;

Hammack et al., 2009b; Ressler et al., 2011b; Mahan and Ressler, 2012). In particular, mice deficient in either BDNF or PACAP or their receptors display blunted responses to stress/fear conditioning (Hashimoto et al., 2001; Otto et al., 2001; Liu, 2004; Rattiner,

2004). Consistent with our previous and present results, both BDNF and PACAP also enhance activity in CNS circuits associated with stress and fear (Rattiner, 2005; Cho et al., 2012). Given the importance of autonomic synapses in gating visceral functions associated with and altered by stress and fear (e.g. cardiorespiratory output) stress disorder symptoms could result from BDNF- and PACAP-induced synaptic modifications such as we have described.

Fig. 2.7: Proposed model of nAChR-mediated autonomic synapses before (Left) and after (Right) PACAP exposure to induce plasticity. Previously undefined abbreviations: Pre (presynaptic), Post (postsynaptic) Peri (perisynaptic). Circles in MAGUK and NOS1 proteins depict PDZ modules. Large rectangles in MAGUKs depict C-terminal SH3/GK domains. In PKA/AKAP complexes, rectangles depict AKAP cores, ovals depict PKA regulatory subunits (R), and triangles depict PKA catalytic subunits (C). The dashed arrows indicate PKA activity resulting in α7-nAChR up-regulation and NOS1 phosphorylation. Small black dots depict ACh release from presynaptic vesicles. See text for other details.

Chapter 3

PACAP Induces Long-term Synaptic Plasticity through

Coordinated Adenylate Cyclase and Phospholipase C

Activation and Gene Transcription.

3.1 Introduction

Cellular signaling associated with neuropeptides can trigger synaptic plasticity at chemical synapses. One route through which neuropeptides exert these neuromodulatory actions is through activation of metabotropic g-PCRs on adjacent postsynaptic targets.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a pluripotent neuropeptide exhibiting neuromodulatory effects on excitatory synapses in the nervous system

(Reviewed in Starr and Margiotta, 2016). As a secretin family neuropeptide, PACAP is recognized by three class B g-PCRs referred to as the VPAC receptors (VPAC1R,

VPAC2R) and the PAC1 receptor (PAC1R). The PAC1R and VPACRs can be distinguished by their relative affinities for PACAP as well as by their potencies for linking peptide bonding to activation of intracellular signaling pathways (Rawlings et al.

1996; Vaudry et al 2009; Dickson et al. 2011; Margiotta and Starr, 2016). At synapses,

PACAP/VPACR/PAC1R signaling couples to AC and PLC-mediated signaling cascades to induce time-dependent alterations in synaptic function resulting in both short-term

(ST) or long-term (LT) synaptic plasticity at glutamatergic and cholinergic synapses

(Reviewed in Dickson and Finlayson, 2009; Blechman and Levkowitz, 2013; Starr and

Margiotta, 2016) suggesting that PACAPs influences on local neuronal targets and behavior in vivo involves synaptic plasticity.

Within parasympathetic ciliary ganglion (CG) neurons PACAP is synthesized, stored, and released from preganglionic synapses in an activity-dependent fashion (Pugh and Margiotta, 2006; Sumner et al. 2010; Pugh et al. 2009). Furthermore, PACAP exhibits a 1000-fold greater binding specificity than VIP as well as mobilizes both cAMP synthesis and IP turnover with greater potency, indicating that CG neurons express the

PAC1R (Margiotta and Pardi, 1995; Pardi and Margiotta 1999; Pugh et al. 2009). As shown in Chapter 2, exogenous PACAP rapidly enhances cholinergic transmission at synapses that form between CG neurons in culture (Jayakar et al. 2014). These synapses exhibit a dependence on cholinergic transmission mediated by activation of postsynaptic heteropentameric α3*-nAChRs or α7 nAChRs and resemble those formed on CG neurons in vivo, making it a useful model for examining nicotinic synapse formation, function and regulation (Ravdin and Berg, 1979; Margiotta and Berg, 1982; Role and Fischbach, 1987;

Chen et al. 2001). Ectopic application of PACAP (100 nM) within minutes enhances cholinergic transmission in CG cultures, increasing the frequency and amplitude of sEPSCs by 100-300% and 30-50% respectively. The ST PACAP-induced synaptic plasticity in CG neurons was characterized by enhancments in presynaptic ACh release and occurred via a PAC1R, AC, cAMP, PKA-dependent signaling pathway. The short- term (ST) PACAP-induced presynaptic plasticity resulted from a concomitant PAC1R and α3*-, α7-nAChR mediated activation of NOS1 resulting in synthesis and retrograde signaling via NO (Pugh et al. 2009; Jayakar et al. 2014).

PACAP is also a potent neurotrophic factor of CG neurons, activating a MAPK dependent pro- survival pathway (Pugh et al. 2006) as well as inducing nuclear translocation of pCREB (Sumner et al. 2009). In conjunction with these findings, PACAP alters the expression of genes relevant to synaptic function and induces a long-term (LT) plasticity detected 48 hours after the transient 15 min. PACAP treatment (Sumner et al.

2009). This LT PACAP-induced synaptic plasticity was characterized by similar enhancments in sEPSC frequency but more robust increases in sEPSC amplitude (Starr and Margiotta, 2014; Starr and Margiotta, 2016). However, the mechanisms underlying this LT PACAP-induced plasticity has never been thoroughly elucidated. Consequently, we decided to investigate the signaling mechanisms underlying the LT PACAP-induced synaptic plasticity at cholinergic synapses. In line with our hypothesis (see section 1.8), we confirm that PACAP does induces a LT synaptic plasticity that is mediated by distinct intracellular effectors and mechanisms from the ST PACAP-induced synaptic plasticity.

3.2 Methods

3.2.1 Neuronal Cultures

Cultures were prepared using ciliary ganglia obtained from embryonic day 8 (E8,

Stage 34-35) chicks (Hamburger and Hamiliton, 1951) using procedures identical to those previously described (Jayakar et al. 2014). Briefly, the cover glass surfaces of 12 mm diameter mini-wells in 35 mm WillCo-dishes (BioSoft international) were pre-coated with 0.2 mg/ml poly-D-ornithine and 12.5 µg/ml mouse laminin (a gift from Salvatore

Carbonetto, McGill University). Dissected ganglia were treated with trypsin (0.025%, 30 min, 37°C), the neurons dissociated by mechanical trituration, and ≈104 neurons (1.5

83 ganglion equivalents) plated per WillCo-dish well. The culture medium consisted of

Eagle’s minimum essential medium (MEM) supplemented with 2 mM glutamine, 100

U/ml penicillin, 10% heat-inactivated horse serum (Invitrogen, Rockville, MD) and with freshly prepared E17 chick eye extract (3% v/v, MEMeye) (Nishi and Berg, 1981).

eye Cultures were maintained at 37°C in 95% / 5% CO2, and MEM replenished every 2-3 days.

3.2.2 Electrophysiology

After 7-8 d in culture, electrophysiological data were acquired from CG neurons in the Willco-dishes using an Axopatch 200B amplifier and Digidata 1322 interface, both controlled by pClamp-9 software (all from Molecular Devices, Sunnyvale, CA).

Recordings were made at room temperature (RT, 22°C) in a recording solution (RS) containing (in mM): 145 NaCl, 5.3 KCl, 5 HEPES, 0.8 MgCl2, 5.4 CaCl2 that was supplemented with 10% heat-inactivated horse serum (RShs; pH 7.4). Patch pipettes (2.0

- 5.0 MΩ) were fabricated from Corning 8161 glass tubing and filled with (in mM):

145.6 CsCl, 0.6 CaCl2, 2.0 EGTA, 15.4 glucose, and 5.0 Na-HEPES (pH 7.3). Synaptic function was assessed from nAChR-mediated EPSCs acquired at -70 mV after establishing a stable whole-cell recording configuration. Spontaneous EPSCs were acquired in 2 min epochs and then analyzed off-line using Mini Analysis (6.0.3,

Synaptsoft, Fort Lee, NJ) as previously described (Chen et al. 2001; Zhou et al. 2004;

Pugh et al. 2009; Jayakar et al. 2009). Only events that activated abruptly and had peak amplitudes of 2-3 X baseline RMS current noise (typically 1.5 - 2.5 pA) were included.

For each neuron, sEPSC frequency (Fs, Hz) was determined from the total number of

84 events divided by the recording epoch duration while sEPSC amplitudes were determined from the average of individual event amplitudes (As, pA).

3.2.3 Drug Treatments

Unless stated otherwise, test reagents were applied from frozen stocks stored at -

20 or -80°C to CG neurons in MEMeye (with sham controls receiving MEMeye alone) and electrophysiological tests performed at 7 or 8 d in culture. PACAP (PACAP38, Bachem,

Bubendorf, Switzerland) was applied at 100 nM for 15 min. The synaptic effects were assessed directly after PACAP treatment and washout (with RShs). To examine LT plasticity, PACAP was applied to cultures after 5 or 6 d, washed out as above, and testing commenced 48 h hours later. ST and LT synaptic effects were compared with results obtained on the same day from sham treated control neurons from the same culture. As a reversible inhibitor of PKA, Cyclic 3',5'-[hydrogen (R)-phosphorothioate] adenosine (Rp- cAMPS) (Van Haastert, 1984) was used to test whether initial PKA activation was necessary to trigger the LT plasticity. Rp-cAMPS (100 μM) was applied 30 min prior to co’treatment with PACAP on d6 CG neuronal cultures. Following PACAP washout, Rp- cAMPS was reapplied for 3 hours to block PKA inhibition. Cultures were then washed 3

X with MEMeye and experiments commenced 45 h later. TTX (1 µM) or D-tubocurarine

(dTC, 100 µM) was used to test whether PACAP triggers synaptic plasticity by an activity-dependent mechanism (Jayakar et al. 2014). Respectively, these antagonists reversibly block impulse-dependent or all spontaneous synaptic activity in the cultures

(Chen et al. 2001). The antagonists were applied for 15 min prior to and then together with PACAP. Following PACAP ± antagonist treatment and washing, the antagonists

85 were reapplied and were present for the entire test (e.g. 48 h) period. Prior to the start of experiments, cultures were washed with RShs (3-6 X) to remove antagonists, and results compared to sham controls, as before from the same culture and obtained on the same day. Actinomycin-D (Act-D) was used to examine whether the LT and ST effects of

PACAP were dependent on gene transcription. For the ST plasticty, d7 CG neuronal were pretreated with 0.5μM Act-D 30 min prior to coapplication with PACAP. Cultures were washed with RShs as described above and experiments commenced immediately thereafter. For the LT plasticity d6 CG neuronal cultures were pretreated for 30 min with either 0.1 μM-1.0 μM Act-D before coapplication with 100 nM PACAP. Due to the cytotoxic effects of Act-D on CG neurons (Brusés and Pilar, 1995), all cultures were washed both immediately following PACAP treatment and an additional 6-7 X 4 h later to ensure complete Act-D washout.

3.2.4 Statistical Evaluation

Parameter values are expressed as mean ± SEM, usually followed by the number of neurons tested (n). In most cases, mean values obtained following drug treatments are presented as a fold change relative to those obtained for sham-treated control neurons from the same culture (1.00) such that a 2-fold change indicates a 100% increase, and statistical comparisons made using Prism software (v 5.0d GraphPad, La Jolla, CA). For single treatment comparisons, statistical significance was assessed using Student’s unpaired, two-tailed t-test with criterion cutoff at p<0.05 following Welch’s correction for unequal variances, when necessary. For multiple treatment comparisons, significance was determined by ANOVA followed by Bonferroni’s multiple comparison post-hoc test.

3.3 Results

3.3.1 PACAP Induces Short-term and Long-term Synaptic Plasticity

We previously demonstrated that PACAP induces a ST plasticity at nicotinic synapses on CG neurons, and elucidated relevant cell signaling mechanisms.

Specifically, both the frequency (Fs) and amplitude (As) of α3*-nAChR-mediated sEPSCs increase minutes after transient PACAP application (100 nM, 15 min). This ST synaptic plasticity involves obligatory PAC1R signaling via AC, cyclic AMP (cAMP) and

PKA that acts through α7-nAChRs, and NOS1 activation in postsynaptic neurons.

Synthesized NO acts retrogradely to enhance quantal release from adjacent presynaptic terminals (Pugh et al. 2009; Jayakar et al. 2014). LT synaptic plasticity is also apparent such that sEPSC frequencies and amplitudes remain significantly elevated 24, 48, and 72 hours after the same transient 15 min PACAP treatment (Fig. 3.1D, E). To assess mechanisms relevant to the LT plasticity, experiments were conducted on neurons from

CG neuronal cultures grown for 7-8 days that received transient PACAP or sham treatments 48 h earlier, compared with those treated with PACAP on the same day

(Figure 3.1A). Neurons from sham-treated (Control) cultures displayed sEPSCs occurring at an average frequency (Fs) of 1.21 ± 0.05 Hz and amplitude (As) of 12.04 ±

0.31 pA (Fig. 3.1B). In accord with previous experiments, transient PACAP treatment on the day of the experiment led to enhanced synaptic activity indicative of ST plasticity, with Fs and As increasing by 3.09-fold and 1.34-fold (Fs* and As*, respectively, n=40) relative to Control neurons from the same cultures tested in parallel. Transient PACAP exposure 48 h earlier revealed LT plasticity that featured an increase in Fs* and As*

(2.78-fold and 2.26-fold, respectively, n=128) with the increase in As* exceeding that

Figure 3.1: PACAP triggers short-term (ST) and long-term (LT) plasticity at nicotinic synapses. A. At times corresponding to down-going arrows, CG neuronal cultures received sham treatments (Control test) or 100 nM PACAP for 15 min followed by washout with MEMeye (Short-term (ST) test and Long-term (LT) test). Up-going arrows depicts the onset of the experiments. The line segments between down-going and up- going arrows depicts the time between PACAP treatment and washout and the onset of experiments. B. Example representative traces of nAChR mediated sEPSCs from CG neurons that received sham treatment (black), ST PACAP test or LT PACAP test. Calibration: 20 pA,100 ms. C. Bar graphs depict normalized sEPSC frequency (Fs*) and amplitude (As*). The LT PACAP (n = 128) and ST PACAP induced plasticity (n = 40) enhances Fs* to a similar degree (2.8 and 3.1 fold respectively), while the LT significantly increases As* in comparison to the ST PACAP treatment in comparison to controls (n =172) (2.2 fold and 1.3 fold respectively). Asterisks (*) indicate a significant difference (p<0.05) relative to Control while carats (^) indicate a significant difference from the ST PACAP condition, assessed by a Students unpaired two-tailed T-test. D., E. Washout kinetics were assessed by comparing Fs* (D) and As* (E) at the indicated times following PACAP washout, relative to their respective Controls (solid line; SEM is depicted by the dashed lines). The red box represents the ST plasticity while blue boxes represent the LT plasticity. These figures were modified from Pugh et al. (2009). Asterisks (*) indicate a significant difference (p<0.05) relative to each experiments respective Controls, assessed by Students unpaired two-tailed T-tests.

