A Dissertation Entitled Regulation of Neuronal L-Type Voltage-Gated

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A Dissertation

entitled

Regulation of Neuronal L-type Voltage-Gated Calcium Channels by Flurazepam and

Other Positive Allosteric GABAA Receptor Modulators

Damien E. Earl

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

Doctor of Philosophy Degree in Biomedical Sciences

______Dr. Elizabeth I. Tietz, Committee Chair

______Dr. Zi-Jian Xie, Committee Member

______Dr. David R. Giovannucci, Committee Member

______Dr. Scott C. Molitor, Committee Member

______Dr. Bryan K. Yamamoto, Committee Member

______Dr. Patricia Komuniecki, Dean College of Graduate Studies

The University of Toledo

August 2011

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

Regulation of Neuronal L-type Voltage-Gated Calcium Channels by Flurazepam and Other Positive Allosteric GABAAR Modulators

Damien E. Earl

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biomedical Sciences

The University of Toledo August 2011

Benzodiazpines (BZs) are clinically useful anxiolytics, sedatives, and anticonvulsants.

Their mechanism of action is positive allosteric modulation of γ-aminobutyric acid type

A (GABAA) receptors, the main inhibitory neurotransmitter receptors in the mammalian

central nervous system. Long-term administration of BZs and other positive allosteric

GABAA receptor modulators, neurosteroids, barbiturates, and ethanol can lead to physical dependence manifested by a characteristic withdrawal syndrome. A common mechanism proposed to contribute to this withdrawal syndrome is functional up-regulation of L-type

voltage-gated calcium channels (L-VGCCs). Our lab models BZ dependence using a 1-

week oral treatment of rats with flurazepam (FZP) followed by 1 or 2 days of withdrawal.

This treatment paradigm resulted in a near doubling of voltage-gated Ca2+ currents in

hippocampal CA1 neurons. Enhanced L-VGCC-mediated Ca2+ influx may activate

Ca2+/calmodulin-dependent protein kinase II (CaMKII), which potentiated excitatory

synaptic function in CA1 neurons correlating with expression of FZP withdrawal anxiety.

The current studies tested three hypotheses: 1) GABAA receptor modulators directly

inhibit recombinantly expressed L-VGCCs containing neuronal α1 subunits, Cav1.2 or

Cav1.3; 2) L-VGCC subunit expression is increased in the rat hippocampal CA1 region;

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and 3) CaMKII enhances CA1 excitatory synaptic function via activation and autophosphorylation at Thr286 and/or enhanced localization to the postsynaptic density

(PSD). The findings suggested that while the barbiturate pentobarbital and ethanol

directly inhibit L-VGCCs at clinically relevant concentrations, the concentrations of BZs

and neurosteroids required to inhibit recombinant L-VGCCs were likely too high to be

clinically relevant. Interestingly, Cav1.2 channels were more sensitive to inhibition by

pentobarbital and FZP and were less sensitive to inhibition by the L-VGCC

benzothiazepine (BTZ) antagonist, diltiazem, than Cav1.3 channels. Selective inhibition

2+ could independently block Ca signaling cascades mediated by Cav1.2 and Cav1.3 L-

VGCCs. Mutation studies revealed that the pentobarbital L-VGCC binding site may overlap that of dihydropyridines, and despite structural similarities amongst BTZs and

BZs, the BZ L-VGCC binding site is distinct from that of BTZs. No alteration in L-

VGCC subunit expression was observed in PSD-enriched CA1 homogenates or immunostained hippocampal slices as a function of FZP withdrawal. Taken together, the data suggested that mechanisms other than direct inhibition of L-VGCCs and increased

L-VGCC subunit expression mediate the enhanced Ca2+ influx observed following long-

term FZP treatment. Post-translational modifications and/or enhanced trafficking of L-

VGCCs to the membrane due to persistent BZ enhancement of GABAA receptors are

alternate possibilities. Additionally, after 2 days of FZP withdrawal, total CaMKIIα

expression was decreased in CA1 PSDs with no alteration in the absolute amount of the

autonomously active Thr286 autophosphorylated form of CaMKII. Alternate mechanisms

of CaMKII activation by L-VGCC-mediated Ca2+ influx and for the loss of CaMKII from

PSDs during FZP withdrawal are proposed.

ACKNOWLEDGEMENTS

I am very grateful to Dr. Elizabeth I. Tietz, who has guided and mentored me through

my four years of graduate training. With her constant encouragement and advice I have

overcome many novel challenges and learned from the mistakes I made along the way,

which will prepare me for the challenges that lay ahead. My time in Dr. Tietz’s lab has

enhanced my passion for science and my desire to pursue an academic career.

I thank each of my committee members for their interesting perspectives and

contributions to my training – Dr. L. John Greenfield, Dr. David R. Giovannucci, Dr. Zi-

Jian Xie, Dr. Bryan K. Yamamoto, and Dr. Scott C. Molitor, who provided a critical

review of my first manuscript.

I also thank Drs. William T. Gunning and Francisco J. Alvarez for their advice and mentorship related to electron microscopy, and the EM core staff, Paula Kramer, Scott

Eagle, and Yana Fedotova for their help processing hundreds of negatives. I thank my

fellow lab members, Dr. Paromita Das, Dr. Guofu Shen, Dr. Kun Xiang, Dr. Nathalie

Boulineau, Dr. Liping Wang, Trisha Dwyer, Krista Pettee, Brian Behrle, and Nikki

Wenzlaff for their help and support with the various techniques required for these studies.

Krista Pettee performed one of the immunofluorescent trials and Dr. Boulineau

assisted with confocal imaging. Dr. Das in collaboration with Dr. Alvarez prepared the

tissues used in for CaMKII EM.

I am very grateful for the hard work by our department secretaries, Anita Easterly,

Debra LeBarr, Lisa Akeman, and Martha Heck, who assisted with ordering lab supplies, attending conferences, my NRSA fellowship application, and manuscript submission.

Finally, I would like to thank my family; my parents, Michael and Gigi, my grandparents, Ernest and Sandra, and my sisters, Heather and Skyler. They have been a source of encouragement and inspiration to achieve my goals and be the best person I can be. I also thank my wife Paromita for all of her advice and support over the years. There is no substitute in life for a friend who listens to your thoughts and understands your troubles.

TABLE OF CONTENTS

ABSTRACT ...... iii

ACKNOWLEDGEMENTS ...... v

TABLE OF CONTENTS ...... vii

LIST OF TABLES ...... xii

LIST OF FIGURES ...... xiii

LIST OF ABBREVIATIONS ...... xv

CHAPTER 1 - INTRODUCTION ...... 1

CHAPTER 2 - LITERATURE REVIEW...... 7

2.1 Benzodiazepines and other positive allosteric GABAA receptor modulators ...... 7

2.2 GABAA receptor structure and function ...... 8

2.3 Positive allosteric modulation of the GABAA receptor ...... 11

2.4 Additional sites of action GABAA receptor modulators ...... 15

2.5 Withdrawal from GABAA receptor modulators...... 18

2.6 The hippocampus and its role in drug dependence ...... 20

2.7 Hippocampal GABAergic network...... 24

2.8 Molecular substrates of drug dependence: Role of GABAA receptor

dysfunction ...... 25

2.9 Molecular substrates of drug dependence: Role of ionotropic glutamate

receptors ...... 32

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2.10 Molecular substrates of drug dependence: Role of L-type voltage gated