89 associated with ST plasticity (p<0.05) (Fig. 3.1 B, C). These findings confirm and extend previous results demonstrating that transient PACAP exposure induces both short- and pronounced LT functional plasticity at nicotinic synapses.

3.3.2 PACAP Induces Long-Term Synaptic Plasticity via PAC1Rs and Adenylate

Cyclase/cAMP and Phospholipase C Mediated Signal Transduction.

PACAP is recognized by two class B g-PCRs that predominately trigger both adenylate cyclase (AC) and phospholipase dependent signal transduction cascades. Type

I receptors (PAC1Rs) bind PACAP with high affinity (KD ≈ 1 nM) compared to closely related vasoactive intestinal peptide (VIP; KD ≈ 0.5-1.0 µM) whereas Type II receptors

(VPAC1Rs and VPAC2Rs) have roughly equal affinity for PACAP and VIP (Gottschall et al., 1990; Lam et al., 1990; Spenghler et al. 1993; Hashimoto et al. 1993;). A role for

PAC1Rs in the LT synaptic plasticity was anticipated because CG neurons exhibit high

125 affinity for PACAP (IC50 ≈ 1 nM for displacing I-PACAP27) but much lower affinity for (VIP; IC50 ≈ 1 µM) and because PACAP couples much more strongly to AC- dependent cAMP production (EC50 ≈ 0.5 nM) than does VIP (EC50 ≈ 300 nM) (Margiotta and Pardi, 1995). Consistent with theses findings, transient application of VIP at 100 nM, a concentration expected to fully engage VPAC1Rs or VPAC2Rs, failed to detectably alter Fs* or As* when neurons were tested 48 h later, while 100 nM PACAP produced significant increases in both parameters (Fig 3.2A) indicating that the LT effects of

PACAP are mediated by PAC1R activation.

Since PAC1Rs on CG neurons efficiently couple to adenylate cyclase (AC) to produce cAMP (Margiotta and Pardi, 1995) we tested whether AC and subsequent cAMP

90 production were necessary and sufficient for PACAP to induce a LT plasticity as we previously demonstrated for the ST plasticity (Pugh et al. 2009). Consistent with a requirement for PAC1R coupling via AC, the increases in Fs* and As* seen 48 h after

PACAP exposure was blocked following 30 min pretreatment with the AC inhibtor MDL

12330A hydrochloride (MDL, 1μM) (Guellan et al. 1977) (Fig. 3.2B). Interestingly, stimulation of endogenous cAMP signaling was sufficient to induce a LT synaptic plasticity as reversible stimulation of cAMP synthesis with the AC agonist forskolin (10 uM, 1 hr) in the absence and presence of the PLC inhibitor U 73122 (U7, 1 uM) resulted in significant elevations in Fs* and As* (Fig. 3.2D). PAC1Rs also couples to Gαq to activate PLC and stimulate IP turnover resulting in intracellular Ca2+ release (Margiotta and Pardi, 1999; Woo and Margiotta, 2007). Despite being refractory for the ST

PACAP-induced plasticity, inhibition of PLC via pretreatment with 1 μM U7, a

2+ concentration demonstrated to inhibit PAC1R-induced IP turnover and [Ca ]in mobilization in CG neurons (Pardi and Margiotta, 1999) blocked the LT PACAP-induced increase in Fs* and As* 48h after PACAP washout (Figure 3.2C). These results suggest that concomitant activation of PLC and AC are required to induce the LT PACAP- induced synaptic plasticity.

We then wanted to determine whether activation of PLC signaling, was sufficient to mimic the LT plasticity. Muscarine application on CG neurons activates M3 receptors to induce IP turnover as well as stimulate release of internal Ca2+ and upregulates PKC activation consistent with canonical PLC activation (Rathouz et al. 1995; Kan et al.

2014). When muscarine was applied (100 μM, 180 min) either with or without MDL to inhibit AC, neither Fs* nor As* values were detectably different from those of controls

91 when neurons were tested 48 h later (Fig. 3.2E). Application of a cell permeable sulfonamide compound that that directly activates all PLC isotypes referred to 2,4,6-

Trimethyl-N-(m-3-trifluoromethylphenyl) benzenesulfonamide (m-3m3FBS; 25μM; Bae et al. 2003) also failed to augment Fs* and As* in the the presence of MDL, verifying the results with muscarine (Data not shown). The capacity for forskolin but neither muscarine nor m-3m3FBS to induce a LT synaptic plasticity suggests that PLC signaling cascades activated via PAC1Rs likely activate a shared downstream effector of cAMP.

3.3.3 The Short-Term and Long-Term PACAP-Induced Synaptic Plasticity are

Mechanistically Distinct.

Following PAC1R and AC-dependent stimulation of cAMP production in CG neurons, PACAP induces ST synaptic plasticity by obligatory PKA and nAChR- activation with concomitant Ca2+ influx stimulating NOS1 and subsequent NO production (Pugh et al. 2009; Jayakar et al. 2014). Consequently, we examined whether similar steps were required for the induction of the PACAP-induced LT synaptic plasticity. PACAP signals via cAMP to induce activation of PKA in CG neuronal cultures (data not shown). To determine whether PKA activation was necessary for the

LT plasticity we pretreated CG neurons with the reversible competitive PKA inhibitor

Rp-cAMPS (100 μM) (Van Haastert et al. 1984). Application of Rp-cAMPS inhibited the

ST PACAP-induced plasticity, blocking the ST PACAP-induced increases in both Fs* and As* (data not shown). Likewise, to determine whether initial activation of PKA was required for the LT PACAP-induced plasticity, we inhibited PKA prior (30 min) to

PACAP application and reapplied it for a duration of three hours after PACAP treatment

Figure 3.2: PACAP-induces LT synaptic plasticity via PAC1R activation and requires AC and PLC. A. The LT PACAP plasticity is PAC1R-depenent. Both FS* and AS* were elevated 48 h after 15’ exposure to PACAP (LT PACAP, n = 7) relative to sham-treated controls (Contro, n = 11) whereas identical treatment with 100 nM VIP (LT VIP, n = 8) failed to alter synaptic function. B. AC inhibition with MDL12330A hydrochloride (MDL) blocked the LT PACAP-induced synaptic plasticity. Pre-incubation with MDL 93

(1 μM, 30 min) followed by 15’ co-incubation with 100 nM PACAP abrogated PACAP- induced increases in FS* and AS* normally seen 48 h after 15 min exposure to 100 nM PACAP. Bar graphs depict average values obtained from 7-11 neurons C. PLC inhibition with U 73122 (U7) blocked the LT PACAP-induced synaptic plasticity. Preincubation with U7 (1 μM, 30 min) followed by 15’ co-incubation with 100 nM PACAP abrogated the increases in FS* and AS* normally seen 48 h after 15 min exposure to 100 nM PACAP. Bar graphs depict average values obtained from 9-16 neurons per condition D., E. AC/cAMP but not PLC signaling is sufficient to induce LT synaptic plasticity. D. Transient incubation with Forskolin (10 μM, 60 min) induced a LT synaptic plasticity significantly increasing FS* and AS* in neurons tested 48 h later relative to Controls (n = 19) and U7 (n = 5) treatment conditions either when Forskolin was applied alone (n = 9) or co-incubated with U7 to block PLC (n = 7) E. By contrast, transient incubation with muscarine (100 μM, 180 min) to mimic PLC signaling had no effect on FS* or AS* in neurons tested 8 h later compared to Controls (n = 11) or MDL treatment alone (n = 7). Muscarine, applied alone (n = 11) or in the presence of 1μM MDL (n =9) failed to block AC/cAMP signaling. + signs connotate addition of a treatment while – signs connotate absence of treatment. Asterisks (*) indicate significance (p<0.05) relative to Control via One-Way ANOVA.

(See Methods). Rp-cAMPs failed to block the LT PACAP-induced increase in Fs* and

As* indicative of independence from initial PKA activation (Fig. 3.3A). To confirm these findings, we conducted experiments in the presence of the myristoylated PKI (14-22)

(PKI), a specific and irreversible inhibitor of PKA (Glass et al. 1989). Pretreatment (30 min) with 1 μM PKI, a concentration shown to inhibit the ST PACAP-induced plasticity

(Pugh et al. 2009) failed to inhibit the LT PACAP- induced synaptic plasticity (Fig.

3.3B). Taken together, these studies demonstrate that unlike the ST PACAP-induced synaptic plasticity, the LT effects of PACAP are independent of initial and persistent

PKA signaling.

The reversible nAChR antagonist dTC blocks both α3*- and α7-nAChRs on CG neurons (Nai et al. 2003) and eliminates all synaptic activity in CG cultures (Chen et al.

2001; Jayakar et al. 2014). Since nAChR blockade with dTC (100 µM) abrogated the ability of PACAP to induce ST plasticity (Jayakar et al. 2014) we tested whether

94 sustained nAChR block with dTC would similarly affect PACAP-induced LT plasticity

(Fig. 3.3C). Test cultures were exposed to dTC (100 µM) 15 min prior to application with PACAP. Following washout, dTC was then re-added for the entire 48 h post treatment period followed by 4-6 washes prior to the onset of experiments. The dTC treatments were effective since synaptic activity was eliminated in the presence of dTC

(data not shown). dTC was also reversible and without detectable homeostatic influence since Fs* and As* were restored to control levels following dTC washout (Fig. 3.3C).

Contrasting with its effects on the PACAP-induced ST synaptic plasticity, dTC failed to abrogate the LT synaptic plasticity (Fig. 3.3C). Neurons treated with PACAP and dTC displayed significant elevations in Fs* and As* when assayed 48 h after PACAP treatment relative to sham controls or those treated with dTC alone from the same experiments

(p<0.05 for both). Moreover, the elevated Fs* levels were not detectably different in neurons treated with PACAP plus dTC compared with neurons in the same cultures that received PACAP alone. While As* was somewhat less elevated in neurons treated with

PACAP plus dTC compared with neurons that received PACAP alone, neither As* nor

Fs* in neurons treated with PACAP plus dTC differed from overall values obtained from all PACAP treated neurons. Experiments of identical design were conducted using TTX

(1 uM) to block voltage-dependent sodium channels and drastically reduce synaptic activity in the cultures (Margiotta et al. 1982; Chen et al. 2001) . As with dTC, the TTX treatments were effective as both Na+ and synaptic currents were abolished. Likewise, washout of TTX restored Fs* and As* and was without homeostatic influence (Fig.

3.3D). Consistent with results obtained using dTC, TTX block of impulse activity failed to abrogate the LT synaptic plasticity (Fig. 3.3D). Neurons treated with PACAP and TTX

95 displayed significantly higher levels of Fs* and As* when assayed 48 h after PACAP treatment relative to sham controls or those treated with TTX alone from the same experiments (p<0.05 for both). Moreover, neither elevated Fs* nor As* levels were detectably different in neurons treated with PACAP plus TTX compared with neurons in the same or all cultures that received PACAP alone. Taken together, these results indicate that the LT PACAP induced plasticity is independent of nAChR activation and synaptic activity.

NOS1 blockade with L-NAME also abrogates the ability of PACAP to stimulate

NO production and induce the ST plasticity (Pugh et al. 2009; Jayakar et al. 2014). We therefore tested whether NOS1 blockade would similarly affect the LT PACAP-induced synaptic plasticity (Fig. 3.3E). Test cultures were exposed to L-NAME (100 µM) 30 min prior to 15 min. PACAP treatment, and synaptic activity assessed in cultures 48 h later.

NOS1 inhibition failed to abrogate the PACAP-induced LT synaptic plasticity (Fig.

3.3E). Neurons treated with PACAP plus L-NAME displayed significantly higher levels of Fs* and As* when assayed 48 h after PACAP treatment relative to sham controls or those treated with L-NAME alone from the same experiments (p<0.05 for both). More over neither elevated Fs* nor As* levels were detectably different in neurons treated with

PACAP plus L-NAME compared with neurons in the same or all cultures that received

PACAP alone. In one experiment L-NAME (100 µM, 30 min) applied 48 hours after transient PACAP treatment failed to abrogate increases in Fs* and As* (data not shown) indicating that the ability of PACAP to induce LT synaptic plasticity is not dependent upon residual NOS1 activation (data not shown). Taken together, these studies indicate that while PACAP induces both short- and LT form of synaptic plasticity that share a

96 dependence on PAC1Rs, AC activation and cAMP accumulation the LT PACAP induced synaptic plasticity is independent of PKA signaling, neuronal activity, and NOS1. Given the efficacy of the inhibitors, the results further indicate that the elaboration of LT synaptic plasticity is independent of prior ST plasticity. Thus, the two forms of synaptic plasticity induced by PACAP are mechanistically distinct.

3.3.4 The LT PACAP-Induced Synaptic Plasticity requires Gene Transcription but is Independent of PAC1R Internalization

Paralleling their ability to induce LT plasticity at nicotinic synapses in CG cultures, 15 min PACAP treatment rapidly activates the transcription factor CREB and alters the expression of numerous genes relevant to cholinergic transmission (Sumner et al. 2008). To determine whether the elaboration of PACAP-induced LT plasticity requires new gene transcription, RNA synthesis was inhibited with 0.1 or 1.0 µM actinomycin D (Act- D) concentrations that were previously shown to inhibit 80 or

97.5%, respectively, of all RNA synthesis in CG cultures (Brusés and Pilar, 1995).

Cultures were treated with Act-D for 20 min prior to the 15 min PACAP treatment, and then washed 7-8 times; once following the 15 minute PACAP incubation period and 6-7 times 4 hours later to remove any residual Act-D. Identical results were obtained with either 0.1 or 1.0 µM Act-D and the data for both have been pooled. The Act-D treatments abolished the LT plasticity (Fig. 3.4A), blocking increases in both Fs* and As* induced by

PACAP treatment 48 h earlier. The effect was specific because PACAP induced ST plasticity was unaffected by Act-D (Fig. 3.4B) and because Fs* and As* values in cultures treated with Act-D alone were not detectably different compared with those obtained

Fig. 3.3: The LT PACAP-induced synaptic plasticity is independent of PKA, neuronal activity, and NOS1. All bar graphs depict normalized sEPSC frequency (Fs*) and amplitude (As*) for each set of experiments (n = 7-18 neurons per condition). A. Reversible inhibition of PKA with Rp-cAMPs (100 µM) both 30 min before and for 3 h after PACAP washout failed to block the LT PACAP-induced enhancement in Fs* and As*. B. Likewise persistent inhibition of PKA with the irreversible PKA inhibitor PKI(14-22) (1 µM, 30 min) also failed to block the LT PACAP-induced synaptic

98 plasticity. C., D. The LT PACAP-induced synaptic plasticity is activity-independent. Neuronal activity was inhibited with either the voltage dependent sodium channel inhibitor Tetrototoxin (TTX, 1 µM, 15 min) or d-tubocurarine hydrochloride (dTC, 100 µM) both before and for the 48 h duration after the 15’ PACAP treatment. Inhibition of neuronal activity with either (C) TTX or (D) dTC failed to block the LT PACAP-induced enhancement in Fs* and As*. E. Inhibition of NOS1 prior to PACAP treatment with L- NAME (100 μM, 30 min) did not block the LT PACAP-induced plasticity. + signs connotate treatment while – signs indicate absence of treatment. Asterisks (*) indicate a significant difference (p<0.05) from Controls and carats (^) indicate a significant difference from the LT PACAP-treatment condition alone, assessed by One-Way ANOVA.

from sham-treated controls in both plasticity experiments. In addition, there were no detectable differences in cell morphology, input resistance or voltage dependent calcium current densities in Act-D relative to Controls and sham treated neurons (data not shown).

Taken together these findings demonstrate that the physiological effects underlying

PACAP-induced LT plasticity depend on new gene transcription.

PAC1R internalization has been implicated in PACAP-induced synaptic plasticity in the cardiac ganglion (Merriam et al. 2013). PAC1R internalization is mediated by clathrin-mediated endocytosis (CME) and occurs within minutes following PACAP treatment (Shintani et al. 2000, Germano et al. 2001, Merriam et al. 2013). In the cardiac ganglion, CME of the PAC1R has been coupled to MEK/ERK signaling and is required for PACAP-induced increases in CdG neuronal excitability (Merriam et al. 2013, Todd et al. 2016, May and Parsons, 2016). These findings suggest that both forms of synaptic plasticity induced by PACAP in the CG might be mediated by a non-membrane delimited mechanism. We investigated this possibility by pretreating neurons with Pitstop 2 (Pit 2), a cell permeable competitive inhibitor of the clathrin terminal domain to selectivity inhibit endocytosis. Cultures were pretreated for 20 min with 15 μM Pit 2, to inhibit

PAC1R internalization (Merriam et al 2013). Pit 2 failed to block the ST and the LT

PACAP-induced enhancement in Fs* and As* (Fig. 3.4C, D). Additionally, Pit 2 did not abrogate PACAP mediated phosphorylation of CREB indicating that Pit 2 does not perturb PACAP signaling in CG neurons (data not shown). Taken together, these findings indicate that the ST and the LT PACAP-induced synaptic plasticity does not require internalization and subsequent endosomal signaling.

3.4 Discussion

In the following Chapter, we demonstrated that PACAP induces a ST as well as a LT synaptic plasticity. While featuring similar increases in sEPSC frequency, the LT

PACAP-induced plasticity exhibited more robust increases in sEPSC amplitude 48 hrs after washout. Furthermore, PACAP sustainably enhanced cholinergic activity in CG neurons 24, 48 and 72 h after PACAP washout suggesting that PACAP could be persistently enhancing cholinergic synaptic activity in these cultures. Subsequent pharmacological studies revealed that the LT PACAP-induced plasticity was dependent upon PAC1R-mediated activation of both AC and PLC and subsequent gene transcription.

We observed several divergences between the LT and the ST PACAP-induced plasticity

(see Chapter 2). The LT effects of PACAP were independent of synaptic activity, PKA signaling and NOS1 activation, indicating that critical signaling effectors underlying the

ST PACAP-induced plasticity were not responsible for mediating the LT PACAP- induced plasticity. Likewise, inhibition of gene transcription did not alter the ST PACAP- induced plasticity. Taken together, these findings indicate that the LT effects of PACAP are mediated via gene transcription through PAC1R-induced AC and PLC mediated signaling cascades.

100

Fig 3.4: The LT- but not the ST PACAP-induced synaptic plasticity requires protein synthesis, while neither requires PAC1R internalization. A. Inhibition of gene transcription blocked the LT PACAP-induced synaptic plasticity. Bar graphs depict results from two separate experiments (n =7-9 neurons per condition) in which 5-6d CG neuronal cultures were pretreated with Act-D (0.1 and 1.0 μM, 30 min) prior to the 15’ PACAP treatment. 4 hour following PACAP treatment CG cultures were washed 6-7 x with MEMeye to ensure Act-D washout (Brusés et al, 1995) and experiments commenced 44 h later. Act-D abrogated the LT PACAP-induced effects on Fs* and As* in comparison to PACAP treatment alone. B. Inhibition of gene transcription does not block the ST PACAP-induced synaptic plasticity. Pretreating 7d CG neurons with Act-D, (0.5 μM, 30 min) followed by 15’ PACAP failed to abrogate the ST PACAP-induced effects on Fs* and As*. Bar graphs depict average Fs* and As* from 3-7 neurons per condition. C., D. Inhibition of PAC1R internalization failed to inhibit the ST and LT PACAP- induced synaptic plasticity. C. Pit 2 application (15μM, 30 min) on 5-6d CG neuronal cultures failed to inhibit the LT-PACAP-induced increases in Fs* and As* (n = 9-15 neurons per condition). D. Preapplication of Pit 2 (15μM, 30 min) to block CME on 7-8d CG neuronal cultures failed to block the ST PACAP-induced increases in Fs* and As* (n = 6-9 per condition). + signs indicate treatments while – signs connotate absence of treatment. Asterisks (*) indicate a significant difference relative to Controls assessed by One-Way ANOVA.

101

PACAP signaling in the CG neurons activates PAC1Rs resulting in both cAMP

2+ synthesis, IP turnover and [Ca ]in mobilization, indicative of activation of AC- and PLC- mediated signaling cascades (Margiotta and Pardi, 1995; Pardi and Margiotta, 1999).

Inhibition of AC or PLC blocked the PACAP-induced enhancement in Fs* and As* 48h after PACAP washout, indicating a requirement for both AC and PLC signaling respectively. The capacity for PACAP to induce synaptic plasticity via bimodal signaling transducing pathways has been demonstrated in the submandibular ganglion (Hayashi et al. 2002; Kamaishi et al. 2004) and in CA1 pyramidal neurons (Macdonald et al. 2005;

Macdonald et al. 2007; Costa et al. 2009). However, it was unknown whether activation of one or the other signaling pathway was sufficient to induce a LT synaptic plasticity.

Stimulating endogenous cAMP synthesis with forskolin significantly elevated Fs* and

As* in the absence and presence of U7 while stimulation of PLC signaling with muscarine or m-3m3FBS in the absence and presence of MDL failed to alter Fs* and

As*. These results indicate that maximal stimulation of AC is sufficient to induce a LT synaptic plasticity that resembles the LT PACAP-induced synaptic plasticity. Therefore, we hypothesize that PLC signal transducing pathways likely amplify shared effectors of

AC signaling pathways such as p38 MAPK and ERK (da Silva et al. 2004; Yanagidate et al. 2006; Blechmann and Levkowitz, 2013). However, further studies investigating the contributory roles of p38 MAPK and ERK as well as effectors of PLC signaling

2+ including IP3, [Ca ]in and PKC are warranted to verify this hypothesis.

The ST synaptic plasticity induced by PACAP couples PAC1R mediated canonical AC, cAMP, PKA signaling with a membrane localized, activity-dependent,

Ca2+ mediated activation of NOS1, resulting in retrograde signaling of NO to

102 subsequently enhance presynaptic quantal release (Margiotta and Pardi 1995; Pardi and

Margiotta, 1999; Pugh et al. 2009; Jayakar et al. 2014). The LT PACAP-induced plasticity was distinct from the ST PACAP-induced synaptic plasticity in that it was independent of both initial and persistent PKA signaling, (Pugh et al. 2009), concomitant nicotinic and neuronal activity (Jayakar et al. 2014) and NOS1 activation. The capacity for the LT PACAP induced plasticity to be independent of PKA indicates a novel role for other targets of cAMP signaling. Epac, a guanine nucleotide exchange factor for the

GTPase Rap, represents an attractive target as PACAP-induced activation of Epac has been shown to activate both p38 MAPK and ERK as well as modulate PACAP-induced synaptic plasticity at CA1 pyramidal neurons (Gerdin and Eiden, 2007; Ster et al. 2009;

Blechman and Levkowitz, 2013). Another cAMP effector activated by PACAP is the newly discovered neuritogenic cAMP sensor (NCS), an effector that was demonstrated to modulate neuritogenesis in neuroscreen-1 cells via activation of ERK (Emery and Eiden,

2012, Emery et al. 2015). Therefore, it is ultimately unknown which downstream effector of cAMP is underlying the LT PACAP-induced synaptic plasticity.

Reversible inhibition of gene transcription with Act-D blocked the LT PACAP- induced synaptic plasticity indicating a requirement for transcription and synthesis of new proteins. This effect is consistent with PACAP’s capacity to activate MAPK- mediated pro-survival pathways as well as increase phosphorylation and nuclear translocation of the transcription factor CREB in CG neruons (Xiangdong et al. 2004;

Pugh et al. 2006; Sumner et al. 2009). Furthermore, PACAP alters the expression of genes relevant to synaptic function (Sumner et al. 2009). Specifically, PACAP significantly up-regulated transcription (i.e. >1.5-fold increase) of six genes encoding

103 presynaptic terminal components (synaptotagmin IV, synaptobrevin 2, choline acetyltransferase, the high-affinity choline transporter, RIMS binding protein 2, and

Neuronal Pentraxin 2 precursor) as well as three encoding postsynaptic components

(TrkB, cortactin binding protein-2, and Contactin-associated protein-like-5) (Sumner et al. 2009). In addition, PACAP significantly down-regulated transcription (i.e. >1.5-fold decrease) of two synapse relevant genes (Nogo-66 Receptor and PDZ domain-containing guanine nucleotide exchange factor). These findings suggest that the LT PACAP-induced synaptic plasticity could be associated with alterations in post- and presynaptic function, physiological parameters that will be examined next, in Chapter 4.

104

Chapter 4

The Long-Term PACAP-Induced Synaptic Plasticity Features

Increases in Post-and Presynaptic Strength through Dynamic

Pre-and Postsynaptic Remodeling.

4.1 Introduction

Neuropeptides trigger synaptic modification via cognate receptors and associated cell signaling pathways. Pituitary adenylate cyclase activating polypeptide (PACAP) has neuromodulatory roles in the nervous system consistent with its widespread localization at synaptic sites throughout the central and autonomic nervous system (CNS, ANS; reviewed by Starr and Margiotta, 2016). We previously found that PACAP is present in cholinergic presynaptic terminals in the avian ciliary ganglion (CG) where it is released by depolarization, and that exogenous PACAP rapidly enhances cholinergic transmission at synapses that form between CG neurons in cell culture (Pugh et al. 2009; Jayakar et al.

2014). These synapses share features with those formed on CG neurons in vivo, including a dependence on cholinergic transmission mediated by activation of postsynaptic nicotinic acetylcholine receptors (nAChRs), making them a useful model for examining nicotinic synapse formation, function and regulation (Chen et al. 2001). Just

105 minutes after treatment, PACAP increases the frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs), >90% of which are mediated by nAChRs composed of α3, β4, α5 ± β2 subunits (α3*-nAChRs), markedly increase (Chen et al.

2001; Pugh et al. 2009). This short-term (ST) synaptic plasticity results from obligatory activation of high-affinity PACAP receptors (PAC1Rs), adenylate cyclase (AC), protein kinase A (PKA), perisynaptic α7-nAChRs, and neuronal nitric oxide synthase (NOS1) in postsynaptic neurons, with subsequent NO production acting retrogradely to enhance vesicular ACh release (quantal content) from adjacent presynaptic terminals (Pugh et al.

2009; Jayakar et al. 2014).

As shown in Chapter 3 transient PACAP exposure induces LT synaptic plasticity via distinctive intracellular signaling mechanisms. In line with PACAPs capacity to activate cyclic AMP response element binding protein (CREB; Zhou et al. 2004; Sumner et al. 2009), the LT PACAP-induced synaptic plasticity was dependent upon gene transcription. Moreover, gene arrays on CG neuronal cultures revealed that PACAP alters the expression of 274 genes including 58 that are relevant to synaptic or cell junction components (Sumner and Margiotta, 2008). Given our hypothesis that the LT PACAP- induced synaptic plasticity can be distinguishd from the ST PACAP-induced synaptic plasticity, we sought to characterize the physiological and synaptic correlates underlying the LT PACAP induced plasticity and distinguish them from the ST PACAP-induced synaptic plasticity.

106

4.2 Methods

4.2.1 Neuronal Cultures

Cultures were prepared using ciliary ganglia obtained from embryonic day 8 (E8,

Stage 34-35) chicks (Hamburger and Hamilton, 1951) using procedures identical to those previously described (Jayakar et al. 2014). Briefly, the cover glass surfaces of 12 mm diameter mini-wells in 35 mm WillCo-dishes (BioSoft international) were pre-coated with 0.2 mg/ml poly-D-ornithine and 12.5 µg/ml mouse laminin (a gift from Salvatore

Carbonetto, McGill University). Dissected ganglia were treated with trypsin (0.025%, 30 min, 37°C), the neurons dissociated by mechanical trituration, and ≈104 neurons (1.5 ganglion equivalents) plated per WillCo-dish well. The culture medium consisted of

Eagle’s minimum essential medium (MEM) supplemented with 2 mM glutamine, 100

U/ml penicillin, 10% heat-inactivated horse serum (Invitrogen, Rockville, MD) and with freshly prepared E17 chick eye extract (3% v/v, MEMeye) (Nishi et al. 1981). Cultures

eye were maintained at 37°C in 95% / 5% CO2, and MEM replenished every 2-3d.

4.2.2 Drug Treatments

Unless stated otherwise, test reagents were applied from frozen stocks stored at -

20 or -80°C to CG neurons in MEMeye (with sham controls receiving MEMeye alone) and electrophysiological tests performed at 7 or 8 d in culture. PACAP (PACAP38, Bachem,

Bubendorf, Switzerland) was applied at 100 nM for 15 min. ST synaptic effects were assessed directly after PACAP treatment and washout (with RShs). To examine LT plasticity, PACAP was applied to cultures after 5 or 6 d, washed out as above, and testing in most cases commenced 48 h hours later. ST and LT synaptic effects were compared

107 with results obtained on the same day from sham treated control neurons from the same culture.

4.2.3 Electrophysiology

- 5.0 MΩ) were fabricated from Corning 8161 glass tubing and filled with (in mM):

Synaptsoft, Fort Lee, NJ) as previously described (Chen et al. 2001; Zhou et al. 2004;

Pugh et al. 2009; Jayakar et al. 2014). Only events that activated abruptly and had peak amplitudes of 2-3 X baseline RMS current noise (typically 1.5 - 2.5 pA) were included.

For each neuron, sEPSC frequency (Fs, Hz) was determined from the total number of events divided by the recording epoch duration while sEPSC amplitudes were determined from the average of individual event amplitudes (As, pA).