calcium channels ...... 38

2.11 Regulation of L-VGCCs ...... 41

CHAPTER 3 - INHIBITION OF RECOMBINANT L-TYPE VOLTAGE-

GATED CALCIUM CHANNELS BY POSITIVE ALLOSTERIC

MODULATORS OF GABA-A RECEPTORS ...... 44

3.1 Abstract ...... 45

3.2 Introduction ...... 47

3.3 Materials and Methods ...... 49

3.3.1 Cell culture and transient transfection ...... 49

3.3.2 Site-Directed Mutagenesis ...... 49

3.3.3 Recording solutions ...... 50

3.3.4 Whole-cell voltage-clamp electrophysiology ...... 51

3.3.5 Drugs ...... 53

3.3.6 Statistical analyses ...... 54

3.4 Results ...... 55

3.4.1 Functional Characterization of Recombinant L-VGCCs ...... 55

3.4.2 GABAAR Modulators Reversibly Inhibit Recombinant L-VGCCs ...... 55

3.4.3 GABAAR Receptor Modulators Enhance L-VGCC Current Decay ...... 57

3.4.4 GABAAR Modulators Induce a Negative Shift in L-VGCC Steady-

State Inactivation ...... 57

3.4.5 Inhibition of Cav1.2 Channels by Desalkylflurazepam is State- and

Frequency-Dependent ...... 59

viii

3.4.6 Two Amino Acids Important for DHP Potency Reduce Pentobarbital

Potency ...... 59

3.4.7 A Single Amino Mutation that Affects Diltiazem Potency does not

Affect Diazepam Potency ...... 61

3.5 Discussion ...... 62

3.5.1 GABAAR Modulator Pharmacology at L-VGCCs: Comparison to

GABAARs ...... 62

3.5.2 GABAAR Modulator Manner of L-VGCC Inhibition: Comparison to

L-VGCC Antagonists...... 62

3.5.3 Cav1.2 and Cav1.3 L-VGCC Differential Sensitivity to GABAAR

Modulators ...... 64

3.5.4 GABAAR Modulator Site of Action ...... 65

3.5.5 Clinical Relevance of L-VGCC Inhibition by GABAAR Modulators

and Implications for Physical Dependence ...... 65

3.6 Footnotes ...... 69

3.7 Acknowledgements ...... 70

3.8 References ...... 71

3.9 Figures and Tables ...... 77

CHAPTER 4 - CA2+/CALMODULIN-DEPENDENT PROTEIN KINASE II

LOCALIZATION AND AUTOPHOSPHORYLATION WITHIN

HIPPOCAMPAL CA1 EXCITATORY POSTSYNAPSES DURING

FLURAZEPAM WITHDRAWAL ...... 98

4.1 Abstract ...... 100

4.2 Introduction ...... 102

4.3 Materials and Methods ...... 106

4.3.1 Long-term FZP treatment ...... 106

4.3.2 Antibodies and specificity...... 106

4.3.2.1 Antibodies used for immunoblot and immunofluorescent

staining ...... 106

4.3.2.2 Antibodies used for post-embedding immunogold electron

microscopic analysis ...... 108

4.3.3 CA1 minislice subcellular fractionation ...... 109

4.3.4 Western blot ...... 110

4.3.5 Hippocampal slice confocal immunofluorescence ...... 110

4.3.6 Post-embedding immunogold electron microscopy ...... 111

4.3.7 Statistical analyses ...... 113

4.4 Results ...... 114

4.4.1 Unaltered L-VGCC subunit expression in CA1 PSD-enriched

fraction during FZP-withdrawal ...... 114

4.4.2 CaMKII-α expression, but not Thr286 autophosphorylation is reduced

in FZP-withdrawn CA1 asymmetric synapses ...... 116

4.5 Discussion ...... 119

4.5.1 Molecular basis of FZP-induced enhancement of CA1 VGCC

function ...... 119

4.5.2 Molecular basis of CaMKII activation during FZP withdrawal ...... 120

4.5.3 Discrepancies with prior studies ...... 122

4.6 Footnotes ...... 124

4.7 Acknowledgements ...... 125

4.8 References ...... 126

4.9 Figures and Tables ...... 134

CHAPTER 5 - CONCLUSIONS AND SUMMARY ...... 149

5.1 Conclusions ...... 149

5.2 Summary and inferences ...... 151

REFERENCES ...... 161

LIST OF TABLES

3.1 Parameters for concentration-dependent inhibition of L-VGCCs by diltiazem

and GABAAR modulators

3.2 GABAAR modulators enhance L-VGCC current decay

3.3 Negative shift in L-VGCC steady-state inactivation by diltiazem and

GABAAR modulators

4.1 CaMKIIα immunogold synaptic labeling

4.2 pCaMKII immunogold synaptic labeling

4.3 CaMKIIα and pCaMKII non-PSD immunogold synaptic labeling

xii

LIST OF FIGURES

3.1 Chemical structures of the compounds used in the current studies (except for

ethanol)

3.2 Functional expression of recombinant L-VGCCs in HEK293T cells

2+ 3.3 Concentration-dependent inhibition of Cav1.3 Ba currents by diazepam, and

normalized, rundown-corrected time-courses of Cav1.2 and Cav1.3 L-VGCC

inhibition by diltiazem and GABAA receptor modulators

3.4 Concentration-response curves representing inhibition of Cav1.2 and Cav1.3 L-

VGCCs by diltiazem and GABAAR modulators

3.5 Diazepam enhances L-VGCC current decay

3.6 L-VGCC voltage-dependent steady-state inactivation curves in the presence and

absence of GABAAR modulators

3.7 Cav1.2 L-VGCCs are inhibited by desalkylflurazepam in a state- and frequency-

dependent manner

3.8 The sensitivity of dihydropyridine-insensitive Cav1.2 (DHPI) L-VGCCs to

GABAAR modulators

3.9 A single amino acid mutation in Cav1.2, I1150A, reduces diltiazem but not

diazepam potency, and diazepam-mediated Cav1.2 inhibition is not antagonized

by diltiazem

4.1 Cav1.2 and Cav1.3 immunoblot analysis of subcellular fractions created from CA1

homogenates

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4.2 Unaltered L-VGCC subunit expression in the PSD-enriched fraction from 1-day

and 2-day FZP-withdrawn rats

4.3 Unaltered Cav1.2 subunit expression detected by immunofluorescent staining of

hippocampal slices from 2-day FZP-withdrawn rats

4.4 CaMKII and Thr286 autophosphorylated CaMKII expression in CA1 SR

asymmetric synapses of control and 2-day FZP-withdrawn rats

4.5 Pre- and postsynaptic distribution of CaMKII and pCaMKII immunogold

particles in CA1 asymmetric synapses of control and 2-day FZP-withdrawn rats

4.6 Distribution histograms of asymmetric synaptic profiles containing different

numbers of CaMKII and pCaMKII immunogold particles within the PSD

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LIST OF ABBREVIATIONS

5-HT serotonin AMPA α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ANOVA analysis of variance BDZ benzodiazepine BTZ benzothiazepine BZ benzodiazepine 2+ 2+ [Ca ]i intracellular Ca concentration CA1-4 cornu ammonis fields 1-4 CaM calmodulin CaMKII Ca2+/calmodulin-dependent protein kinase II CNS central nervous system DHP dihydropyridine DMSO dimethyl sulfoxide EDTA ethylenediaminetetraacetic acid EGTA ethylene glycol tetraacetic acid E-LTP early phase long-term potentiation FZP flurazepam GABA γ-aminobutyric acid GCL granule cell layer Gmax maximal conductance HEK human embryonic kidney HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HVA high voltage-activated IC50 half-maximal inhibitory concentration ID integrated density KCC2 K+-Cl− cotransporter 2 LB Luria-Bertani L-LTP late phase long-term potentiation LTP long-term potentiation L-VGCC L-type voltage-gated calcium channel mEPSC miniature excitatory postsynaptic current mIPSC miniature inhibitory postsynaptic current ML molecular layer mRNA messenger ribonucleic acid NKCC1 Na+-K+-Cl− cotransporter 1 NMDA N-methyl-D-aspartate PAA phenylalkylamine PDZ postsynaptic density-95/Discs large/zona occludens-1 PEG poly(ethylene) glycol

PKA protein kinase A or cyclic adenosine monophosphate-dependent protein kinase PKC protein kinase C PKG protein kinase G or cyclic guanosine monophosphate-dependent protein kinase PP2A phosphatase 2A PP2B phosphatase 2B or calcineurin PSD postsynaptic density SLM stratum lacunosum-moleculare SP stratum pyramidale SO stratum oriens SR stratum radiatum TBS tris-buffered saline TBST tris-buffered saline with 0.1% Tween 20 TEA tetraethylammonium TSPO translocator protein 18 kDa V50 membrane potential of half-maximal activation or inactivation Vh holding potential Vrev reversal potential Vt test potential WT wild-type

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CHAPTER 1

INTRODUCTION

Benzodiazepines (BZs) are clinically useful anxiolytics, sedative-hypnotics, and

anticonvulsants attributable to positive allosteric modulation of γ-aminobutyric acid type

A (GABAA) receptors, the primary inhibitory neurotransmitter receptors in the

mammalian central nervous system (CNS). However, the clinical usefulness of BZs is

limited during long-term treatment by the development of tolerance to their sedative and

anticonvulsant effects (File, 1985; Rosenberg & Chiu, 1985; Rosenberg et al., 1991) and physical dependence manifested by a characteristic withdrawal syndrome (Griffiths &

Johnson, 2005). BZ withdrawal involves a number of symptoms including anxiety and

insomnia (Griffiths & Johnson, 2005). Though with a lesser safety margin, similar

anxiolytic, sedative-hypnotic, and anticonvulsant effects are by and large seen with other

less selective positive allosteric GABAA receptor modulators including neurosteroids,

barbiturates, and ethanol. Further, although the withdrawal syndrome associated with

each of these modulators includes symptoms similar to that of BZ withdrawal such as

anxiety, withdrawal symptoms are often much more severe for barbiturates and ethanol

(Hodding et al., 1980; Smith, 2002).

In animal models of the drug withdrawal phenomenon, anxiety and drug-induced

seizure susceptibility are commonly measured outcomes (Emmett-Oglesby et al., 1990;

Chaix et al., 2007). Our lab studies tolerance and withdrawal phenomena in rats using a

model in which the water-soluble BZ flurazepam (FZP) is administered orally for 1 week followed by drug withdrawal. This treatment paradigm consistently results in withdrawal

anxiety after 1 day of FZP withdrawal (Van Sickle et al., 2004; Xiang & Tietz, 2007).

Due to the interaction of BZs with a binding site on GABAA receptors, many studies of BZ tolerance and withdrawal mechanisms have focused on the GABAergic system.

These studies have shown that while dysfunction of GABAergic transmission may underlie tolerance to BZ effects (Tietz et al., 1986; Xie & Tietz, 1992; Zeng et al., 1995;

Zeng & Tietz, 2000), dysfunction of both GABAergic and glutamatergic systems may be involved in the mechanisms of BZ physical dependence (Izzo et al., 2001; Allison &

Pratt, 2003). Glutamate is the main excitatory neurotransmitter in the CNS and activates postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N- methyl-D-aspartate (NMDA) receptors, which respond by depolarizing the postsynaptic membrane and mediating Ca2+ influx. FZP-induced withdrawal anxiety correlated with

the progressive potentiation of AMPA receptor-mediated currents in hippocampal CA1

neurons (Van Sickle et al., 2004; Xiang & Tietz, 2007). AMPA receptor potentiation was

observed after 1 and 2 days of withdrawal, but not immediately after FZP treatment (Van

Sickle et al., 2004). A concomitant increase in GluA1, but not GluA2 subunit expression

in the soma and proximal apical dendrites of CA1 neurons and postsynaptic incorporation of homomeric GluA1 AMPA receptors was observed during FZP withdrawal (Van Sickle et al., 2004; Song et al., 2007; Das et al., 2008; Shen et al., 2010).

In models of long-term potentiation (LTP) in the CA1 region, synaptic strength is enhanced in an activity-dependent manner requiring Ca2+ influx via NMDA receptors or

L-type voltage-gated calcium channels (L-VGCCs) dependent on the stimulation intensity and pathway (Grover & Teyler, 1990; Remondes & Schuman, 2003).

Interestingly, high voltage-activated (HVA) Ca2+ channel currents were enhanced in CA1

neurons immediately after FZP treatment and continued through 2 days of withdrawal

(Xiang et al., 2008). Moreover, systemic pre-injection on an L-VGCC antagonist, but not

an NMDA receptor antagonist prevented withdrawal anxiety and AMPA receptor

potentiation after 1 and 2 days withdrawal (Van Sickle et al., 2004; Xiang & Tietz, 2007;

Xiang et al., 2008). Thus, Ca2+ influx through L-VGCCs appears to contribute to AMPA

receptor potentiation and withdrawal anxiety following long-term FZP treatment. A role

for up-regulated L-VGCC function has also been proposed to contribute to physical

dependence on barbiturates (Rabbani et al., 1994; Rabbani & Little, 1999) and ethanol

(Messing et al., 1986; Whittington et al., 1991; Katsura et al., 2006).