Data for conducting quantal analyses were acquired from individual neurons by a two-step process that allowed quantal size and quantal content to be assessed in the same

108 neuron. First, spontaneous activity was sampled from a neuron for 1 min in whole-cell mode as above, and small EPSCs that appear after bursts of impulse-dependent sEPSCs were acquired for ≈100 ms. Such trailing events have been identified as mEPSCs at CG synapses in situ and result from asynchronous ACh release that follows impulse-driven presynaptic output (Sargent, 2009; Kaeser and Regehr, 2014). Functional comparisons between spontaneous and asynchronous mEPSCs collected from CG neurons are compared in Table 4.2. Next, a neurite fasicle converging on the same neuron was stimulated 30-50X @ 0.1 Hz via a theta-glass bipolar suction electrode fabricated to 1-3

MΩ tip resistance and mounted in a ported holder (THS-F15PH, Warner Instruments,

Hamden, CT) as previously described (Pugh et al. 2009). Minimal stimulation criteria were used to recruit single axons such that evoked EPSCs (eEPSCs) activated fully with brief, constant delay without indication of a secondary component, and had kinetics matching those of sEPSCs. mEPSCs and eEPSCs were assessed off-line using Mini

Analysis (6.0.3, Synaptsoft, Fort Lee, NJ) employing the same selection criteria as those for sEPSCs and relevant parameters compiled in Excel spreadsheets for analysis. For each neuron, quantal content (m) was assessed as before (Pugh et al. 2009) both empirically (me) from the ratio of average eEPSC amplitudes (Ae, including failures where amplitude = 0) to quantal size (me = Ae/ae) and from the Poisson relationship between the total number of stimulus trials (NT) to response failures (N0) given by mf = ln(NT / N0).

Analysis of total membrane α3*-nAChR population on individually selected CG neurons was selectively sampled by focally applying nicotine (20 μM in RS) via pressure microperfusion (5-10 psi) after pre-treating the cultures with 50 nM αBgt to block α7-

109 nAChRs for 1 h (Nai et al. 2003; Zhou et al. 2004; Pugh et al. 2009). The resulting nicotine-induced α3*-nAChR currents were analyzed using Clampfit (pClamp 9.0, Axon

Instruments, Burlingame, CA). Peak response amplitudes were normalized to neuron soma membrane capacitance (pA/pF). Tetrodotoxin (TTX, 1 μM) was included in some cases to test the effect of action potential blockade on α3*-nAChR responses. No significant differences between non-TTX treated cells and TTX treated cells was observed and the data was pooled.

Analysis of neuronal excitability was assessed in current clamp mode after attaining the whole-cell configuration using patch pipettes filled with 110 KMeSO4, 10

NaCl, 5 MgCl2, 0.6 EGTA, and 10 HEPES as previously described (Pugh et al. 2009).

Action potentials were elicited by applying 200 ms depolarizing current pulses from 5-

100 pA in 5 pA steps at 0.1 Hz. Both the number of action potentials and inter-spike intervals were computed and analyzed as a function of the applied current using Clampfit

(pClamp 9.0, Axon Instruments, Burlingame, CA). Neurons displaying resting potential values > -40 mV or input resistances <500 MΩ were excluded from analysis.

4.2.4 Synaptic Architecture

The relationship between α3*-nAChRs and adjacent input terminals on CG neurons in control and PACAP-treated cultures was assessed by antibody co- immunolabeling followed by laser scanning confocal image acquisition, and image analysis. Immunolabeling was conducted as previously described (Jayakar et al. 2011) except that manipulations were performed on CG cultures grown on 35 mm glass-bottom

WillCo-dishes instead of on removable glass coverslips. Briefly, cultures were incubated

110 with rat anti-α3/α5 mAb35 (1:500; a gift from Dr. D. K. Berg, University of California,

San Diego, CA) to label α3*-nAChRs and, after fixation (2% paraformaldehyde, 20 min) and permeablization, with mouse anti-SV2 mAb10h (1:25; Developmental Studies

Hybridoma Bank, University of Iowa, Iowa City, IA) to label presynaptic terminals. For fluorescent detection, labeled cultures were incubated with Cy3-conjugated rat and Alexa

Fluor 488-conjugated mouse monoclonal secondary antibodies (at 1:200 and 1:400 dilutions respectively, Jackson ImmunoResearch Laboratories). After washing, the cultures were then treated with anti-fade mounting medium (Vectashield, Vector

Laboratories, Burlingame, CA) and 25 mm diameter glass coverslips applied and sealed in place with nail polish.

Images were acquired from labeled cultures using a using a Leica TCS SP5 laser scanning confocal microscope (Leica Microsystems, Bannockburn, IL) equipped with conventional solid state and a Ti-sapphire tunable multi-photon laser (Coherent, Santa

Clara, CA). 8-bit images were acquired with a 63X objective (NA 1.40) at 0.136

µm/pixel resolution, and 20 to 30 optical Z-sections (0.5 µm thick) were collected using a motorized focus unit using the sequential scan mode to eliminate any spectral overlap in the individual fluorophores. Laser power and gain were set to minimize saturation and avoid detectable bleed-through between the channels. For SV2 immunoreactivity, Alexa

Fluor 488 was excited at 488 nm laser and emission collected at 501-548 nm, while for

α3*-nAChR immunoreactivity Cy3 was excited at 561nm and emission collected at 572-

648 nm. Images are a 2D representation of the 3D LSCM image stack as labeled. To enable comparison of control and test conditions, exposure settings were held constant within a given experiment. Image stacks were saved as TIFF files.

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Image analysis was conducted using ImageJ (version 1.45s, http://rsbweb.nih-

.gov/ij/). The relationship between presynaptic (mAb10H-positive, SV2) and postsynaptic (mAb35-positive, α3*-nAChR) sites was assessed from single optical sections or from projections of 1-2 adjacent sections acquired both from the neuron’s upper surface (en-face) and from its perimeter. In both cases, a region of interest (ROI) was first drawn to surround the extent of detectable labeling on the α3*-nAChR image and copied to the SV2 image. As previously described (Jayakar and Margiotta, 2011) the degree of α3*-nAChR (and SV2) labeling intensity was obtained from the mean fluorescence intensity within the ROI minus the mean background fluorescence outside the ROI (ΔF = Fm - Fb) divided by Fb (ΔF/Fb). Clusters of α3*-nAChR and SV2 labeling

(containing at least > 3 pixels; 53.4 nm2) were quantified by establishing an intensity

-2 2 threshold (8 - 10 X Fb) and their densities (# µm ), areas (nm ), and intensities (ΔF/Fb) determined after automated particle counting. The extent of α3*-nAChR and SV2 colocalization was assessed from Manders’ coefficients (RM). Portions of α3*-nAChR clusters that mapped to adjacent SV2 clusters were considered postsynaptic, and the densities, areas, and relative intensities of these synaptic α3*-nAChR sites were quantified as above.

4.2.5 Statistical Evaluation

Parameter values are expressed as mean ± SEM (n). In most cases, mean values obtained following drug treatments are presented as a fold change relative to those obtained for sham-treated control neurons from the same culture (1.00) such that a 2-fold change indicates a 100% increase, and statistical comparisons made using Prism software

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(v 5.0d GraphPad, La Jolla, CA). For single treatment comparisons, statistical significance was assessed using Student’s unpaired, two-tailed t-test with criterion cutoff at p<0.05 following Welch’s correction for unequal variances, when necessary. For multiple treatment comparisons, significance was determined by ANOVA followed by

Bonferroni’s multiple comparison post-hoc test. In some cases, cumulative frequency histograms were produced and subsequently analyzed using a Kolmogorov-Smirnov test.

4.3 Results

4.3.1 The Long-Term PACAP-Induced Synaptic Plasticity Features no Changes in

Neuronal Excitability.

In Chapter 3, we show that PACAP induces a LT synaptic plasticity in CG neuronal cultures, an effect accompanied by similar increases in sEPSC frequency (Fs) and significant elevations in sEPSC amplitude (As) relative to the ST PACAP-induced plasticity (see Fig 3.1 in Section 3.3.1). We therefore, investigated whether the LT

PACAP-induced synaptic platicty featured alterations in three synaptic parameters. The first was neuronal excitability. In CG neurons, action potentials (AP) can be elicited with current pulses (Fig 4.1A). In current-clamp mode, the ST PACAP-treated CG neurons were significantly depolarized (Vrest) (Vrest ≈ -44.40 ±2.0 mV) in comparison to Control treated cultures (Vrest ≈ -48.82 mV ±1.5 mV). No significant differences were observed in

Vrest between the Controls and the LT-PACAP condition (Vrest ≈ -46.70±1.4 mV) (Table

4.1). To elicit APs, CG neurons were stimulated with 200 ms depolarizing currents from

0-100 pA in 5 pA steps at a frequency of 0.1 Hz. Both Control neurons and LT PACAP treated neurons fired low frequency APs when sufficiently stimulated under current

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clamp, such that the number of spikes evoked initially increased linearly between 5 - 30

pA stimulation range before plateauing at an average of 3.33 (±0.05) spikes for both

conditions between the +35 pA and +100 pA stimulation range. The ST PACAP

conditions also exhibited an initial linear increase in AP production between 5-20 pA

stimulation range. However, in comparison to both controls and the LT PACAP treatment

the ST PACAP-induced plasticity was characterized by a significant reduction (p<0.05,

One-way ANOVA) in repetitive firing, plateauing at an average of 1.87 (±0.034) spikes

across the same stimulation range (Fig. 4.1B, C). In conjunction with these findings, the

threshold potential (Vthresh) for the acute PACAP treatment group was significantly

depolarized (Vthresh = -28.32 mV (±1.11) in comparison to Controls (Vthresh = -31.42 mV

±0.62) and the LT PACAP treatment condition (Vthresh = -32.21 ±0.61 mV) (Table 4.1).

No significant difference was observed in the inter-spike intervals between Controls, the

ST PACAP-induced plasticity and the LT PACAP-induced plasticity treatment

conditions (Table 4.1).

Table 4.1: CG Neuron Excitability Parameters. Cells depict the average values (±SEM) for Control (n = 11), ST PACAP (n =11), and LT PACAP (n = 10) treatment conditions. Asterisks * indicates a significant difference (p<0.05) relative to Control and Carats (^) indicate a significant difference from the ST PACAP treatment analyzed via a Students unpaired two-tailed T-test.

Control ST PACAP LT PACAP

-48.8 -44.4 -46.7 Resting potential (±1.5) (±2.0)* (±1.4) (mV)

Threshold -28.4 -32.2 -31.2 (±0.6) potential (mV) (±1.1)* (±0.6)

Inter-spike 42.4 (±2.2) 36.5(±1.6) 49.2 (±3.3)^ Interval (ms)

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4.3.2 The Long-term PACAP-Induced Synaptic Plasticity Features Increases in

Postsynaptic Strength.

All CG neurons express heteropentameric α3*- nAChRs and homopentameric α7- nAChRs. In CG neuronal cultures α3*-nAChRs are localized within postsynaptic densities and underlie ≈ 90% synaptic activity where as α7-nAChRs are localized extrasynaptically and are responsible for ≈ 10% of synaptic activity (Chen et al. 2001;

Jayakar et al. 2011). Given that the LT effects of PACAP are characterized by a 2 fold increase in As relative to controls (As*) (see Fig. 3.1), we decided to investigate whether the LT PACAP-induced plasticity was characterized by increases in postsynaptic strength. Isolation of spontaneous miniature excitatory postsynaptic currents (mEPSCs) are indicative of nAChRs activated in postsynaptic densities by ACh released from single exocytosed vesicles from adjacent presynaptic terminals (Lin and Bennet, 1994; Bekkers and Stevens, 1995; Chen et al., 2001). Consequently, if the LT and ST PACAP-induced synaptic plasticity was associated with alterations in mEPSC amplitude, it would be indicative of increases in postsynaptic nAChRs.

Spontaneous mEPSCs can be reliably measured following impulse dependent transmission blockade with the sodium channel inhibitor TTX (1 uM) (Chen et al. 2001,

Nai et al. 2003, Pugh et al. 2009). As can be seen in figure 4.2A, criterion mEPSCs (2.0-

2.5X above RMS noise) from Control neurons are readily detectable upon application of the sodium channel inhibitor TTX (1 μM) and are characteristically smaller in amplitude

(as = 8.54±0.22 pA) and occur with much lower frequency (fs) (fs = 0.28 ± 0.02 Hz) than

2+ impulse-dependent sEPSCs in cultures. Furthermore, as and fs are independent of [Ca ]out

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Figure 4.1: The LT PACAP-induced synaptic plasticity stabilizes CG neuronal excitability. A. Example records depict changes in action potential firing associated with application of progressively larger depolarizing currents (20, 50, 75 pA) in sham- treated Control neurons (black), neurons tested immediately after PACAP treatment (ST PACAP, red) and 48 h after (LT PACAP, blue) transient PACAP treatment (100 nM, 15 min). Traces show voltage responses to 200 ms duration depolarizing current pulses delivered at 0.1 Hz. Calibration bar: 10 mV and 50 ms. B. The number of action potentials increased linearly with similar current sensitivity up to 20 pA in control neurons (black circles and line; 0.11 pA-1, n = 11) as well as in neurons tested 48 h after PACAP treatment (blue circles and line; 0.09 pA-1, n = 10) and 15 min after PACAP treatment (red circles and line; 0.11 pA-1, n = 11). In the latter case, spiking sensitivity attenuated to a steady state value in response to currents >20 pA, whereas in sham- treated neurons and in LT PACAP treated neurons action potentials continued to increase up to 35 pA. C. For the steady-state range of 35-75 pA, the number of action potentials in sham treated control neurons and neurons tested 48 h after PACAP treatment were indistinguishable (3.2 ± 0.4 and 3.3 ± 0.4, respectively, p = 0.9) and significantly larger than that for neurons tested 15 min after PACAP treatment (1.8 ± 0.3, p<0.006 for both). Asterisks (*) indicate a significant difference from Controls while carats (^) indicate a significant difference from the ST PACAP-induced synaptic plasticity assessed by One-Way ANOVA.

116 and α7-nAChR activation because inhibition of α7-nAChRs with αBgt (50 μM, 1 h) fails to alter as and fs relative to controls (Nai et al. 2003; Pugh et al. 2009), indicating that detectable mEPSCs are mediated by activation of α3*-nAChRs. No significant differences were observed in either as and fs for the ST PACAP treated group (fs = 0.27

± 0.01 Hz; as = 8.31 ± 0.18 pA) when compared to the Control group, confirming our previous findings (Pugh et al. 2009). Interestingly, the LT PACAP induced plasticity was associated with a significant 34% increase in as (as = 11.36 ± 0.71 pA) (Fig. 4.2B) that corresponded to a significant rightward shift (data not shown, p<0.0001, Kolmogorov-

Smirnov). There was also a significant 25% increase in fs (fs = 0.34 ± 0.03 Hz) relative to

Controls (Fig. 3.2B) however, no significant rightward shift was observed (data not shown, p > 0.05, Kolmogorov-Smirnov). No significant differences were observed in half decay times and half-rise times of individual mEPSCs as a function of treatment group indicating that neither ST nor LT PACAP treatment altered α3*-nAChR gating properties in postsynaptic densities (Table in 4.2).

We then sought to identify whether the LT PACAP-induced plasticity was characterized by enhanced α3*-nAChR sensitivity. Previous studies indicate that the ST

PACAP-induced synaptic plasticity, despite being refractory for quantal size, still augments α3*-nAChR sensitivity (Pardi and Margiotta 1995, Margiotta and Pardi

1999;.Pugh et al. 2009; Jayakar et al. 2014). Utilizing localized microperfusion, nicotine

(20 uM) was rapidly perfused onto individual neurons for 1.5s following pretreatment with α7-nAChR inhibitor αBgt (50 nM, 30 min) to isolate α3*-nAChR mediated currents

(Fig. 4.2C). The LT PACAP treatment condition was characterized by a significant increase in peak α3*-nAChR currents (Ipeak ≈ 17.42 ± 2.14 pA/pF) in comparison to

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Controls (Ipeak ≈ 9.18 ±1.53 pA/pF), indicating that the LT effects of PACAP are associated with increased α3*-nAChR sensitivity. No detectable alterations were observed in decay times, indicaing that the increase observed in Ipeak was not attributable to alterations in α3*-nAChR gating properties (fig 4.2D). Therefore, the increases in quantal size coupled with increased α3*-nAChR sensitivity indicates that the LT

PACAP-induced synaptic plasticity is associated with enhanced postsynaptic strength.

4.3.3 Long-Term PACAP-Induced Synaptic Plasticity is Associated with Increased

Quantal Content.

In addition to enhancing as, the LT plasticity was also characterized by significant elevations in fs, indicative of alterations in presynaptic transmitter release. To examine this possibility experiments were conducted to measure mean quantal content (m); a value representing the average number of quanta (vesicles) exocytosed following a single presynaptic impulse (Johnston and Wu, 1995; Pugh et al. 2009). In culture, CG neurons receive direct presynaptic inputs from axons traveling in neurite fascicles. Suction and stimulation of these convergent fascicles via a bipolar stimulating electrode induces short-latency currents that can be detected in patch-clamped neurons (Fig. 4.3A,B). It is presumed that these evoked EPSCs (eEPSCs) are indicative of evoked transmitter release in that application of TTX or the global nAChR antagonist d-tubocurare (dTC) abolished the effect (Pugh et al. 2009).

Stimulation of converging fascicles at a stimulation intensity to reliably evoked

EPSCs, as well produced failures for both Control and LT PACAP treatment conditions

(Fig. 4.3 A-C). Consequently, this setup allowed for the measurement of quantal content

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Figure 4.2. The LT PACAP-induced synaptic plasticity enhances quantal size and α3*- nAChR sensitivity. A., B. Unitary synaptic current amplitude (quantal size) was assessed by analyzing α3*-nAChR-mediated mEPSCs (See METHODS) acquired at -70 mV from CG neurons in the presence of 1 µM TTX to block impulses. A. Exemplar records illustrate larger mEPSC amplitudes from neurons treated with PACAP (100 nM, 15 min followed by washout) 48 h prior to recording (LT PACAP, blue) when compared with those from sham-treated neurons (Control, black) or neurons treated with PACAP on the same day (ST PACAP, red). Calibration bars: 10 pA and 5 ms. B. Scatterplot quantification of mEPSC frequency and amplitude (with lines depicting mean ± SEM) reveal significant increases for both in CG neurons receiving LT PACAP treatments compared to control ST PACAP treatments (n = 19-20 cells per condition). C., D. The cell surface α3*-nAChR population was selectively activated by focally applying 20 µM Nicotine to CG neurons following incubation with αBgt (50 nM, 30 min) to block α7- nAChRs. C. Exemplar records illustrate larger peak α3*-nAChR whole-cell currents are induced by 1.5s perfusion with 20 µM nicotine from a neuron treated with 100 nM PACAP 48 h prior to recording (blue trace) when compared with currents from a sham- treated control neuron (black trace). Calibration bars indicate 200 pA and 300 ms. D. Quantification of peak α3*-nAChR currents (normalized to cell capacitance, pA/pF) reveals significantly larger responses in CG neurons receiving LT PACAP (n = 4) treatment compared to sham treated control neurons (n = 5) in the same experiment (Left bar graphs). No detectable alterations in average decay constants (Right) was observed in either condition.

119 through two methodologies. The first is empirical in which me can be calculated from the average evoked response amplitudes (Ae) including the failures (Ae = 0) relative to the average amplitude of ansynchronous mEPSCs (ae) (me= Ae/ ae) (del Castillo and Katz,

1954, Johnston and Wu, 1995). In all the experiments, spontaneous asynchronous mEPSCs from each neuron were collected for 1 minute prior to suction and stimulation of an adjacent converging fascicle. Asynchronous mEPSCs were collected following a burst of sEPSCs. The average amplitude obtained for asynchronous mEPSCs in Control neurons (n=12) compared to those collected in the presence of TTX (see Table 4.2) differed by ≈ 1% (ae = 8.47 ± 0.08 pA) and exhibited similar half rise (thalf =1.42 ± 0.04 ms), and half-decay times (thalf= 2.81 ± 0.11 ms) in comparison to spontaneous mEPSCs

(see table 4.2). mEPSCs collected for the LT PACAP treatment (n=13) condition were characterized by a significant 21% increase in mEPSC amplitude (ae = 9.93 ± 0.16 pA) in comparison to ansychronous mEPSCs collected from Controls (p<0.001) consistent with our findings with quantal size described above (Table 4.2). In the same comparisons, half-decay times of asynchronous mEPSCs were nominally longer than those of spontaneous mEPSCs (2.93 ± 0.28 and 2.57 ± 0.12 ms) but were not significantly different. Consequently, we considered the asynchronous mEPSCs as quantal events and their average amplitudes (ae) indicative of quantal size. Analysis of me identified that the

LT PACAP-treatment condition significantly increased the number of quanta released per prsynaptic impulse from ≈1.14 ±0.09 to ≈ 2.44 ±0.27. In conjunction with this finding the

LT PACAP treatment condition was characterized by a significant elevation in Ae (Ae ≈

23.81 ± 2.74 pA) in comparison to Controls (Ae ≈ 9.34 ± 0.66 pA).

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Table 4.2: Parameter Values for Spontaneous mEPSCs and Asynchronous mEPSCs. Cells depict the average values associated with spontaneous mEPSCs (± SEM) collected in the presence of TTX (Spontaneous mEPSCs) or during ansynchronous release following a burst of sEPSCs (Ansynchronous mEPSCs). For Spontaneous mEPSCs (n = 19-20 cells per condition), * indicates a significance difference (p<0.001) relative to Control and the ST PACAP treatment condition analysed via One-Way Anova. For asynchronous mEPSCs (n=12-13 cells for each condition), asterisks (*) indicate a significant difference (p<0.05) relative to their respective Controls analysed via a Student’s unpaired two-tailed T-test.

Spontaneous mEPSCs Asynchronous mEPSCs

Control ST PACAP LT PACAP Control LT PACAP mEPSC 8.54 8.30 11.36 8.30 9.93 amplitude (pA) (±0.22) (±0.18) (±0.71)* (±0.22) (±0.16)*

Half rise time 3.29 3.44 3.35 3.38 3.15 (ms) (±0.06) (±0.09) (±0.12) (±0.16) (±0.07)

Half decay 2.57 2.81 2.78 2.93 2.99 time (ms) (±0.12) (±0.15) (±0.15) (±0.28) (±0.09)

The second methodology used to measure m was the Poisson method of failures

(mf). This methodology predicts that under conditions of low release probability mf can be

determined by taking the natural log of the total number of delivered stimuli (N T) relative

to the number of failures (N0) [mf = ln(NT/N0)] (del Castillo and Katz, 1954; Martin and

Pilar, 1964; Johnston and Wu, 1995; Pugh et al. 2009). Similar to the results obtained for

me, the LT PACAP treatment group significantly elevated mf from ≈ 1.21 ±0.09 quanta to

≈ 2.48 ±0.23 quanta released per presynaptic action potential. Consistent with an increase

in mf, LT PACAP treated neurons exhibited a significant reduction in the probability of

evoking a failure (Pfailure = 10.54 ± 1.60%) in comparison to Control cultures (Pfailure =

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31.00 ± 2.42%). We therefore conclude that the LT PACAP-induced synaptic plasticity in addition to enhancing postsynpatic strength, augments presynaptic ACh release as indicated by increases in fs, me, and mf.

4.3.4 The Long-Term PACAP-Induced Synaptic Plasticity is Characterized by Pre- and Postsynpatic Remodeling.

The preceding electrophysiological data revealed that the LT PACAP-induced synaptic plasticity was associated enhanced post- and presynaptic strength. Previous studies conducted in our lab have identified that PACAP upregulates transcripts that can affect synaptic stability as well as presynaptic components (Sumner et al. 2009).

Consequently, we decided to examine whether PACAP induces synaptic remodelling of postsynaptic α3*-nAChR clusters, presynaptic puncta, and synaptic sites (Fig. 4.4).

Utilizing double immunocytochemistry, we surface labelled α3*-nAChRs with mAb 35 and presynaptic terminals with mAb 10h to label synaptic vesicle-associated protein 2

(SV2) either immediately after PACAP treatment (ST), or 48 hours (LT) after PACAP treatment. In neurons from control cultures, en-face surface confocal sections displayed non-uniform mAb35 labelling indicative of α3*-nAChR clusters (Fig 4.4Aa-f). Control neurons showed considerable overlap between SV2-positive presynaptic terminals and

α3*-nAChR clusters (Merge). In such cases, the size of the immediate postsynaptic membrane was determined from the portion of α3*-nAChR area in direct apposition with

SV2-labeled presynaptic terminals. The pattern of en face α3*-nAChR and SV2 labelling were similar between control neurons and the ST PACAP treated neurons. ST application of PACAP did not detectably alter the density, size, and intensity

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Figure 4.3: The LT PACAP-induced synaptic plasticity enhances presynaptic quantal release. A. Measurements of evoked EPSCs was acquired while recording from a CG neuron (blue circle) under voltage-clamp (Rec). eEPSCs were elicted via limited intensity current pulses from a single bipolar stimulating suction pipette (Stim) to a convergent neurite fascicle (orange) (Pugh et al. 2009). An axon (black line) and its presynaptic terminals (black circles) are shown contacting the postsynaptic neuron. B., C. Representative responses from a Control neuron and a neuron treated with PACAP 48 h earlier (LT PACAP) to a neurited stimulated 30 to 50 times (0.125 Hz) at a limited stimulation intensity, either evoked EPSCs (+) or failures (-). Calibration bar:10 pA and 10 ms. D. Quantal analysis summary for sham-treated neurons (Control, n = 12) and neurons treated with PACAP 48 h earlier (LT PACAP, n = 13) from 3 experiments. At LEFT, mean quantal content (m) values were calculated empirically from the ratio of average eEPSC and mEPSC amplitudes (me = Ae/ae) with mEPSCs generated by asynchronous release acquired in the same neurons prior to fascicle stimulation as described in Methods. At right, mean quantal content values were calculated using the Poisson method of failures (mf = ln (NT/N0) where NT and N0 are the total number of stimuli and the number that failed to evoke a detectable response, respectively. Lines depict mean m values ± SEM .

123 of α3*-nAChR clusters, SV2-labeled puncta, or merged synaptic sites indicating that the

ST PACAP-induced plasticity was not associated with changes in pre- and postsynaptic remodelling (Fig 4.4B, Table 4.3). For the LT PACAP-induced effects alterations in

α3*-nAChR clusters, presynaptic puncta, and synaptic sites were observed. Analysis of en face sections revealed that CG neurons 48 h after PACAP washout exhibited a significant (p<0.05) ≈ 1.70 fold (±0.31) increase in α3*- fluorescence intensity, as well as a ≈1.51 fold ±0.16 and 1.57 fold ±0.20 increase in the density and size of α3*-nAChR clusters (Table 4.3; Figure 4.4B). Furthermore, the LT PACAP-induced plasticity significantly increased the size of presynaptic terminals by ≈ 37% (1.37 ±0.15) without significantly altering the intensity (1.21 ±0.20) and density of presynaptic puncta (1.22 ±

0.17). Examination of colocalized synaptic sites revealed that the LT effects of PACAP significantly enhanced the density (2.56 ±0.38) and size (1.809 ± 0.26) in comparison to

Controls (Table 4.3; Fig. 4.4B), an effect consistent with significant elevatiosn in manders coefficient (Rm) between both conditions (Rm; Table 4.3). Both findings for the

ST and LT synaptic plasticity remained consistent when presynaptic puncta and postsynaptic α3*-nAChRs were examined at midline portions of the same CG neurons

(data not shown). Taken together, these results indicate that unlike the ST PACAP- induced plasticity, the functional changes associated with the LT PACAP-induced plasticity are accompanied by dynamc synaptic remodelling in the alignement, size and density of pre- and postsynaptic terminals.

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Table 4.3: Structural Correlates of PACAP-induced LT and ST Synaptic Plasticity. Cells depict the quantified values obtained from onfocal images of CG neurons immunolabeled with mAb35 and mAbh10 to detect α3*-nAChR clusters and SV2 containing presynaptic terminals, respectively. CG neuron cultures at 8 d in vitro were immunolabeled 48 h or 15 min after treatment with PACAP (100 nM, 15 min), times corresponding to LT and ST PACAP induced plasticity, and results compared with those from sham-treated control sister cultures labelled in parallel. nAChR clusters and SV2 puncta were identified and analyzed within regions of interest (ROIs) from soma surface (en-face) optical sections. As indicated in Methods, overall intensity applies to the mean relative mAb35 (α3*-nAChR) fluorescence intensity within selected ROIs. Other parameters apply to α3*-nAChR clusters and α3*-nAChR cluster/SV2 puncta overlap sites (synapses) including size, density and relative intensity. Rm indicates Manders coefficient for quantifying α3*-nAChR/SV2 colocalization (0 ≤ Rm ≤ 1). The number of neurons evaluated is given in parentheses, and asterisks (*) indicate a significant difference (p < 0.05) in the indicated parameter compared to that for sham-treated control neurons.