The mechanism of enhanced neuronal L-VGCC function following long-term

treatment with various GABAA receptor modulators is currently unknown. It may be

related to the ability of BZs (Yamakage et al., 1999; Xiang et al., 2008) and other

GABAA receptor modulators, such as the neurosteroid, allopregnanolone (Hu et al.,

2007), the barbiturate, pentobarbital (ffrench-Mullen et al., 1993), and ethanol (Messing

et al., 1986) to directly inhibit L-VGCCs. Direct inhibition during FZP treatment would

also attenuate downstream consequences of L-VGCC-mediated Ca2+ influx, explaining

why AMPA receptor function is unaffected until withdrawal even though L-VGCC

currents are enhanced during treatment. Although a direct interaction has been proposed,

no studies have investigated the effects of GABAA receptor modulators on channels

composed of either of the two subtypes of neuronal L-VGCCs, Cav1.2 and Cav1.3. In

particular, expression in a heterologous system would isolate L-VGCC subtypes from

GABAA receptors and other VGCCs, such as N-, P/Q-, R- and T-type (Catterall et al.,

2005). Moreover, Cav1.2 channels were found to be more sensitive than Cav1.3 channels

to inhibition by the well-characterized organic L-VGCC antagonists, dihydropyridines

(DHPs), benzothiazepines (BTZs), and phenylalkylamines (PAAs) (Koschak et al., 2001;

Xu & Lipscombe, 2001; Tarabova et al., 2007), suggesting that GABAA receptor modulators might also differentially inhibit L-VGCC subtypes. In particular, there are structural similarities amongst BZ and BTZ compounds. Therefore, the first aim of the current studies was to test the hypothesis that GABAA receptor modulators inhibit recombinantly expressed Cav1.2 and/or Cav1.3-containing L-VGCCs at concentrations achieved in vivo during long-term drug exposure.

The mechanistic basis of enhanced voltage-dependent Ca2+ influx is also currently

unknown. It could be due to increased expression of L-VGCC subunits, trafficking of L-

VGCCs to the plasma membrane from intracellular compartments, or post-translational

modifications, such as phosphorylation that enhance L-VGCC currents. Previous studies indicated an increased number of radiolabeled L-VGCC antagonist binding sites on neuronal membranes following long-term exposure to BZs (Katsura et al., 2007),

barbiturates (Rabbani & Little, 1999), and ethanol (Messing et al., 1986; Whittington et al., 1991; Katsura et al., 2006), supporting increased plasma membrane expression of L-

VGCCs. Moreover, one lab detected increased Cav1.2 and Cav1.3 subunit expression in

cerebral cortical neurons following prolonged BZ or ethanol exposure in vitro and in vivo

(Katsura et al., 2006; Katsura et al., 2007). Studies from our lab revealed a negative shift

in the voltage-dependence of HVA Ca2+ currents following long-term FZP exposure

(Xiang et al., 2008), suggesting that Cav1.3 in particular might be up-regulated, since it activates at relatively negative membrane potentials compared to other HVA Ca2+ channels (Koschak et al., 2001; Xu & Lipscombe, 2001). Additionally, since Cav1.2 and

Cav1.3 L-VGCCs are expressed near PSDs (Zhang et al., 2005; Day et al., 2006; Tippens

et al., 2008; Zhang et al., 2008; Leitch et al., 2009), increased Cav1.3 subunit expression

near PSDs could mediate the AMPA receptor potentiation seen during FZP withdrawal.

Accordingly, the second aim was to test the hypothesis that Cav1.3 subunit expression is

increased near hippocampal CA1 PSDs during FZP withdrawal. Based on evidence of

increased Cav1.2 expression in cerebral cortex following long-term BZ exposure (Katsura et al., 2007) and the fact that α1 subunit phosphorylation can also enhance L-VGCC

currents (Oliveria et al., 2007), the current studies additionally assessed Cav1.2 subunit expression and phosphorylation state in the hippocampus during FZP withdrawal.

One of the downstream consequences of up-regulated L-VGCC function near PSDs may be enhanced Ca2+-mediated activation of Ca2+/calmodulin-dependent protein kinase

II (CaMKII), a holoenzyme formed from up to 12 CaMKIIα and CaMKIIβ molecules

(Bennett et al., 1983). In particular, after homomeric GluA1 AMPA receptors are inserted

into CA1 PSDs during the first day of FZP withdrawal, increased AMPA receptor

conductance was observed after 2 days of withdrawal attributable to phosphorylation of

GluA1 at Ser831 by CaMKII (Shen et al., 2009; Shen et al., 2010). During NMDA

receptor-mediated LTP, Ca2+ influx through NMDA receptors leads to autophosphorylation of CaMKIIα at Thr286 and CaMKIIβ at Thr287. CaMKII

autophosphorylation results in Ca2+-independent, autonomous CaMKII activity, release

of the CaMKII holoenzyme from the actin cytoskeleton, and translocation to binding

partners in the PSD where it mediates phosphorylation of GluA1 homomers (Fink &

Meyer, 2002). However, during FZP withdrawal, although total CaMKIIα, but not

CaMKIIβ expression was increased in CA1 homogenates, including a PSD-enriched

subcellular fraction, there was no corresponding alteration in the level of Thr286/287 autophosphorylated CaMKII (Shen et al., 2010). This suggested that Ca2+ influx through

L-VGCCs may lead to autonomous CaMKII activation and/or translocation to the PSD

via a different upstream mechanism compared to Ca2+ influx through the NMDA

receptor. It is also possible that similar CaMKII autophosphorylation and translocation

does occur during FZP withdrawal, but that it was not detectable in PSD-enriched

homogenates. Thus, the third aim tested the hypothesis that total and autophosphorylated

CaMKIIα translocates to the PSD of asymmetric synapses located in CA1 neuron

proximal apical dendrites during the second day of FZP withdrawal.

CHAPTER 2

LITERATURE REVIEW

2.1 Benzodiazepines and other positive allosteric GABAA receptor modulators

The first BZ, chlordiazepoxide, was discovered in the 1950’s by Leo Sternbach as a psychoactive compound with sedative-hypnotic, anticonvulsant, and muscle relaxant effects (Sternbach, 1972). Following clinical trials that verified its safety and efficacy as an anxiolytic and sedative-hypnotic drug, it was introduced into clinical practice in 1960.

In the years that followed, numerous other BZs with varying pharmacokinetic and pharmacodynamic properties were introduced for the treatment of anxiety disorders, insomnia, muscle spasms, seizures, as well as alcohol dependence. Due to a combination of sedative and amnestic effects, BZs are also used as premedication for medical and surgical procedures (Dailey, 1990).

Similar behavioral effects are observed for neurosteroids, barbiturates, and ethanol.

Like BZs, these drugs are positive allosteric modulators of GABAA receptors. Thus, they enhance the inhibitory action of GABA in the CNS, attributing to their clinical depressant effects. These agents are classified as “sedative-hypnotics” or “CNS depressants,” although numerous other drugs with mechanisms of action distinct from modulation of

GABAA receptors are also grouped into this category. Prior to the introduction of BZs into clinical practice, barbiturates were the treatment of choice for anxiety disorders,

insomnia, and seizures. However, like ethanol, barbiturates have a high incidence of

severe side effects, which may be related to their greater number of molecular targets at

therapeutic concentrations. Due to the relative safety and efficacy of the more selective

BZs, they have largely supplanted barbiturates in clinical practice.

2.2 GABAA receptor structure and function

GABA is the major inhibitory neurotransmitter in the mammalian CNS. It is

synthesized from glutamate in GABAergic neurons by the enzyme glutamate

decarboxylase. Release of GABA from nerve terminals activates both pre- and postsynaptic receptors. GABA is removed from the extracellular space by reuptake into both neurons and glial cells via high affinity transporters and is broken down by GABA- transaminase. GABA activates both ionotropic GABAA and metabotropic GABAB receptors. GABAB receptors are expressed both pre- and postsynaptically. They are G

protein-coupled receptors that inhibit presynaptic N- and P/Q-type VGCCs (Bussieres &

El Manira, 1999), enhance postsynaptic L-VGCCs (Bray & Mynlieff, 2009), or

hyperpolarize the membrane via activation of K+ channels (Innis et al., 1988). However,

GABAB receptors are structurally distinct from GABAA receptors and not considered

pharmacologic targets of GABAA receptor modulators (Chebib & Johnston, 1999).

GABAA receptors are members of a Cys-loop superfamily of ligand-gated ion channels, which also includes inhibitory glycine receptors, and excitatory nicotinic acetylcholine and serotonin (5-HT3) receptors. Each subunit of a Cys-loop receptor

consists of a large extracellular N-terminal domain, which contains the conserved

disulfide-linked cysteine residues separated by a 13-amino acid spacer, four α-helical

transmembrane domains (M1-4), and a short extracellular C-terminal domain. The large

N-terminal region is an asymmetric domain made of β-sheets with minus and plus sides connected by the disulfide-linked Cys-loop. Each side is divided into three loops. Loops

A-C form the plus side and loops D-F form the minus side. The minus side of one subunit always faces the plus side of an adjacent subunit. Ligands of Cys-loop receptors bind to a conserved region within loops A-F at the interface between two adjacent subunits (Sigel,

2002; Ernst et al., 2005; Miller & Smart, 2010). The structurally conserved M2

transmembrane helix of each subunit forms an anion or cation channel pore (Ernst et al.,

2005).