α3*-nAChR Labeling α3*-nAChR / SV2 Overlap  

Test Overall Cluster Cluster Cluster Rm Synapse Synapse Synapse Condition Intensity Density Size Intensity Density Size Intensity -2 2 -2 2 (ΔF/Fb) (µm ) (µm ) (ΔF/Fb) (µm ) (µm ) (ΔF/Fb)

LT 9.9* 0.32* 0.31* 41.4 0.63* 0.18* 0.24* 45.6 PACAP ± 0.3 ± 0.04 ± 0.04 ± 6.2 ± 0.03 ± 0.03 ± 0.03 ± 5.9 (n = 12) Control 5.8 0.21 0.20 38.2 0.50 0.07 0.13 41.0 (n = 12) ± 1.2 ± 0.02 ± 0.03 ± 6.9 ± 0.04 ± 0.01 ± 0.01 ± 6.6

ST PACAP 3.8 0.22 0.19 22.4 0.79 0.13 0.14 27.2 (n = 8) ± 0.5 ± 0.04 ± 0.02 ± 1.6 ± 0.02 ± 0.03 ± 0.03 ± 2.2

Control 3.7 0.25 0.19 21.6 0.75 0.15 0.11 26.7 (n = 7) ± 0.5 ± 0.04 ± 0.02 ± 1.1 ± 0.04 ± 0.03 ± 0.02 ± 2.4

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Figure 4.4: The LT PACAP-induced synaptic plasticity features dynamic post- and presynaptic remodeling. A. 63X confocal images of CG neuron somas from 7d cultures immunolabeled with mAb 35 to detect α3*-nAChRs (red), and mAb h10 to detect SV2 (green) in presynaptic terminals. The left panel (a-c) shows α3*-nAChR (Red) labeling in somatic hemispheric Z-stack projections (10-20, 0.5 um optical sections) with ROIs delimiting the cell surface (en-face) views in the middle section that was used to analyze both α3*-nAChR clusters, and presynaptic puncta (SV2, green) (d-l). Merged regions show colocalized regions (yellow). The far right panel shows colocalization along the mid-region of the cells (perimeter, m-o). Calibration bars: 5 µm. B. Bar graphs depict immunostaining parameter values for LT PACAP condition (black bars n=12) and the ST PACAP treatment condition (gray bars, n=8) relative to control CG neurons (Dashed line, n=20) analyzed using Image J (1.37c). Asterisks (*) depict a significant difference (p<0.05) relative to Controls using a Students unpaired two-tailed T-test.

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4.4 Discussion

In this chapter, we sought to determine the physiological and synaptic hallmarks underlying the LT PACAP-induced synaptic plasticity on cholinergic synapses and to distinguish these effects from the ST PACAP-induced plasticity. We have previously demonstrated that the ST PACAP-induced plasticity is associated with reductions in CG neuronal excitability and increases in presynaptic transmitter release and α3*-nAChR sensitivity (Margiotta and Pardi, 1995; Pugh et al. 2009, Jayakar et al. 2014). Like the ST

PACAP-induced plasticity, the LT PACAP-induced plasticity was also characterized by significant elevations in presynaptic transmitter release and α3*-nAChR sensitivity.

However, we also observed divergences in the electrophysiological and synaptic profiles between the ST and LT plasticity (Table 4.4). Unlike the ST PACAP treatment, no alterations in AP generation was observed for the LT PACAP-induced plasticity indicating that the LT effects are not associated with changes in CG neuronal excitability.

Likewise, the LT PACAP-induced synaptic plasticity was characterized by enhancemetns in as and fs, both of which were not significantly altered in the ST PACAP treatment condition. Finally, the LT PACAP-induced plasticity exhibited increases in the size, density and alignment of pre- and postsynaptic terminals while no such effects were observed for the ST PACAP treated CG neurons. Taken together these findings reveal both distinguishable differences between the ST and LT plasticity and demonstrates that

PACAPs capacity to enhance both pre- and postsynaptic strength occurs via dynamic synaptic remodeling of pre- and postsynaptic terminals.

In the autonomic nervous system, PACAP alters the excitable properties of neurons in the cardiac ganglion (Braas et al., 1998, Parsons et al. 2000, De Haven and

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Table 4.4: The LT and ST PACAP Exhibit Distinguishable Physiological and Synaptic Hallmarks. Table summarizing the key findings obtained from Chapter 4 relative to Controls. The table also extrapolates findings published in Pugh et al. (2009) and Jayakar et al. (2014).

Parameter ST PACAP LT PACAP Neuronal excitability Decrease No change

Quantal size No change Increase α3*-nAChR sensitivity Increase Increase mEPSC frequency No change Increase

Quantal Content Increase Increase

Size of α3*-nAChR clusters No change Increase

Density of α3*-nAChR clusters No change Increase

Size of presynpatic terminals No change Increase

Density of synaptic sites No change Increase

Size of synaptic sites No change Increase

Cuevas, 2004, Merriam et al., 2004, Hardwick et al. 2006, Tompkins et al., 2007,

Tompkins and Parsons, 2008, Merriam et al., 2013, Tompkins et al. 2015), the superior cervical ganglion (May et al. 1998, May et al. 1998, Beaudet et al. 2000), the submandibular ganglion (Hayashi et al. 2002, Kamaishi et al. 2004), the major pelvic ganglion (Tompkins et al., 2010) and the ciliary ganglion (Pugh et al. 2009). Consistent with our previous findings, the ST effects of PACAP were associated with a reduction in neuronal excitability (Pugh et al. 2009). While, the mechanisms underlying this reduction in neuronal excitability have not been thoroughly uncovered we have demonstrated that the ST PACAP-induced plasticity reduces Ca2+ entry through voltage-dependent Ca2+

128 channels (Pugh et al. 2007, Starr and Margiotta, unpublished), an effect similar to

PACAP-induced synaptic plasticity in the submandibular ganglion (Hayashi et al. 2002;

Kamaishi et al. 2007). However, given that no change AP propagation was observed for the LT PACAP induced plasticity, these findings suggest that the LT effects of PACAP were not characterized by alterations in neuronal excitability.

We observed that the LT PACAP-induced plasticity was characterized by significant increases in αs, and peak α3*-nAChR sensitivity, effects that corresponded to increases in the overall intensity, size and density of α3*-nAChR clusters. It is not known whether the effects of PACAP on α3*-nAChR upregulation is mediated exclusively by gene transcription or other post-translational modifications. In gene microarrays, no changes were observed in mRNAs encoding α3, β4, α5, or β2 subunits comprising the stoichiometry of α3*-nAChRs 96 hours after PACAP treatment (Sumner et al. 2009).

However, given that we observe the sharpest increase in sEPSC amplitude 48 hours after

PACAP treatment, it is possible that increased transcription and translation of α3*- nAChR subunits occurs at an earlier time poit then was examined (Kim et al. 2013;

Rogers and Gahring, 2015). Additionaly, in cholinergic neurons nAChR assembly and maturation occurs in the ER and golgi and upregulation of nAChRs is linked to nAChR subunit composition, decreased nAChR subunit degradation, increased rates of nAChR maturation and subunit assembly, decreased rates of nAChR turnover on the plasma membrane, and increased rates nAChR exocytotic trafficking and lateral mobility on cells surface (Jacob et al. 1986; Peng et al. 1994; Jeanclos et al. 2001; Huebsch and Maimone,

2003; Christianson and Green, 2004; Vallejo et al. 2005; Corringer et al. 2006; Rezvani et al. 2007; Millar and Harkness, 2008; Albequrque et al., 2009; Fernandes et al. 2010).

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Given this wide array possibilities regarding how α3-nAChRs can be upregualated, future studies are warranted to investigate how PACAP ultimately increases α3*-nAChR surface expression.

We next assessed whether the LT PACAP-induced plasticity was associated with changes in mean quantal content. Given that PACAP was characterized by a 25% increase in fs this seemed plausible. Quantitative transmission studies conducted via stimulation of presynaptic fascicles to evoke EPSCs in a target neuron confirmed that a presynaptic mechanism was involved. Calculations of quantal content via the empirical method [me = Ae/ae] and the Poisson method of failures [mf = ln(N/n0)] revealed that

PACAP doubled the amount of vesicles exocytosed per presynaptic stimulus. We are confident that acquisition of ae following current bursts were authentic mEPSCs as they exhibited similar values in amplitude, rise time and decay time as those collected in the presence of TTX. Quantal content is a direct function of two factors; the probability (p) of vesicular release and the number (n) of vesicles stored within presynaptic terminals

[m=n(p)] (del Castillo and Katz, 1954; Johnston and Wu, 1995). Our imaging experiments indicated that the LT effects of PACAP increased the size of SV2 labeled presynaptic puncta by ≈ 37%. Given that SV2 is presynaptic marker for synaptic vesicles, these findings show that PACAP increases n. However, while PACAP does decrease the rate of evoking failures, a variable correlated with probability of release, it still ultimately unknown whether the LT PACAP-induced synaptic alters the probability of presynaptic ACh release (Branco and Staras, 2009). Interestingly, our gene array data supports a role for gene transcription in mediating the PACAP-induced increases in presynaptic strength. PACAP increases expression of mRNAs encoding choline

130 acetyltransferase (ChAT) and the high-affinity choline transporter (HaChT) presynaptic terminals, two essential enymes required for ACh synthesis (Balazokova and Blakely,

2006; Nicholls et al. 2012). Increased expression of both HaChT and ChAT could underlie PACAP-induced increases in the size of SV2 labeled presynaptic puncta.

Likewise, PACAP also increased the expression of vesicular priming components on vesicles and in active zones including synaptobrevin, synaptotagmin IV and rims binding protein 2 (Sumner et al. 2009). Thus, increased expression of transcripts relevant to ACh synthesis and release are consistent with a LT PACAP-induced enhancement in quantal content and would need to be investigated in the future to determine their roles in mediating the LT PACAP-induced synaptic plasticity.

The enhanced size and density of synaptic sites indicates that PACAP signaling promotes synaptic colocalization between pre- and postsynpatic terminals. It is clear from our data, that postsynaptic modifications are critical as we observed significant increases in the overall intensity, size and density of α3*-nAChR clusters. Likewise, while the size of presynaptic terminals became larger, the enhancement in synaptic sites was not attributable to synaptogensesis as we observed no changes in the density of presynaptic terminals. Consequently, the joint increases in both pre- and postsynaptic alignment suggests that the PACAP is inceasing synaptic stability. In CG neurons, nAChRs interact with complex scaffolding proteins and cytoskeletal networks to maintain their stability synaptically and extrasynaptically (Conroy et al. 2003, Jones et al. 2010, Fernandes et al.

2010). In support of this hypothesis, gene arrays have verified that acute PACAP application in CG neuronal cultures regulates transcripts associated with synaptic stability including contactin associated protein like 5, cortactin binding protein 2, and neuron

131 pentraxin 2 and the reticulon 4 receptor (O’brien et al. 1999; Sumner et al. 2009; Schwab,

2010; Shi et al. 2014; Varea et al. 2015; Pelkey et al. 2015). Taken together these results suggest that PACAP is functionally enhancing synaptic stability between cholinergic neurons.

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Chapter 5

Discussion of Findings

5.1 Summary of Findings

A 15 min transient exposure to PACAP induces a ST and LT synaptic plasticity characterized by elevations in Fs* and As* in CG neuronal cultures both immediately and

48 hrs after PACAP washout (Pugh et al. 2009). While featuring similar increases in

Fs*, the LT PACAP-induced plasticity at 48h was characterized by more robust increases in As*. Furthermore, PACAP-induced elevations in synaptic activity was observed 24h,

48h, and 72 h after PACAP treatment showing that PACAP could be sustainably enhancing cholinergic transmission. However, the mechanisms underlying this PACAP- induced LT plasticity was unknown. Therefore, the primary goal of this dissertation was to examine the biochemical signaling pathways, electrophysiological profile, and synaptic hallmarks underlying the LT and to distinguish these effects from the ST

PACAP-induced synaptic plasticity.

The experiments presented in Chapter 2 highlight critical intracellular mechanisms mediating the ST PACAP-induced synaptic plasticity. One essential effector underlying the ST synaptic plasticity is NOS1 (Pugh et al. 2009). PACAP elevated NO synthesis in CG neurons, an effect that required PKA signaling, extracellular Ca2+ entry, and activation of α7-nAChRs. Inhibition of PLC with U7 had no effect on PACAP

133 induced activation of NOS1, confirming previous findings that the ST effects are independent of PLC signaling (Pugh et al. 2009). Further, scavenging extracellular NO with cPTIO blocked the ST PACAP-induced elevations in Fs* and As* indicating that

NO acts as a retrograde signaling messenger. Activation of α7-nAChRs was essential for the ST plasticity as treatment with αBgt abrogated PACAP-induced elevations in Fs* and

As*. Reversible inhibition of α3*-nAChRs with αCTx-AuIB also had a modest effect on the ST PACAP-induced synaptic plasticity, significantly reducing As*, but nominally reducing Fs*. In concert with a role for nAChRs to coordinate the ST PACAP-induced synaptic plasticity, NOS1 existed in a protein complex with α3*- and α7-nAChRs.

Additionally, localized PKA signaling via interactions with AKAP was required for the

ST plasticity as inhibition of AKAP-RIIβ interactions with Ht31 blocked the PACAP- induced elevations in Fs* and As*. Interestingly, Ht31 had no effect on the ST PACAP- induced increases in α3*-nAChR sensitivity confirming that AKAPs target PKA signaling to functionally enhance NO synthesis within close proximity to synaptic sites.

Having previously characterized the biochemical signaling mechanisms underlying the ST PACAP-induced synaptic platicity, the experiments in Chapter 3 were aimed at examining the intracellular mechanisms underlying the LT PACAP-induced plasticity. Specifically, the LT PACAP-induced effects were dependent upon PAC1R- mediated activation of AC and PLC. Stimulation of AC with forskolin induced a LT synaptic plasticity significantly elevating both Fs* and As* in the presence and absence of U7. However, mimicking Gαq with muscarine or PLC signaling with m-3m3FBS both in the presence and absence of MDL, failed to mimic the LT PACAP-induced synaptic plasticity, indicating that AC signaling was sufficient to cause a LT syanptic plasticity. In

134 addition to a dependency on PLC, other distinguishable differences were observed between the ST and LT PACAP-induced synaptic plasticity. Unlike the ST effects of

PACAP, the LT plasticity was independent of PKA, impulse dependent and nAChR activity, and NOS1 but required gene transcription. Taken together the results from

Chapter 3 demonstrate that PACAP induces a novel form of LT synaptic plasticity on cholinergic synapses that requires concomitant PAC1R-induced stimulation of AC and

PLC mediated signalling pathways as well as gene transcription. Further, because inhibition of PKA, nAChR activation, synaptic activity, and NOS1 had no effect on the

LT PACAP-induced synaptic plasticity and inhibition of gene transcription did not block the ST PACAP-induced plasticity, these results identify that the intracellular mechanisms underlying the LT PACAP-induced synaptic plasticity are distinct from the ST PACAP- induced synaptic plasticity.