GABAA receptors are assembled from a combination of 19 subunits encoded by

separate genes: α1-6, β1-3, γ1-3, δ, ε, θ, π, and ρ1-3. ρ subunits form receptors sometimes referred to as GABAC receptors based on their unique pharmacology, but are classified as

a type of GABAA receptor (Barnard et al., 1998; Olsen & Sieghart, 2008). The large majority of GABAA receptors are formed from two copies of an α, two copies of a β, and

one copy of either a γ2 or δ subunit, with (α1)2(β2)2γ2 representing the most common subunit composition in the brain (McKernan & Whiting, 1996). The primary differences between γ2 and δ subunit-containing GABAA receptors are their subcellular localization,

affinity for GABA, and decay kinetics. The γ2 subunit mediates synaptic expression, usually when combined with α1, α2, or α3 subunits (Olsen & Sieghart, 2008). Synaptic receptors have a lower affinity for GABA and fast desensitization (channel closure with ligand still bound), appropriate for phasic responses to the millimolar GABA concentrations that occur in the synaptic cleft during neurotransmission (Clements, 1996;

Jones & Westbrook, 1996). The γ2 subunit is also expressed extrasynaptically, especially when combined with α4, α5, or α6 subunits (Olsen & Sieghart, 2008). These receptors

may have similar or higher affinity for GABA compared to synaptic GABAA receptors,

sometimes dependent on the β subunit expressed (Ebert et al., 1994; Saxena &

Macdonald, 1996; Wafford et al., 1996). The δ subunit is expressed exclusively

extrasynaptically and combines with mainly α4 and α6 subunits (Olsen & Sieghart,

2008). In addition, GABAA receptors containing the δ subunit display an even higher

affinity for GABA (5- to 25-fold) and slower desensitization kinetics compared to

equivalent receptors containing a γ2 subunit (Saxena & Macdonald, 1994; Saxena &

Macdonald, 1996; Brown et al., 2002; Wallner et al., 2003), which is appropriate for

tonic responses to ambient micromolar concentrations of GABA (Lerma et al., 1986).

Most GABAA receptors contain two binding sites for GABA, at the interface between

the α and β subunits. Binding of both GABA molecules dramatically increases the open

− − probability of the associated anion channel, which is permeable to both Cl and HCO3 , among other anions. The relative permeability to these anions varies depending on the

− − subunit composition ranging from 0.2 − 0.4 (HCO3 /Cl ) (Bormann et al., 1987; Kaila,

1994). In most mature neurons, this results predominantly in Cl− influx and

− hyperpolarization of the postsynaptic neuron, though a small HCO3 efflux accounts for a

− GABAA receptor reversal potential more positive than that for Cl alone (Kaila & Voipio,

1987; Kaila et al., 1993). In some cases, such as in embryonic neurons and during certain

− − pathologic conditions, a relatively large intracellular Cl concentration ([Cl ]i) shifts the

− − reversal potential in a depolarizing direction resulting in Cl /HCO3 efflux and neuronal

− depolarization (Staley & Proctor, 1999; Blaesse et al., 2009). The [Cl ]i is maintained by

cation-Cl− cotransporters (CCCs). During early postnatal development there is a

physiologic switch in the GABA reversal potential from depolarizing to hyperpolarizing

due to decreased expression of the Cl−-accumulating CCC, Na+-K+-Cl− cotransporter 1

(NKCC1) and increased expression of the neuron-specific Cl−-extruding CCC, K+-Cl− cotransporter 2 (KCC2) (Plotkin et al., 1997; Rivera et al., 1999; Dzhala et al., 2005).

Any alteration in the expression or activity of these transporters can result in GABAA receptor dysfunction via a shift in the Cl− reversal potential (Staley et al., 1995; Dzhala et al., 2005).

2.3 Positive allosteric modulation of the GABAA receptor

Although the clinical effects of BZs were discovered in the 1950’s, it was not until

the early 1980’s that a high affinity CNS BZ site was identified (Mohler & Okada, 1977)

and a connection with the GABA receptor was established (Gavish & Snyder, 1981). The

BZ binding site has since been characterized as homologous to the GABA binding site, but between the α and γ subunits (Sigel, 2002; Olsen & Sieghart, 2008). Upon binding,

BZs enhance the affinity of the receptor for GABA, which results in an increase in the

frequency of channel opening and mean channel open time (Study & Barker, 1981;

Lavoie & Twyman, 1996). BZs are allosterically coupled to GABA, in that GABA

binding also enhances the affinity of BZs (Klein & Harris, 1996). Studies using

photoaffinity labeling identified a conserved histidine residue in α1 (His101), α2 (His101),

α3 (His126), and α5 (His105) subunits that plays a critical role in BZ binding (Sigel, 2002).

The analogous amino acid in α4 and α6 subunits is an arginine residue, making GABAA receptors formed from these subunits insensitive to BZ agonists (Mohler et al., 2004).

Consistently, mutation of this histidine residue in α1/α2/α3/α5 subunits to an arginine renders the GABAA receptor insensitive to BZ agonists (Benson et al., 1998). Knock-in

mice containing this mutation in α1 subunits showed reduced sedative, anticonvulsant,

and amnestic effects by diazepam, but are still sensitive to its anxiolytic and muscle

relaxant effects (Rudolph et al., 1999; McKernan et al., 2000). α2-H101R knock-in mice

lacked sensitivity to the anxiolytic and muscle relaxant effects by diazepam, but retained

sensitivity to its sedative and anticonvulsant effects (Low et al., 2000; Crestani et al.,

2001). α3-H126R knock-in mice showed a partial reduction in muscle relaxant effects of

diazepam at high doses, but retained sensitivity to the anxiolytic, sedative, and

anticonvulsant effects (Low et al., 2000; Crestani et al., 2001). α5-H105R knock-in mice

displayed reduced muscle relaxant effects, but retained anxiolytic, sedative, and

anticonvulsant effects of diazepam (Crestani et al., 2002). In addition, α5-H105R knock-

in mice showed a deficit in α5-containing extrasynaptic GABAA receptor expression in

hippocampal dendrites and a corresponding enhancement in associative learning

(Crestani et al., 2002), and α5 knock-out mice displayed enhancement in hippocampus-

dependent spatial memory tasks (Collinson et al., 2002). Collectively, the findings

strongly support a role for α1 subunits mediating the sedative and some of the

anticonvulsant effects, α2 subunits in mediating the BZ anxiolytic effects, α2, α3, and α5

subunits mediating the muscle relaxant effects, and α1 and possibly α5 subunits

mediating the amnestic effects of BZs.

Other GABAA receptor modulators exert their actions at sites distinct from BZs, and

these sites are allosterically linked to each other. For example, neurosteroids and

barbiturates enhance BZ binding (Klein & Harris, 1996). Amino acid residues within the

transmembrane domains of α and β subunits are important for GABAA receptor sensitivity to neurosteroids (Mitchell et al., 2008), barbiturates (Olsen & Sieghart, 2008), and ethanol at higher concentrations (Wallner et al., 2006a). Moreover, δ-containing

GABAA receptors displayed greater efficacy to neurosteroids and barbiturates (Brown et

al., 2002) and greater potency to ethanol (Wallner et al., 2003) compared with γ2-

containing receptors. The greater sensitivity of δ-containing receptors likely stems from

the fact that these receptors display low GABA efficacy compared to γ2-containing receptors allowing more room for positive modulation of GABA efficacy (Olsen &

Sieghart, 2008).

Despite the commonalities, these compounds appear to have distinct binding sites within GABAA receptor subunits. Neurosteroids produce GABA-enhancing effects at

lower, nanomolar concentrations and directly activate GABAA receptors at higher,

micromolar concentrations (Lambert et al., 1987; Hosie et al., 2006). The mechanism of

the neurosteroid GABA-enhancing effect involves increased time spent in longer duration

openings, without increasing single channel conductance or GABA affinity (Peters et al.,

1988; Twyman & Macdonald, 1992). The neurosteroid binding pocket that mediates the

GABA-enhancing action is likely located in M1 (Gln241) and M4 (Asn407 and Tyr410) of α subunits. Direct activation of GABAA receptors requires binding of a second neurosteroid

molecule to a site within M1 (Thr236) of α subunits and M3 (Tyr283) of β subunits (Hosie

et al., 2006). Interestingly, these studies were performed using γ-containing GABAA receptors. The presence of the δ subunit enhanced neurosteroid efficacy, but not apparent affinity (Brown et al., 2002), which is likely related to enhanced allosteric modulation of

GABA efficacy rather than neurosteroid binding.

Barbiturates also produce GABA-enhancing effects at lower, micromolar concentrations and directly activate GABAA receptors at higher, sub-millimolar

concentrations (Schulz & Macdonald, 1981). Also like neurosteroids, the GABA-

enhancing action involves increased time spent in longer duration openings, without

increasing single channel conductance or GABA affinity (Peters et al., 1988; MacDonald

et al., 1989; Steinbach & Akk, 2001; Feng et al., 2004). The barbiturate binding sites are likely located in the β subunit transmembrane domains, since mutation of Pro228 in M1 of

β, but not α subunits reduces the GABA-enhancing effects of barbiturates, but not diazepam or the neurosteroid, alphaxalone (Greenfield et al., 2002). The direct activation of GABAA receptors by barbiturates is dependent upon residues downstream from the

middle of M2 of β subunits based on chimeric studies (Serafini et al., 2000). It should be

noted that as with mutations affecting neurosteroid actions, it cannot be ruled out that

these mutations affect the ability of barbiturates to allosterically modulate channel

function, rather than affecting barbiturate binding. Binding studies are also not

conclusive, as mutations can allosterically affect ligand binding affinity without being

part of the binding site themselves (Greenfield et al., 2002). However, the α and β subunit

transmembrane mutations affecting neurosteroid affinity are ideally positioned to

accommodate hydrogen-bonding predicted to mediate neurosteroid binding (Hosie et al.,

2006; Mitchell et al., 2008). In addition, the lack of evidence supporting a role for α

subunits in mediating barbiturate affinity would seem to indicate a site of action solely

within β subunits. Thus, the neurosteroids and barbiturates seem to act at distinct GABAA receptor binding sites, consistent with results of earlier studies that found a lack of competitive interaction (Gee et al., 1988; Peters et al., 1988).