Chapter 4 both characterized the electrophysiological and synaptic hallmarks underlying the LT PACAP-induced synaptic plasticity. The LT PACAP-induced synaptic plasticity was not characterized by alterations in neuronal excitability as no changes in

AP propagation were observed. However, the LT effects did feature increases in quantal size and α3*-nAChR sensitivity, an effect consistent with increases in postsynaptic strength. Further, the LT plasticity featured elevations in fs and quantal content indicative of elevations in presynaptic transmitter release. Consistent with elevations in postsynaptic strength, confocal analysis of pre- and postsynaptic sites identified that the T PACAP- induced synaptic plasticity featured increases in the intensity, size, and density of surface

α3*-nAChR clusters. Additionally, the LT plasticity also featured elevations in the size of presynaptic terminals as well as drastic increases in the density and size of colocalized

135 synaptic sites. While not tested, we have previously shown that the ST plasticity also features similar elevations in α3*-nAChR sensitivity and quantal content to the LT effects (Margiotta and Pardi, 1995; Pardi and Margiotta, 1999; Pugh et al .2009; Jayakar et al. 2014). However, several of our findings show distinguishable electrophysiological differences between ST PACAP and LT PACAP-induced plasticity. Specifically, the ST effects were associated with a reduction in neuronal excitability and exhibited no alterations in quantal size and mEPSC frequency. Analysis of synaptic sites, as well as pre- and postsynaptic terminals revealed no changes in the intensity, size and density of postsynaptic α3*-nAChR clusters, presynaptic puncta and colocalized synaptic sites.

These findings demonstrate that the physiological and synaptic hallmarks underlying the

LT PACAP induced synaptic plasticity are distinguishable from the ST effects.

Consequently, our hypothesis predicting that the LT PACAP induced plasticity would exhibit distinguishable biochemical signaling, electrophysiological characteristics and synaptic hallmarks from the ST PACAP-induced plasticity was verified.

5.2 General Discussion

The findings presented in this dissertation demonstrates that PACAP induces both

ST and LT synaptic plasticity at cholinergic synapses. While both types of synaptic plasticity in CG neuronal cultures exhibited increases in Fs*, the LT PACAP- induced synaptic plasticity featured a more robust and significant enhancement in As* compared to the ST PACAP-induced synaptic plasticity. Significant distinctions were also observed between the ST and LT PACAP-induced synaptic plasticity biochemically, electrophysiologically and morphologically at synapses. Consequently, in addition to

136 characterizing the cellular and physiological underpinnings mediating the LT PACAP- induced synaptic plasticity we conclude that ST and LT PACAP-induced synaptic plasticity are mechanistically distinct.

We demonstrated previously that the ST PACAP induced synaptic plasticity was characterized by a PAC1R, PKA-induced enhancement in the sensitivity α3*- and α7- nAChRs as well as a PAC1R, AC, cAMP, PKA, NOS1 mediated increase in presynaptic quantal release (Margiotta and Pardi, 1995; Pardi and Margiotta, 1999; Pugh et al. 2009).

The experiments presented in Chapter 2 clarified the contributory roles of NOS1, PKA, and identified a novel role for α3*- and α7-nAChRs in mediating the ST PACAP-induced synaptic plasticity. PACAP stimulated NO production in CG neurons, an effect inhibited by blocking NOS1, nAChRs and PKA and by chelating extracellular Ca2+. Scavenging of extracellular NO attenuated the ST PACAP-induced plasticity indicating that NO, generated in postsynpatic CG neurons, acts in retrograde-like fashion to enhance presynaptic transmitter release. In conjunction with the role of nAChRs in blocking NO synthesis, inhibiting nAChRs with dTC blocked the ST PACAP-induced plasticity.

However, a larger role for α7-nAChR activation was observed as inhibition of α7- naChRs with αBgt blocked both PACAP induced NO synthesis and elevations in Fs* and

As*. Inhibition of α3*-nAChRs however, had no effect on PACAP induced NO synthesis and only partially inhibited the ST PACAP induced synaptic plasticity.

PKA signaling played a significant role in the ST PACAP-induced synaptic plasticity on CG neuronal cultures. Inhibition of PKA blocks both the ST-PACAP induced increases in NO synthesis and elevations in Fs* and As*, supporting the conclusion that PKA “sensitizes” NOS1 to prolong NO synthesis (Bredt et al. 1992; Pugh

137 et al. 2009; Hurt et al. 2012; Jayakar et al. 2014). Targeted PKA signaling via AKAPs was crucial in mediating the ST PACAP-induced synaptic plasticity. CG neurons express

AKAPs, and disruptions in PKA interactions with AKAP following application of Ht31 blocked the ST PACAP-induced elevations in Fs* and As* without affecting PKA- mediated increases in α3 nAChR sensitivity (Jayakar et al. 2014). The inability of Ht31 to block nAChR upregulation in concert with findings identifying that PKA inhibition blocks NO synthesis, suggests that AKAP localizes PKA signaling to NOS and synaptic sites. Such a possibility could be achieved through interactions with PDZ containing scaffolding proteins that associate with AKAPs localized within proximity to post- and presynaptic terminals (Reviewed in Sanderson and Dell’acqua 2011).

Co-immunoprecipation experiments verified that NOS1 was present in a protein complex with both α3* and α7 nAChRs. Given that the α3*-nAChRs exhibit a moderate

2+ + 2+ + Ca to Na permeability (PCa/PNa ≈ 1.0-1.5) and α7 nAChR exhibits a high Ca to Na permeability(PCa/PNa ≈10-20), the formation of a protein complex between both subtypes of nAChRs and NOS1 in concert with the role for nAChR activation and inhibition in mediating both the ST PACAP-induced effects on Fs* and As* as well as NO synthesis suggests that Ca2+ entry via nAChRs underlies PACAP-induced NOS1 activation

(Séguéla et al. 1993; Vernino et al. 1994; Role and Berg, 1996; Jayakar et al. 2014).

Scaffolding proteins could link nAChR activation with NOS1 in a similar manner as identified in glutamatergic synapses where Ca2+ permeable NDMARs form ternary protein complexes with NOS1 via PSD 95 to efficiently couple NDMAR activation with

NOS1-induced NO synthesis (Paarman et al. 2008). Experiments from CG homogenates show that CG neurons express all four subytpes of PSD95 proteins and that α3*-nAChRs

138 form protein complexes with PSD-95 while α7 nAChRs formed complexes with an unidentified PDZ containing protein (Conroy et al. 2002; Neff III et al. 2009). Given the localization of somatic α7 and α3*-nAChRs with MAGUKs as well as the direct association of MAGUK proteins with localizing NOS1 in glutamatergic synapses

(Paarman et al. 2008), Ca2+ entry via perisynaptic α7 nAChRs and perisynaptic and synaptic α3*-nAChRs could underlie the PACAP-induced activation of NOS1.

In addition to expanding upon the contributory roles of PKA, nAChRs and NOS1 in mediating the ST PACAP-induced plasticity, the experiments presented in Chapter 2

(Jayakar et al. 2014) as well as in Pugh et al. (2009) provided a clear platform by which to compare the signal transducing mechanisms underlying the LT PACAP-induced synaptic plasticity to the ST PACAP-induced synaptic plasticity. Given our knowledge on the mechanisms underlying ST PACAP-induced synaptic plasticity, we concluded in

Chapter 3 that the LT PACAP-induced synaptic plasticity is mechanistically distinct from the ST PACAP-induced synaptic plasticity. First, despite a requirement for AC, the LT

PACAP-induced effects did not require activation of PKA. Second, the LT effects of

PACAP were completely independent of synaptic and nAChR activities. Third, in concert with an independence from both PKA and nAChR activation, inhibition of NOS1 failed to block the LT PACAP-induced synaptic plasticity. Fourth, the LT PACAP induced synaptic plasticity required PLC activation and gene transcription. However, The ST

PACAP-induced elevations in Fs* and As* was unaffected following inhibition of gene transcription. As shown in figure 5.1, these findings demonstrate that principal intracellular effectors underlying the ST PACAP-induced synaptic plasticity were not mediating the LT PACAP-induced synaptic plasticity.

139

Fig 5.1: The ST and LT PACAP-induced synaptic plasticity are mediated by distinct intracellular signaling mechanims. The diagram details the proposed signal transducing pathways underlying the ST-PACAP induced synaptic plasticity (Left, outlined in Red) and the LT PACAP-induced synaptic plasticity (Right, outlined in Blue) extrapolated from Pugh et al. (2009), Chapter 2, and Chapter 3.

140

In concert with our cnclusion that the ST- and LT PACAP-induced plasticity are mechanistically distinct, we identified that that the LT effects were mediated via bimodal,

PAC1R-induced AC and PLC signaling cascades. While this dual requirement for AC and

PLC has been identifide for PACAP-induced synaptic plasticity in CA1 pyramidal neurons and the submandibular ganglion (Hayashi et al. 2002; Kamaishi et al. 2004;

Macdonald et al. 2005; Macdonald et al. 2007; Costa et al. 2009) it was unknown whether activation of these pathways functionally enhanced shared downstream effectors, or acted independently from one another. Because AC activation was both necessary and sufficient to induce a LT plasticity, while PLC activation was required, but insufficient to induce a LT plasticity, these results support the hypothesis that PLC likely amplifies shared effector(s) of AC (See Fig. 5.1). Consequently, these findings support a novel signal transducing mechanism by which PACAP/PAC1R bimodal AC and PLC singaling cascades induces synaptic plasticity in the nervous system. For example,

PACAP/PAC1R-mediated mediated activation of either PLC (through PKC) or AC signaling pathways (via PKA, Epac or NCS) respectively, can result in activation of ERK and MAPK-dependent signaling pathways Filippatos et al. 2001; Sakai et al. 2002;

Dziema and Obreitan, 2002; Gerdin and Eiden, 2007; Emery and Eiden, 2012;

Blechmann and Lekowitz, 2013; May et al. 2014; Clason et al. 2017). Therefore, these findings signify the importance for future studies to investigate potential downstream effectors underlying the LT PACAP-induced synaptic plasticity in CG neurons and in other systems.

While our experiments with Pitstop 2 verify that both the ST and LT PACAP- induced synaptic plasticity on CG neurons do not require CME, our results do not rule

141 out a possible involvement for β-arrestin. In the cardiac ganglion, PACAP/PAC1R- induced synaptic plasticity requires clathrin-mediated endocytosis (CME). Furthermore,

CME of the PAC1R directly modulates PACAP-induced activation of MEK/ ERK signaling pathways (Merriam et al. 2013; Tompkins et al. 2016; Clason et al. 2016). In addition to desensitizing GPCRs and providing a platform to bind adaptor protein complexes (see section 1.4) necessary for CME, β-arrestins can activate non-traditional

GPCR signaling pathways associated with Src family tyrosine kinases, ERK1/2, and

MAPK (reviewed in DeWire et al. 2007; Lutrell and Gesty-Palmer, 2010). For example, while no direct association between PAC1R signaling and β-arrestin has been established, knock down of β-arrestin 1 in pancreatic β cells blocked PACAP-induced sustained

ERK1/2 phosphorlyation revealing a requirement for β-arrestin in mediating PACAP- induced activation of ERK (Broca et al. 2009). Consequently, in addition to AC and PLC, it is possible that the LT PACAP-induced plasticity could also be partially mediated by β- arrestin signaling.

Given PACAP’s capacity to persistently enhance cholinergic synaptic activity as well as alter the expression of genes relevant to synaptic function we also examined the electrophysiological and synaptic hallmarks underlying the LT PACAP-induced synaptic plasticity at cholinergic synapses. We first examined whether the LT PACAP-induced plasticity enhanced cholinergic activity by increasing CG neuronal excitability. For the

LT PACAP-induced plasticity, no significant changes in repetitive firing, inter-spike interval, resting potential, and threshold potential were observed 48h after PACAP treatment indicating that the LT PACAP-induced plasticity was not mediated by alterations in neuronal excitability. However, consistent with previous findings (Pugh et

142 al. 2009; Pugh et al. 2007), the ST PACAP-induced plasticity was characterized by a significant reduction in CG neuronal excitability an effect accompanied by a reduction in repetitive firing, and more depolarized resting membrane potentials and threshold potentials. The cause for the acute PACAP mediated reduction in CG neuronal excitability remains unknown, however reductions in calcium current density influx has been observed (Pugh et al. 2007; Pugh et al. 2009; Starr and Margiotta, unpublised). The reduction of CG neuronal excitability could be mediated by PACAP-induced regulation of Ca2+ dependent K+ channels which are known to reduce repetitive firing. In cerebellar granule neurons, for example, PACAP application augments BK channel activation via concomitant ePAC-mediated calcium release, Rap and p38 MAPK signaling cascades, significantly hyperpolarizing cerebellar granule neurons and elevating afterhyperpolarization potentials (Ster et al. 2007). Whatever the cause, the reduction in

CG neuronal excitability is not consistent with the enhancments in Fs* and As* observed for the ST PACAP-induced synaptic plasticity.

The inability of the LT PACAP-induced plasticity to be associated with changes in excitability led us to examine whether the PACAP-induced effects were characterized by increases in the number of α3*-nAChRs contained in postsynaptic densities. In concert with increases in As*, the LT PACAP-induced synaptic plasticity featured a significant 34% elevation in quantal size indicative of increases in the number of α3*- nAChRs contained within postsynaptic densities. Additionally, the LT PACAP-induced effects featured a 90% increase in global α3*-nAChR sensitivity consistent with an increase in the number of functional α3*-nAChRs somatically, similar to that found for the ST PACAP-induced synaptic plasticity (Pugh et al. 2009, Jayakar et al. 2014). No

143 changes in the half decay times underlying individual mEPSCs or nicotine evoked responses were observed between experimental groups, signifying that the enhancement in quantal size and sensitivity was not due to gating properites of α3*-nAChRs. Instead, the LT PACAP-induced enhancement in postsynaptic strength was observed to be associated with postsynaptic remodelling. Specifically, the LT PACAP-induced synaptic plasticity significantly elevated the intensity, size and density of α3*-nAChR clusters localized synaptically and perisynaptically on CG neurons. No changes in any of these parameter values were observed for the ST PACAP-induced plasticity supporting our previous conclusion that despite increasing α3*-nAChR sensitivity, the ST effects of

PACAP are refractory for α3*-nAChRs localized to postsynaptic densities (Pugh et al.

2009).

To our knowledge this is the first demonstration that PACAP-induced synaptic plasticity has been directly linked to increases in the number of α3*-nAChR clusters localized both perisynpatically and within postsynaptic densities. In cholinergic neurons nAChR assembly and maturation occurs in the ER and golgi and upregulation of nAChRs is linked to nAChR subunit composition, decreased nAChR subunit degradation, increased rates of nAChR maturation and subunit assembly, decreased rates of nAChR turnover on the plasma membrane, and increased nAChR exocytotic trafficking to the surface (Jacob et al. 1986; Peng et al. 1994; Jeanclos et al. 2001; Huebsch and Maimone,

2003; Christianson and Green, 2004; Vallejo et al. 2005; Corringer et al. 2006; Rezvani et al. 2007; Millar and Harkness, 2008; Albequrque et al., 2009). Consequently, PACAP could be affecting several cellular processes to upregulate α3*- nAChRs on postsynpatic

CG neurons. One possibility is that PACAP is functionally upregulating transcripts

144 encoding functional subunits comprising α3*- nAChRs. While plausible, previous gene arrays conducted 96 h after PACAP treatment reveal no alterations in the expression of transcripts encoding subunits for α3*- nAChRs (Sumner et al. 2009). However, given that we observe the sharpest increase in sEPSC amplitude 48 h after PACAP treatment, it is possible that PACAP could be functionally, upregulating nAChRs at an earlier time point. Likewise, PACAP could also modulate nAChR assembly and trafficking to the surface by enhancing nAChR subunit stability within the ER via second messenger signaling cascades. Within cholinergic synapses, approximately 80% of nAChRs in the

ER are assembled incorrectly (Wanamaker et al. 2003), and 65%-85% of total nAChRs are localized to the endoplasmic reticulum in a cell (Whiteaker et al. 1998; Fenster et al.