Similar to neurosteroids and barbiturates, ethanol increased mean GABAA receptor

open time via increased time spent in longer duration openings without a change in single channel conductance, but additionally increased the frequency of channel opening

(Tatebayashi et al., 1998). In addition to a low affinity ethanol binding site, likely within

M2 and M3 of α and β subunits (Mihic et al., 1997; Ueno et al., 1999; Wallner et al.,

2006a), a high affinity ethanol binding site may overlap that of the BZ inverse agonist,

Ro15-4513. This was demonstrated by the fact that Ro15-4513 antagonized ethanol’s

low-dose intoxicating and GABA-enhancing effects, and ethanol antagonizes [3H]-Ro15-

4513 binding to recombinant α4β3δ, but not α4β3γ GABAA receptors (Wallner et al.,

2006b). Additionally, α6-R100Q mutation resulted in enhanced sensitivity of GABAA receptors to ethanol and enhanced low-dose intoxicating effects of ethanol (Hanchar et al., 2005), suggesting that Arg100 of α4/6 subunits, in conjunction with adjacent residues

in the δ subunit, may be important for high-affinity ethanol binding and/or allosteric effects. However, there are numerous labs that were unable to replicate the enhancement of δ-containing GABAA receptors by low ethanol concentrations (Borghese & Harris,

2007; Harris et al., 2008), and the existence of a high-affinity ethanol site that explains

threshold intoxication at low millimolar concentrations remains controversial.

2.4 Additional sites of action of GABAA receptor modulators

Though many of the clinical effects of BZs can be explained by their action at

GABAA receptors, other sites of action are also implicated. At clinically relevant free

plasma concentrations (nanomolar range), some BZs may also act at a peripheral BZ

receptor (Szewczyk & Wojtczak, 2002), now known as translocator protein 18 kDa

(TSPO). TSPO is located in the outer mitochondrial membrane and, among other things,

is involved with steroid transport from the outer to inner mitochondrial membrane, the

rate-limiting step in the synthesis of steroid hormones and neurosteroids (Rupprecht et al., 2010). In the CNS, TSPO is known to play a critical role in the formation of

neurosteroids (Papadopoulos et al., 2006), and long-term diazepam treatment in rats

resulted in decreased stress-induced production of the neurosteroid, allopregnanolone

(Wilson & Frye, 1999), which may have been mediated by diazepam’s interaction with

TSPO.

At higher, micromolar concentrations BZs inhibit VGCCs in both neuronal (Reuveny

et al., 1993; Ishizawa et al., 1997; Xiang et al., 2008) and non-neuronal cells (Yamakage

et al., 1999; Hara et al., 2001). Inhibition of L-VGCCs present in cardiac myocytes and

arterial smooth muscle cells could be the cause of reduced ventricular force and

hypotension observed during intravenous injection of BZs (Hara et al., 2001). L-VGCCs

have several functions in neuronal cells, including regulation of firing frequency,

intracellular signaling cascades affecting activity of other receptors and channels and

transcriptional activity (Jones, 1998; Deisseroth et al., 2003). Therefore, inhibition of

neuronal L-VGCCs by BZs could affect neuronal excitability and gene expression.

In addition to their action at the GABAA receptor, neurosteroids, barbiturates, and

ethanol also modulate activity of other receptors and channels at various concentrations.

Like BZs, allopregnanolone does not appear to have many alternate molecular targets

other than GABAA receptors at physiologically relevant nanomolar brain concentrations.

At higher, sub-micromolar concentrations neurosteroids have been shown to inhibit

VGCCs (ffrench-Mullen et al., 1994; Hu et al., 2007), possibly via activation of a G protein-coupled signaling cascade leading to activation of protein kinase C (PKC)

(ffrench-Mullen et al., 1994). Inhibition by neurosteroids could target presynaptic

VGCCs and release of neurotransmitter, as well as postsynaptic L-VGCCs and corresponding effects on neuronal excitability and gene expression as noted above. The

activation of PKC may be an additional mechanism to modulate GABAA receptors,

which might even be inhibitory to receptor function (Brandon et al., 2000) and would

affect numerous other ion channels as well. Thus, a greater number of non-selective

effects would be expected, but further studies are needed to elucidate the role of G

protein-coupled receptors in mediating neurosteroid actions.

Unlike BZs and neurosteroids, both barbiturates and ethanol have numerous other sites of action at clinically relevant brain concentrations. The barbiturates have been

shown to inhibit excitatory glutamate receptors, including AMPA, NMDA, and kainate

receptors (Hoffman & Tabakoff, 1993; Grasshoff et al., 2006), as well as pre- and

postsynaptic VGCCs (Blaustein & Ector, 1975; Gross & Macdonald, 1988; ffrench-

Mullen et al., 1993). Ethanol has been shown to affect activity of all Cys-loop receptors,

including inhibition of excitatory nicotinic acetylcholine and 5-HT3 receptors and

enhancement of inhibitory glycine receptors (Crews et al., 1996; Aguayo et al., 2002;

Harris et al., 2008). Various potassium channels and VGCCs are also inhibited by ethanol

(Messing et al., 1986; Harris et al., 2008). Ethanol is also considered a potent inhibitor of

NMDA receptors (Crews et al., 1996). A commonality in ethanol’s putative site of action

is amphipathic α-helices containing residues like serine and threonine, where it likely

displaces water molecules to affect protein function (Harris et al., 2008). Interestingly,

the effects of ethanol on GABAA receptors and some other channels depends on the

activity of intracellular signaling cascades (Woodward, 1999; Aguayo et al., 2002), and a

role of G protein-dependent signaling cascades in mediating ethanol’s actions has also

been proposed (Harris et al., 2008), similar to neurosteroids.

2.5 Withdrawal from GABAA receptor modulators

Long-term exposure to GABAA receptor modulators can lead to the development of tolerance to their sedative and anticonvulsant effects, requiring higher doses to achieve the same level of effect (File, 1985). Higher doses of drug coupled with long-term exposure may also lead to physical dependence, manifested by withdrawal symptoms

(Hodding et al., 1980; Woods & Winger, 1995; Kan et al., 2004). Although there are some similarities between the withdrawal syndromes of CNS depressants, there is substantial variability in the range of symptoms present and their severity, likely attributable to their different molecular targets. The BZ withdrawal syndrome includes anxiety and insomnia, as well as headaches, muscle aches, nausea, depression, increased sensitivity to light and sound, paresthesias, perceptual changes, depersonalization, and, rarely, seizures or psychosis (Lader, 1994; Griffiths & Johnson, 2005). Synthetic neurosteroids are currently used for surgical anesthesia, but not for chronic conditions; thus, withdrawal effects have not been observed following clinical treatment. However, withdrawal of endogenous neurosteroids occurs following prolonged physiologic elevation during the menstrual cycle or pregnancy. This physiologic withdrawal may result in perimenstrual catamenial epilepsy as well as the symptoms of premenstrual syndrome and postpartum depression (Smith, 2002; Reddy & Rogawski, 2009). These symptoms relate mainly to mood alterations and include depression, fatigue, anxiety, irritability, sleep disturbances, and decreased experience of pleasure (Amin et al., 2006), many which overlap BZ withdrawal symptoms.

Withdrawal symptoms are often far more severe for the non-selective CNS depressants, barbiturates and ethanol. Barbiturate withdrawal symptoms range in severity

from anxiety, insomnia, irritability, restlessness, depression, headaches, nausea, tremors,

and diaphoresis to hallucinations, myoclonus, delirium, grand mal seizures, and severe

fever (Hodding et al., 1980). The ethanol withdrawal syndrome is similar to that of

barbiturates, including the rare appearance of the most severe condition, delirium

tremens, which is a combination of a severely confused state, hallucinations, and

symptoms of sympathetic overactivity such as diaphoresis, hypertension, and tachycardia

(Hodding et al., 1980; McKeon et al., 2008).

The barbiturates and ethanol also have a greater abuse liability in terms of their

reinforcing properties (Griffiths & Johnson, 2005). The mechanisms for this are not

entirely clear, but ethanol’s reinforcing effects may relate to its ability to enhance activity of the mesolimbic dopaminergic reward pathway (Soderpalm et al., 2009). Patients taking

BZs less seldom become addicted, and BZ abuse is generally limited to polydrug abusers

(O'Brien C, 2005). Correspondingly, BZs have a limited abuse liability in terms of their reinforcing effects based on both preclinical data and studies in humans (Woods et al.,

1992; Griffiths & Johnson, 2005), although some patients abuse BZs in a chronic quasi- therapeutic manner (i.e., self-administration) in order to relieve or avoid withdrawal symptoms (Griffiths & Johnson, 2005). The greater withdrawal severity and abuse liability of the barbiturates and ethanol likely pertains to their larger number of molecular targets compared to BZs.

Withdrawal phenomena observed in animal models includes measurable effects such as anxiety and susceptibility to pharmacologically induced seizures. The most commonly used drugs to measure seizure susceptibility are kainic acid, a glutamate receptor agonist; pentylenetetrazol, a GABAA receptor antagonist; and β-carboline compounds, methyl-

6,7-dimethoxy-4-ethyl-ß-carboline-3-carboxylate and methyl-β-carboline-3-carboxylate,

BZ inverse agonists (Chaix et al., 2007). Among the variety of tests used to measure

anxiety, such as the open field test and the light-dark box, one of the most common and

well-accepted measures is the open arm test, also known as the elevated plus maze. These behavioral tests take advantage of the natural exploratory tendencies of rodents. Animals

with anxiety reliably spend less time exploring open and well-lit areas, presumably to

avoid potential dangers. In the case of the elevated plus maze, anxious animals avoid the

two open arms and spend more time in the two closed arms (Ramos, 2008). Anxiety is

perhaps the most commonly measured outcome in animal models of withdrawal from

CNS depressants and other drugs of abuse (Emmett-Oglesby et al., 1990), but increased

seizure susceptibility is also frequently observed (Izzo et al., 2001; Smith, 2002).