1999; Paakanan et al. 2006; Rogers et al. 2006; Albequrque et al. 2009). Second messenger signaling cascades can dynamically modify nAChR assembly. For example, stimulation of AC with forskolin increases surface expression of torpedo AChRs expressed in muscle and in transfected fibroblasts (Green et al. 1991). Interestingly, these effects were independent of gene transcription and PKA signaling, and were associated with enhancements in the stability of partially assembled nAChR subunits via heterologous subunit-subunit interactions, doubling the efficiency of nACR assembly

(Green et al. 1991; Ross et al. 1991; Shiranthi et al. 1994). Given the dependence of the

LT PACAP-induced plasticity on AC signaling and forskolins capacity to mimic the LT effects of PACAP suggests that PACAP could be enhancing nAChR assembly by increasing subunit stabilization.

Within the CG, onset of synaptic transmission is detected at embryonic day 5, with 100% of neurons exhibiting ACh-dependent transmission by day 7 (Landmeisser

145 and Pilar, 1972). Clustering of nAChRs on CG neurosn represents an important developmental milestone during cholinergic synapse formation as CG neurons undergo drastic synaptic remodelling throughout embryonic development (Landmeisser and Pilar,

1972). Unfortunately, the mechanisms underlying neuronal nAChR clustering in the CNS and ANS remain unknown. Primarily studied at neuromuscular junctions, clustering of muscle-type nAChRs is triggered via the contact of a presynaptic terminal to a postsynaptic neuron, resulting in a redistribution of surface nAChRs to form clusters at the synaptic junction (Reviewed in Huh and Fuhrer, 2002). Consequently, our discovery that PACAP increases the size and density of α3*- clusters shows that PACAP promotes

α3*- nAChR cluster formation. One potential mechanism underlying this effect is that

PACAP could be functionally altering cytoskeletal dynamics to destabilize extrasynaptic

α3*- nAChRs, promoting lateral mobility to postsynaptic densities. Indeed, extrasynaptic

α3*--nAChRs associate with cytoskeletal scaffolds, actin and microtubules and exhibit higher mobility rates following polymerization of actin and microtubules verse α3*- nAChRs localized to postsynaptic densities (Conroy et al. 2003; Neff III et al. 2009;

Fernandes et al. 2010). Given PACAPs well established roles in affecting cytoskeletal dynamics to promote axonal growth, axonal regeneration, and neurite outgrowth

(Gonzalez et al. 1997; Sakai et al. 2004; Fukiage et al. 2007; Leemhuis et al. 2007;

Tsuchida et al. 2014; Manecka et al. 2013; Ogata et al. 2015) supports the hypothesis that

PACAP could be altering lateral mobility of surface α3*-nAChRs .

Likewise, another hypothesis is that PACAP could be functionally increasing the number of “active” nAChRs contained in synaptic sites. Margiotta and Berg (1987) demonstrated that application of 8Br-cAMP, while not altering the expression, synthesis

146 or gating properties of nAChRs, still enhanced overall nAChR sensitivity, suggesting that cAMP signalling pathways could convert silent α3*-nAChRs to “active” α3-nAChRs on the cells surface (Margiotta and Berg 1987; Berg et al. 1989). Likewise, acute PACAP treatment also augments α3*- and α7-nAChR sensitivity via an ACcAMPPKA dependent pathway (Margiotta and Pardi, 1995; Pardi and Margiotta, 1999; Liu et al.

2000; Pugh et al. 2009; Jayakar et al. 2014). This capacity for PACAP to enhance nAChR function is hypothesized to be critical in mediating the ST PACAP-induced synaptic plasticity (See figure 5.1). However, given that the LT PACAP-induced synaptic plasticity is independent of PKA signalling, as well as concomitant neuronal activity makes this hypothesis seem unlikely.

Because only 20-35% of the elevation in As* could be attributed to elevations in postsynaptic strength we examined whether the LT PACAP-induced plasticity altered presynaptic ACh release. Quantitative transmission studies, in which presynaptic fascicles converging onto a single CG neuron were stimulated at a limited stimulated intensity, definitively confirmed the involvement of a presynaptic mechanism. In these experiments, calculations of quantal content (m) calculated via the empirical method (me) and the Poisson method of failures (mf) confirmed that the LT PACAP-induced synaptic plasticity doubled the number of vesicles released per presynaptic action potential in comparison to Controls. Subsequent immunocytochemical analysis of presynaptic puncta labelled with SV2 indicated the LT PACAP induced plasticity increased the size of SV2 labelled presynaptic puncta by 37% with no alterations observed in the intensity or density. Theoretically, the number of vesicles released per action potential is a product of two parameters: 1) the number of readily releasable vesicles (푛) contained within

147 presynaptic terminals and 2) the probability (푝) of vesicular release [푚 = 푛(푝)]

(Johnston and Wu, 1995). Synaptic vesicle protein 2 (SV2) is expressed on all synaptic vesicles (Custer et al. 2006) and, therefore the LT PACAP-induced elevations in the size of SV2 labelled presynaptic terminals indicates that PACAP elevates 푛. Even though

PACAP reduced the probability to evoke failures as well as increases the size SV2 labeled presynpatic puncta, both of which are correlated with increased probability of vesicular release (Custer et al. 2006; Branco and Starass, 2009), it is still unknown whether PACAP functionally alters probability of ACh release. Likewise, it is unknown whether the actual physical size of presynaptic terminals or number of active zones were modified by PACAP signaling.

Given that the LT PACAP-induced plasticity is independent from retrograde NO signaling, suggests that these effects are largely mediated by gene transcription. Increased expression of the high affinity choline transporter (HaChT) and choline acetyltransferase

(ChAT) could underlie the LT PACAP induced elevations in n. In gene arrays, PACAP upregulates transcription of both HaChT and ChAT, two enzymes critical for maintaining

ACh levels within presynaptic cholinergic terminals (Nicholls et al. 2012; Purves et al.

2012; Bazalakova and Blakely, 2006). Additionally, PACAP also up-regulates transcription (i.e. >1.5-fold increase) of other presynaptic proteins contained on synaptic vesicles and in active zonesthat mediate vesicular release including synaptotagmin IV, synaptobrevin 2, and RIMs binding protein 2 (Sumner et al. 2009). Taken together, we hypothesize that PACAP-induced elevations in quanal content are likely a product of gene transcription. Thus, future experiments are warranted to examine the relative contributions of these proteins in mediating the LT PACAP-induced synaptic plasticity.

148

In line with PACAPs capacity to functionally induce presynaptic remodelling as well as enhance α3*-nAChR cluster localization to synaptic sites suggests that PACAP signaling directly modulates synapse stability. In CG neuronal cultures, synaptic stability is dependent upon associations between α3*-nAChRs and scaffolding proteins, such as

PSD-95 family members, cytoskeletal stabilizing proteins including contactin binding protein and cortactin, and trans-synaptic proteins (Conroy et al. 2003; Parker et al. 2004;

Neff III et al. 2009; Raab et al. 2010). Gene arrays have verified that acute PACAP application in CG neuronal cultures upregulates transcripts associated with synaptic stability including contactin associated protein like 5, cortactin binding protein 2, and neuron pentraxin 2 precursor as well as downregulates expression of transcripts that promote synaptic destabilization such as the reticulon 4 receptor (Sumner et al. 2009).

Because these respective gene products are all involved in processes related to synapse function, and/or modification (O’brien et al. 1999; Schwab, 2010; Shih et al. 2014; Varea et al. 2015; Pelkey et al. 2015) bolsters the hypothesis that PACAP functionally strengthens synapse stabilization.

Taken together, the results from this dissertation have several implications regarding the functional role of PACAP/PAC1R signaling on the CG and in the nervous system. Ciliary neurons in the avian CG control pupillary light reflex by innervating the iris sphincter muscle, as well as lens accommodation by innervating the ciliaris muscle.

In response to light, CG-mediated neuronal activity causes the iris sphincter muscle to contract, causing the pupil to constrict (Reiner et al. 1983). PAC1R knock out mice exhibit reductions in pupillary light reflex following exposure to medium and high intensity light (Engelund et al. 2012). This effect is specific to the PAC1R as PACAP

149 deficient mice exhibit no alterations in pupillary light reflex (Kawaguchi et al. 2010). In addition to the pupilary light reflex, the CG controls lens accommodation of the eye, in which increased CG neuronal output increases convexivity of lens via contraction of the ciliary muscle “to increase refractive power,” and allow for the lens to focus on nearby objects (McDougal and Gamelin, 2015). Lens accommodation requires rapid stimulation of neurons localized in the the edinger westphal nucleus (EDW) (the accessory oculomotor nucleus in birds), the nucleus that projects preganglionc cholinergic efferents via CN III that innervate the CG. Maximal accommodation achieved via stimulation of the CG was found to occur at a frequency of 20-50 Hz in cats (Marge et al. 1954; Ripps et al. 1961; Gamlin et al. 1994) and in one study, examination of randomly sampled

αEDW neurons during “close accommodation”, were identified to fire at frequencies of between 20-80 Hz, a frequency sufficient to release PACAP from preganglionic terminals

(Tompkins et al. 2009). PACAPs capacity to rapidly enhance α3*- and α7-nAChR sensitivity (Margiotta and Pardi, 1995; Pardi and Margiotta, 1999; Pugh et al. 2009;

Jayakar et al. 2014) suggests that ST PACAP signalling could be maintaining or enhancing local nAChR populations on preganglionic cholinergic synapses, reducing rundown, desensitization, or functionally enhanciong nAChRs-mediated responses on CG neruons during the initiation of the pupillary light reflex or lens accommodation.

Consequently, one implication of rapid PACAP signalling, consistent with PAC1R mediated effects on pupillary light reflex, is to increase the activation of CG neurons in order to strengthen CG neuronal output to muscle targets in the eye.

While the functional implications of PACAP signalling in the CG on pupillary light response and lens accomodation support a role for the ST PACAP-indued synaptic

150 plasticity, the research from this dissertation highlights a developmental implication for

LT PACAP-induced synaptic plasticity on the CG. We have shown that PACAP is expressed throughout avian embryonic development and is an endogenous neurotransmitter to ciliary neurons in the CG (Pugh et al 2007; Pugh et al. 2009). We have demonstrated that CG neurons express PAC1Rs, the activation of which promotes pro-survival signaling pathways, gene transcription and neuromodulatory actions that sustainably potentiate cholinergic synaptic function (Margiotta and Pardi, 1995; Pardi and Margiotta 1999; Pugh et al. 2007; Sumner et al. 2009; Pugh et al., 2009; Jayakar et al. 2014; Starr and Margiotta 2014, 2016). Indeed, our findings described above show that PACAP-induced LT synaptic plasticity at cholinergic synapses results in dynamic synaptic remodelling of both postsynaptic α3*-nAChR clusters and presynaptic terminals. Indeed, PACAP’s capactity to enhance the α3*-nAChR clustering, and localization to peri- and presynpatic sites, indicates that PACAP signaling promotes cholinergic synapse formation, stabilization and development in the CG. Understanding the mechanisms underlying these effects is critical in elucidating potential effectors underlying cholinergic synapse regulation. Likewise, future studies, are also warranted to investigate the role of PACAP/PAC1R signaling in modulating CG synapse function during development and in post-hatched chickens in situ.

In addition to functional and developmental implications on the CG, the capacity for PACAP-induced LT synaptic plasticity has implications for PACAP-induced central nervous system as well. PACAP signalling in the central nervous system has a well- documented capacity to persistently modify behaviour. For example, microinfusions of

PACAP to the ventral medial nucleus of the hypothalamus results in increased

151 thermogenesis, and locomotion as well as decreased appetite for up to 4-6 hours (Resch et al. 2011, 2013, 2014). A single microinfusion of PACAP to the bed nucleus of the stria terminalis enhanced acoustic startle responses of rats for up to seven days as well as reduced appetite for up to 24 h (Hammack et al. 2009; Kocho-Schellenberg et al. 2014).

Intrathecal injection of Maxidilan, a PAC1R agonist, produced long-term mechanical allodynia for 84 days without affecting thermal nociceptive threshold (Yokai et al. 2016).

Likewise, PACAP induces synaptic plasticity in the amygdala, cerebellum, hypothalamus, hippocampus as well as several other regions in the CNS. What the findings from this dissertation show is that PACAP has the capacity to induce LT synaptic plasticity, and thus one implication from these findings is that LT plasticity could underlie these functional PACAP-induced effects following microinfusion/injection in the CNS. While links between upregulation of NMDARs and

AMPARs have been hypothesized (Macdonald et al. 2005, 2007; Costa et al. 2009; Cho et al. 2012; Michel et al. 2006), our study represents the first to demonstrate that PACAP actually does induce long-term synaptic plasticity and that it occurs through physical structural remodelling at cholinergic synaptic sites. Thus, the implications from my studies highlights a clear need for future studies to determine whether PACAP-induced

LT plasticity occurs and is responsible for mediating PACAP-induced effects on behavior in the CNS.

In conclusion, we have identified that PACAP induces a novel LT synaptic plasticity in CG neuronal cultures. We demonstrated that the LT effects were mediated via PAC1R-induced activation of both AC and PLC signalling cascades and required gene transcription. Further, the LT PACAP-induced synaptic plasticity was characterized by

152 increases in both postsynaptic strength and presynaptic transmitter release. In concert with increases in quantal release and quantal size the LT PACAP-induced synaptic plasticity featured significant dynamic synaptic remodelling associated with elevations in the intesnsity, size, and density of α3*-nAChR clusters, increases in the size of presynaptic puncta, and increases in the density and size of synaptic sites (Fig. 5.2).

Consequently, our findings demonstrate that PACAP induces LT synaptic plasticity and suggests that PACAP-induced synaptic plasticity in the CG as well as in other regions in the autonomic and the central nervous system could potentially play a role in functional, and behavioural outcomes.

153

154

Figure 5.5 Proposed hypothetical model of the LT PACAP-induced synaptic plasticity. The cartoons depict a CG neuron projecting its axon and forming three cholinergic synapse with a second CG neuron. A. While untreated CG neurons (Top) exhibit less localization between pre- and postsynpatic densities, PACAP via PAC1R-induced activation of AC and PLC signaling and subsequent gene transcription induces a LT synaptic plasticity (Bottom) that results in functional enalargements in the size of presynaptic terminals, increases in presynaptic transmitter release as well as increases the size of α3*-nAChR clusters and localization of postsynpatic densities to presynaptic puncta 48 hours later.

155

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