2.6 The hippocampus and its role in drug dependence

The hippocampal formation is part of the limbic system, which functions to regulate

learning, memory, and emotions. Anatomically, the limbic system consists of the limbic

lobe, a phylogenetically primitive subdivision of the cerebral cortex, as well as other

functionally and anatomically related structures. The limbic lobe consists of the

parahippocampal gyrus (including entorhinal cortex), the cingulate gyrus, and the

hippocampal formation. The additional structures of the limbic system include parts of

the hypothalamus, the septal area, the nucleus accumbens, certain neocrtical areas (e.g.,

orbitofrontal cortex) and the amygdala (Iversen et al., 2000). The hippocampal formation

is subdivided into the hippocampus proper, the dentate gyrus, and the subiculum. The

hippocampus proper is divided into four fields with distinct cytoarchitecture: CA1, CA2,

CA3, and CA4 (abbreviated from cornu ammonis, or “Ammon’s horn”). The dentate

gyrus contains a layer of principle cells that forms a V-shaped pair of joined blades and a

polymorphic hilar region located between the blades, which surrounds field CA4. These

fields can be further subdivided into somatic, dendritic, and axonic layers. The alveus is

the deepest layer, a white matter tract formed by the myelinated axons of the principle

(pyramidal) cells in CA1 and subiculum. Next is stratum oriens (SO), formed by the

basal dendrites of pyramidal cells in CA1-3, then stratum pyramidale (SP), the pyramidal

cell bodies, then stratum radiatum (SR), the apical dendrites, and stratum lacunosum-

moleculare (SLM), the distal extensions of the apical dendrites. Immediately adjacent to

the SLM is the molecular layer (ML), containing the apical dendrites of the principle cells of the dentate gyrus, the granule cells. The SLM of the hippocampus proper is separated from the ML of the dentate gyrus by the hippocampal fissure. The granule cell layer (GCL) is formed by the somata of granule cells, and as mentioned above, constitutes a compact layer of cells forming two blades facing opposite directions

(Amaral & Witter, 1989). Between the two blades is the hilar region, which is a layer containing various types of polymorphic GABAergic interneurons that innervate the nearby granule cells (Houser, 2007). This polymorphic region merges with the CA4 pyramidal cells, which also appear to innervate the nearby granule cells (Amaral &

Witter, 1989).

The rat hippocampus is also divided into two domains along the longitudinal

(septotemporal) axis, referred to as the dorsal and ventral hippocampus. These domains

are different in both connectivity and putative function, but the intrinsic circuitry is

maintained along the entire length of the hippocampus. In particular, the hippocampus

can be viewed as a trisynaptic circuit, which represents unidirectional information flow.

The first path in this circuit and the main input to the hippocampal formation comes from

the entorhinal cortex via the perforant pathway. These inputs synapse primarily onto the

dendrites of granule cells (ML), but also onto the distal dendrites of CA3 and CA1

pyramidal neurons (SLM, some SO). Granule cell axons project to the dendrites of

pyramidal neurons in CA3 (SR/stratum lucidum) via the mossy fiber pathway, and axons from CA3 (and CA2) pyramidal neurons project to the dendrites of pyramidal neurons in

CA1 (SR/SO) via the Schaffer collateral pathway, completing the trisynaptic circuit. CA3 also projects to contralateral CA1 via the anterior commissure. Axons from CA1 pyramidal neurons project mainly to the subiculum, as well as pre- and postsubiculum

(Amaral & Witter, 1989; Somogyi & Klausberger, 2005). The combined CA1 and subicular axons form the alveus and project to entorhinal cortex (among other cortical regions, discussed below), mammillary body, amygdala, hypothalamus, thalamus, lateral septal nucleus, bed nucleus of the stria terminalis, nucleus accumbens, and anterior olfactory nucleus (Swanson & Cowan, 1977). Although this unidirectional circuit seems to carry information in a plane transverse to the hippocampus, significant information transfer also occurs along the longitudinal axis (Amaral & Witter, 1989).

The hippocampus is best known for its role in memory formation, but also functions in anatomic circuits linked to the expression of anxiety (Degroot & Treit, 2004) and seizures (Griesemer & Mautes, 2007), both symptoms of withdrawal from GABAA receptor modulators. The circuits that mediate these various functions may be anatomically separated in the dorsal and ventral hippocampus. In particular, the dorsal hippocampus receives visuospatial input and projects to retrosplenial and anterior cingulate cortex (bidirectional connection), mammillary nuclei (via the fornix), anterior

thalamic nuclei, lateral septal nucleus, nucleus accumbens, and rostral caudate-putamen,

which are collectively involved in spatial memory and environmental exploration,

especially via connections to ventral tegmental area and the reticular part of substantia

nigra. In contrast, the ventral hippocampus receives mainly olfactory, gustatory, and visceral input and projects to olfactory bulb, anterior olfactory nucleus, amygdala

(bidirectional connection), medial prefrontal cortex (bidirectional connection), periventricular and medial zones of the hypothalamus, lateral septal nucleus, and bed nucleus of the stria terminalis, which are collectively involved in the regulation of emotions, motivated behavior, and affective state. A third intermediate region may be distinguished as receiving a more widespread mix of inputs and projecting to anterior

olfactory nucleus, medial prefrontal cortex, amygdala (bidirectional connection), anterior

hypothalamic nuclei, supramammillary nucleus, medial mammillary nucleus, and lateral

septal nucleus (Cenquizca & Swanson, 2007; Fanselow & Dong, 2010). These

projections are similar to the ventral hippocampus and may additionally function to

translate spatial inputs into motivated behaviors (Fanselow & Dong, 2010).

In addition to the intermediate region mediating overlapping function, longitudinal information processing and widespread connections between hippocampal inputs and outputs (Fanselow & Dong, 2010) suggest that the function of dorsal hippocampus may

not be restricted to encoding spacial information. In particular, some studies implicate a

role of dorsal hippocampus in mediating anxiolytic or anxiogenic effects of various drugs

(Bitran et al., 1999; File et al., 2000) and BZ-induced withdrawal anxiety directly

correlates with hyperexcitability of dorsal hippocampal CA1 neurons (Xiang & Tietz,

2007). Thus, the hippocampus represents an important node in anatomic circuits linked to

the expression of anxiety, a common symptom observed following withdrawal from CNS depressants.

2.7 GABAergic interneurons and hippocampal network activity

Network activity of the hippocampus is complex and determined by both intrinsic glutamatergic and GABAergic signals and a variety of extrinsic inputs. Of particular

relevance to the study of BZs and other GABAA receptor modulators are the location of

GABAergic interneurons and their synaptic connections within the hippocampus.

Numerous classes of interneurons exist based on the location of their cell bodies and the

arborization of their dendrites and axons. Within the CA1 field, GABAergic interneuron

cell bodies are located mainly in the avleus, SO, SP, and SR-SLM border. Their dendritic

trees and axonal projections vary greatly. Some project locally, while others project to

different regions, such as the dentate gyrus, CA3, subiculum, or septum. A few types of

interneurons specifically project to other interneurons. Many of these interneurons are

also distinguished based on the expression pattern of various calcium-binding proteins

and neuropeptides. Specifically, GABAergic interneurons express in differing

combinations parvalbumin, calretinin, calbindin, cholecystokinin, somatostatin,

neuropeptide Y, and vasoactive intestinal peptide (Somogyi & Klausberger, 2005). Each

interneuron of a particular class appears to be activated in a similar manner and may even

be connected by similar inputs, as well as gap junctions (Mann & Paulsen, 2007). Thus, the various classes of interneurons can be differentially activated depending on the source

and firing frequency of the input, which will then define the output firing rate of CA1

pyramidal neurons or other neuronal projections (Somogyi & Klausberger, 2005; Mann

& Paulsen, 2007).

GABA released from the axonal projections of interneurons generally targets synaptic

γ2-containing GABAA receptors, but GABA spillover also activates extrasynaptic δ- or

γ2-containing GABAA receptors, as well as GABAB receptors. Inhibitory GABAergic

synapses can be morphologically distinguished from excitatory glutamatergic synapses.

Specifically, inhibitory synapses have symmetric pre- and postsynaptic densities and are generally located on the axon, soma, or dendritic shaft, whereas excitatory synapses

contain thicker postsynaptic densities and are mainly located on dendritic protrusions or

spines. The greatest density of GABAergic synapses on CA1 pyramidal neurons occurs

in the perisomatic region in the proximal SR and SO, followed by SLM, then distal SR

and SO. The percent of inhibitory input in these regions is 98% in proximal SR, 48% in

proximal SO, 15% in SLM, and 2-3% in both distal SR and SO. Synaptic inputs on the

soma and axon initial segment are exclusively inhibitory. Thus, except for a relatively

high inhibitory component in SLM, which would specifically modulate excitatory input

from entorhinal cortex (and thalamus), synaptic inhibition occurs mainly perisomatically.

This strong perisomatic inhibition serves as a veto function for the wide array of

excitatory input, and synchronous GABAergic interneuron firing can therefore regulate

the output frequency of CA1 neurons (Megias et al., 2001).

2.8 Molecular substrates of drug dependence: Role of GABAA receptor dysfunction

Dysfunction of GABAergic transmission has been studied as a putative molecular

mechanism underlying drug tolerance and dependence. This can occur presynaptically via changes in the frequency or amount of GABA release, or postsynaptically via changes in receptor number, channel kinetics, channel conductance, or anionic gradients, as well

as altered GABA or modulator sensitivity. Although a few studies have found changes in

the frequency or amount of GABA release following long-term treatment with GABAA receptor modulators, especially following administration of the non-selective CNS depressants, e.g., barbiturates and ethanol (Hunt, 1983; Saunders & Ho, 1990; Yu & Ho,

1990), the most dramatic alterations occur postsynaptically. Thus, the following discussion will focus on the role of dysfunctional postsynaptic GABAA receptors in

mediating drug tolerance and dependence.

The mechanisms and expression of GABAA receptor dysfunction vary depending on the dose and particular drug administered, as well as the route and duration of administration (Uusi-Oukari & Korpi, 2010). One of the first changes to appear and the most common finding during long-term exposure to various GABAA receptor modulators

in vitro (Hu & Ticku, 1994; Friedman et al., 1996) and in vivo (Gallager et al., 1984;

Morrow et al., 1988; Allan et al., 1992) is allosteric uncoupling of modulator binding

sites from the GABA binding site and from each other. Uncoupling following long-term

treatment with various modulators or even GABA itself can be observed in binding

studies as a reduced ability of GABA, neurosteroids, or barbiturates to enhance

radiolabeled BZ binding (Friedman et al., 1996). Functionally, uncoupling is manifested

as a decreased efficacy of allosteric modulators to enhance GABA-mediated Cl− flux

(Morrow et al., 1988; Allan et al., 1992; Hu & Ticku, 1994). Heterologous uncoupling

also occurs in which long-term exposure to one modulator uncouples other modulator

binding sites from the GABA or BZ binding sites (Buck & Harris, 1990; Hu & Ticku,

1994; Friedman et al., 1996). The mechanism for uncoupling may be due to receptor

subunit changes, as the time course for both GABAA receptor turnover and uncoupling

occur with a t1/2 of about 1 – 3 days with maximal uncoupling after 7 – 10 days of drug

exposure (Borden et al., 1984; Hu & Ticku, 1994). However, uncoupling has also been

observed in heterologous expression systems using recombinant GABAA receptors,

suggesting that post-translational modifications such as phosphorylation are also plausible mechanisms (Klein & Harris, 1996). Uncoupling likely contributes to the rapid tolerance that develops to particular drug effects, especially sedation. Heterologous

uncoupling could also contribute to cross-tolerance that occurs between some GABAA receptor modulators (Harris, 1990; Smith & Stoops, 2001).

Subsequent to an initial uncoupling event, long-term FZP treatment results in decreased miniature inhibitory postsynaptic current (mIPSC) amplitude, synaptic and unitary conductance, and the number of mIPSC events, including an absence of events in about one-third of CA1 pyramidal neurons at 2 days, but not 7 days of withdrawal. These changes were region specific as they were not observed in dentate granule cells (Poisbeau et al., 1997; Tietz et al., 1999b; Zeng & Tietz, 1999; Xiang & Tietz, 2008). Long-term barbiturate or ethanol treatment may also result in decreased GABA-mediated currents

2+ (Klein & Harris, 1996). A role for increased [Ca ]i in mediating the reduced GABA

sensitivity during FZP withdrawal has been proposed (Zeng & Tietz, 1997; Xiang &

2+ Tietz, 2008) based on the known ability of Ca to negatively regulate GABAA receptor function (Stelzer et al., 1988; Houston et al., 2009). Recent findings have also shown that chronic blockade of neuronal activity or L-VGCC-mediated Ca2+ influx reduced synaptic

GABAA receptor expression by enhancing proteasome-dependent turnover and reducing

synaptic insertion (Saliba et al., 2009). These findings suggest that basal L-VGCC-

2+ mediated Ca influx is required to maintain synaptic GABAA receptor expression.

Therefore, reduced L-VGCC activity due to prolonged enhancement of GABA-mediated

inhibition by BZs may result in reduced synaptic GABAA receptor expression during

chronic treatment. Subsequently, enhanced Ca2+ influx partly due to impaired GABA-

mediated inhibition could further disrupt GABAA receptor function during BZ

withdrawal.

In addition to reduced synaptic conductance, a slight depolarizing shift in the GABAA receptor reversal potential from −72 mV in control to −68 mV in 2-day FZP-withdrawn

CA1 neurons was observed, implicating a potential reduction in the Cl− driving force

+ + (Zeng & Tietz, 1997). When interfering Na and K ion fluxes were blocked, a GABAA receptor-mediated depolarization was observed in FZP-withdrawn but not control CA1 neurons following high intensity stimulation, which typically occurred later than the early inhibitory postsynaptic potential (Zeng et al., 1995). The requirement for high intensity stimulation and delayed response suggests that large amounts of GABA release may have been needed, which could result in spillover and activation of extrasynaptic receptors.

Interestingly, it was previously suggested that dendritic extrasynaptic receptors in CA1 pyramidal cells are depolarizing (Alger & Nicoll, 1982). However, whether extrasynaptic receptors are up-regulated in the hippocampus as a consequence of long-term FZP administration remains unknown (see below). It should be noted that a slight depolarizing response can still be inhibitory to membrane excitability due to the shunting effect of reduced membrane resistance when the channels are open (Alger & Nicoll, 1979).

Depolarizing shifts in the GABA reversal potential have also been observed following prolonged use of GABAA receptors (Staley et al., 1995). This effect primarily

occurs in the dendrites, where the large surface area to volume ratio allows for faster

changes in ion concentration. In particular, repeated GABAA receptor activation leads to

− − chronic Cl and HCO3 fluxes, neither of which is able to reach its respective reversal

− potential. Although intracellular and extracellular HCO3 levels are rapidly buffered by

carbonic anhydrase enzymes, intracellualar Cl− accumulation can transiently overcome

the maximal velocity of Cl− transporters, like KCC2. This results in a reduced Cl− driving

− − force and transient GABA-mediated depolarization via HCO3 and possibly Cl efflux

(Staley & Proctor, 1999). While this would explain a transient depolarizing response due

to prolonged BZ-mediated GABA current enhancement, it does not explain the continued

presence of a depolarized GABA reversal potential or the GABA-mediated

depolarization observed after 2 days of FZP withdrawal. It is currently unknown whether

dysfunctional Cl− transport is responsible for the GABA-mediated depolarization, or

whether this was a normal dendritic response unmasked by the decrease in IPSP

amplitude (Zeng et al., 1995). It is unlikely due to increased expression of GABAA

− − receptors with higher relative permeability to HCO3 , since the HCO3 conductance is

selectively reduced during FZP withdrawal (Zeng & Tietz, 2000).

Changes in the expression of GABAA receptor subunits could explain reduced IPSC

amplitudes and decreased sensitivity to GABA and the allosteric modulators, but these

changes are often inconsistent depending on the treatment model. One- and 4-week oral

treatment with FZP (100 – 150 mg/kg/day) resulted in a down-regulation of BZ receptors

across several brain regions measured by autoradiographic localization of flunitrazepam

binding (Tietz et al., 1986). In contrast, once daily intraperitoneal (i.p.) injection of rats

with diazepam (2.5 – 5 mg/kg) for 3 weeks did not affect BZ affinity or number of sites measured by radiolabeled flunitrazepam binding to cerebral cortical membranes

(Gallager et al., 1984). Immediately after 1-week FZP treatment there was decreased α1

and β3 mRNA and protein in the hippocampus and cortex (Chen et al., 1999; Tietz et al.,

1999a). These changes were largely reversed after 2 days of withdrawal (Tietz et al.,

1999a; Tietz et al., 1999b). Thus, while down-regulation of α1/β3-containing GABAA receptors may explain tolerance to the sedative effects of FZP, dysregulation of GABAA receptor expression does not appear to adequately explain the manifestation of withdrawal anxiety or anticonvulsant tolerance up to 2 days after FZP withdrawal

(Rosenberg et al., 1991; Van Sickle et al., 2004). Other studies using either i.p. injection or continuous infusion of rats with diazepam (5 – 20 mg/kg/day or 0.25 µg/g, ~0.9 µM, respectively) have also found decreased α1 mRNA or protein expression in hippocampus and cortex (Heninger et al., 1990; Wu et al., 1994; Impagnatiello et al., 1996). The decreased hippocampal α1 mRNA expression returned to baseline levels after 2 days of withdrawal (Wu et al., 1994). Thus, several but not all studies support decreased α1 subunit expression following long-term BZ treatment. However, there is no consistent finding that a particular subunit is up-regulated which might replace α1-containing

GABAA receptors (Uusi-Oukari & Korpi, 2010). Although only a limited number of

studies have analyzed BZ withdrawal or followed up on changes in mRNA expression by

confirming changes at the protein level, overall the data seem to support changes in

GABAA receptor subunit expression as a mechanism of BZ tolerance, but not withdrawal

symptoms.

Unlike the effects observed for BZs, 2 to 3 days of continuous progesterone treatment

in rats, the precursor to allopregnanolone, resulted in increased α4 subunit protein

expression in the CA1 region. This correlated with decreased GABA-mediated current

decay time and BZ sensitivity. The faster decay time of GABA currents in CA1 neurons

30 would reduce GABA-mediated inhibition, which may have contributed to the expression of anxiety that was also observed at these time points. These effects were dependent on allopregnanolone synthesis and were reversed after 5 to 6 days of treatment (Gulinello et al., 2001). However, all of these effects re-emerged after continued treatment for 3 weeks, followed by 8 to 24 hrs of withdrawal (Smith et al., 1998).

Measurement of subunit mRNA immediately after long-term treatment with pentobarbital revealed decreased α1 expression in the hippocampus (Tseng et al., 1994) and increased α6 and δ expression in the cerebellum (Ito et al., 1996; Lin & Wang, 1996).

During withdrawal there was increased α1, β3, and γ2 expression in cerebral cortex

(Tseng et al., 1993; Tseng et al., 1994) and increased α1 (Tseng et al., 1994) but decreased δ (Lin & Wang, 1996) expression in the cerebellum. Although changes in subunit protein expression were not confirmed, similar to BZs, GABAA receptor subunit regulation would appear to explain tolerance, but not the withdrawal phenomenon associated with barbiturate use.

Several alterations in GABAA receptor subunits have been observed following long- term ethanol treatment, many of which persist during withdrawal. In most brain regions decreased α1 subunit protein expression was observed. This was accompanied by increased α4, β2/3, and γ1 protein expression in the cerebral cortex (Devaud et al., 1997), decreased δ with increased α4 and γ2 expression in the hippocampus (Cagetti et al.,

2003), increased α4 and γ2 in the basolateral amygdala (Diaz et al., 2011), and increased

α6 expression in the cerebellum (Sanna et al., 2004a). The regulation of α1 subunit in the hippocampus required intermittent ethanol treatment, as it was not regulated by continuous treatment (Matthews et al., 1998). The subunit changes in the hippocampus

were also accompanied by a decrease in CA1 neuron mIPSC decay time and BZ sensitivity (Cagetti et al., 2003), similar to that of neurosteroid withdrawal. Interestingly, ethanol increased de novo synthesis of allopregnanolone in hippocampal slices (Sanna et al., 2004b), suggesting that some of ethanol’s long-term effects may be mediated by endogenous neurosteroids.

2.9 Molecular substrates of drug dependence: Role of ionotropic glutamate receptors

Dysfunction of GABAA receptors like that described above, as well as enhancement

of ionotropic glutamate receptors, the main excitatory neurotransmitter receptors in the

CNS, may contribute to withdrawal hyperexcitability (Izzo et al., 2001; Allison & Pratt,

2003; Van Sickle et al., 2004; Xiang & Tietz, 2007). Glutamate release from presyanptic

terminals acts on several classes of pre- and postsynaptic receptors. Like GABA

receptors, glutamate receptors can be divided into ionotropic and metabotropic

categories. The ionotropic receptors include AMPA, kainate, NMDA, and ‘orphan’

receptors. As suggested by their names, the ionotropic receptors were initially classified

pharmacologically by their agonists. Orphan receptors were classified as ionotropic

receptors based on homology, but do not fit into a particular agonist class. It has

subsequently been discovered that each class of ionotropic glutamate receptor is formed

by a specific subset of genetically related subunits. These subunits assemble in the

endoplasmic reticulum as a dimer of dimers to form functional homomeric or heteromeric tetramers. AMPA receptors are constructed from GluA1, GluA2, GluA3, or GluA4 subunits; kainate receptors from GluK1, GluK2, GluK3, GluK4, or GluK5 subunits;

NMDA receptors from GluN1 (present in all NMDA receptors), GluN2A/B/C/D, or

GluN3A/B subunits; and orphan receptors from GluD1 or GluD2 (Collingridge et al.,

2009). The metabotropic glutamate receptors are G protein-coupled receptors that are linked to different second-messenger signaling cascades. These receptors can be divided into 3 groups based on genetic and pharmacologic similarity: Group I receptors, mGluR1 and mGluR5, activate phospholipase C, and both Group II, mGluR2 and mGluR3, and

Group III receptors, mGluR4, mGluR6, mGluR7, and mGluR8 inhibit adenylyl cyclase activity (Conn & Pin, 1997). Recent evidence suggests that orphan receptors may also paradoxically be G protein-coupled receptors rather than functional ion channels (Schmid

& Hollmann, 2008).

The other ionotropic glutamate receptors can be distinguished as having different cationic permeability and kinetic properties depending on subunit composition. The conductive properties are determined by a specific Q/R/N site located in the pore-forming region of the subunit. For AMPA and kainate receptors, GluA2, GluK1, and GluK2 subunits undergo post-transcriptional editing of the genetically encoded glutamine to an arginine codon, which is developmentally regulated for kainate receptor subunits.

Receptors composed entirely of unedited subunits containing a glutamine are permeable to Na+, K+, and Ca2+ and are sensitive to voltage-dependent block by intracellular

polyamines. Receptors containing any edited subunits that have an arginine display

reduced permeability to Ca2+ and channel conductance, and lack sensitivity to

intracellular polyamines. NMDA receptor subunits contain an asparagine residue at the

corresponding site and are permeable to Na+, K+, and Ca2+ and susceptible to voltage-

dependent block by extracellular Mg2+ (Burnashev, 1998; Seeburg & Hartner, 2003).

The kinetics of excitatory postsynaptic currents (EPSCs) are determined by the particular subunit composition of the glutamate receptors assembled in vivo. AMPA receptors have a lower affinity for glutamate and fast activation and desensitization kinetics, mediating the fast component of EPSCs (Jonas & Spruston, 1994). In mature hippocampal neurons, the predominant population of AMPA receptors are composed of mainly Ca2+-impermeable GluA1/GluA2 and GluA2/GluA3 heteromers and only about

8% are Ca2+-permeable GluA1 homomers (Wenthold et al., 1996). Application of kainate to hippocampal neurons revealed a rapidly activating, non-desensitizing current that was not seen with AMPA application. It was later discovered using AMPA-selective antagonists that kainate activated AMPA receptors without desensitizing, and that kainate receptors themselves rapidly desensitize. Although kainate receptors mediate cation fluxes both pre- and postsynaptically, they do not appear to play a major role in shaping hippocampal EPSCs (Lerma et al., 1997). NMDA receptors exhibit higher affinity for glutamate and activate and desensitize slowly, contributing to the slow component of

EPSCs (Jonas & Spruston, 1994). Although under normal conditions NMDA receptors represent the primary source of glutamate-mediated Ca2+ influx into dendritic spines, this can only occur if the Mg2+ block is removed by postsynaptic depolarization. Thus,

NMDA receptors are Ca2+ signaling complexes that serve as coincident detectors of simultaneous pre- and postsynaptic activation.

NMDA receptors in CA1 pyramidal neurons are mainly composed of GluN1/GluN2A or GluN1/GluN2B subunit combinations (Monyer et al., 1994). GluN2B-containing

NMDA receptors are expressed embryonically and postnatally, whereas GluN2A expression occurs during the first two weeks of postnatal development (Sheng et al.,

1994; Zhong et al., 1994). GluN2A- and GluN2B-containing NMDA receptors are

expressed both synaptically and extrasynaptically in hippocampal neurons (Thomas et al.,

2006). GluN2B-containing NMDA receptors are susceptible to lateral mobility into and

out of PSDs (Tovar & Westbrook, 2002; Groc et al., 2004) and additionally serve as

CaMKII binding partners (Strack et al., 2000; Robison et al., 2005) to regulate synaptic

plasticity.

CaMKII serves an important role in both drug-induced and physiologic glutamatergic

plasticity. CaMKII forms oligomeric holoenzymes containing up to 12 subunits (Bennett

et al., 1983). The CaMKIIβ isoform mediates binding of CaMKII holoenzymes to the

actin cytoskeleton within dendritic arbors (Shen et al., 1998). Upon elevation of

2+ 2+ 2+ 2+ intracellular Ca concentrations ([Ca ]i), Ca binds to all four Ca -binding sites on

calmodulin (CaM), resulting in association with and activation of CaMKII via

2+ displacement of an autoinhibitory domain. If [Ca ]i remains elevated for sufficient time,

CaMKII enzymes phosphorylate neighboring CaMKII molecules within the holoenzyme at Thr286 on α or Thr287 on β isoforms. This autophosphorylation results in Ca2+/CaM

2+ trapping and autonomous kinase activity without the need of elevated [Ca ]i. Elevations

2+ in [Ca ]i also result in dissociation of CaMKII holoenzymes from the actin cytoskeleton,

promoting binding with other proteins (Fink & Meyer, 2002).

Alterations in CaMKII activity and glutamate receptor function may contribute to the

drug withdrawal phenomenon. While enhanced AMPA receptor function in the ventral

tegmental area is associated with behavioral sensitization and addiction to drugs of abuse

such as cocaine, amphetamine, morphine, ethanol, and nicotine (Wolf, 2003; Kauer &

Malenka, 2007; Mameli et al., 2011), enhanced AMPA receptor function in other brain

35 regions, such as the cortex, hippocampus, amygdala, and thalamus may underlie BZ withdrawal symptoms (Izzo et al., 2001; Van Sickle & Tietz, 2002; Allison et al., 2005).

Notably, potentiation of AMPA receptors in the dorsal hippocampal CA1 region significantly correlated with the severity of FZP withdrawal anxiety (Van Sickle et al.,

2004; Song et al., 2007; Xiang & Tietz, 2007). Specifically, increased miniature EPSC

(mEPSC) amplitude via enhanced AMPA receptor function was observed after 1 and 2 days of FZP withdrawal, with increased unitary conductance after 2 days of withdrawal

(Van Sickle & Tietz, 2002; Van Sickle et al., 2004). The increased AMPA receptor current amplitude is initiated by the incorporation of GluA1 homomers during the first day of FZP withdrawal (Song et al., 2007; Das et al., 2008), followed by increased conductance via CaMKII-mediated phosphorylation of GluA1 during the second day of withdrawal (Shen et al., 2009; Shen et al., 2010). However, anxiety was not observed after 2 days of FZP withdrawal, likely attributable to a compensatory decrease in

GluN1/GluN2B subunit expression and NMDA receptor function (Van Sickle et al.,

2004; Shen et al., 2009; Das et al., 2010; Shen & Tietz, 2011). Moreover, prior systemic injection with MK-801 (an NMDA receptor antagonist) prevented the NMDA receptor down-regulation and unmasked anxiety on day 2 of withdrawal (Van Sickle et al., 2004).

In contrast to BZs, decreased kainate and increased NMDA receptor ligand binding was observed following long-term barbiturate treatment (Hoffman & Tabakoff, 1993).

Similarly, decreased AMPA and increased NMDA receptor ligand binding with enhanced

NMDA receptor function was observed following long-term ethanol administration or repeated ethanol withdrawal episodes (Hoffman & Tabakoff, 1993; Ulrichsen et al.,

1996). In particular, GluN2A and GluN2B mRNA and/or protein expression were up-

regulated both in vitro and in vivo following long-term ethanol exposure (Nagy, 2004).

The variability in glutamatergic regulation by GABAA receptor modulators may be due to the different molecular targets of these compounds.

Interestingly, FZP-induced regulation of AMPA receptors is similar to that described in the early phase of LTP (E-LTP) following Schaffer collateral stimulation. Induction of

E-LTP requires high frequency (25 - 100 Hz) or theta-burst (5 Hz) stimulation of CA1 afferents resulting in Ca2+ influx through postsynaptic NMDA receptors and subsequent

activation of CaMKII and other kinases like PKC. The initial Ca2+ influx and resultant

change in kinase activity leads to insertion of GluA1 homomers into the PSD enhancing

synaptic strength. Further activation of NMDA receptors and corresponding increases in

2+ [Ca ]i results in CaMKII autophosphorylation and translocation to binding partners in

the PSD, like the GluN2B subunit, where it mediates phosphorylation of GluA1

homomers on Ser831 increasing synaptic conductance (Fink & Meyer, 2002; Boehm &

Malinow, 2005). In contrast, late-phase LTP (L-LTP, >1 hr) induced by repeated high

frequency (100 Hz) stimulation or theta-burst (5 Hz) stimulation requires gene expression

and protein synthesis or dendritic protein translation, respectively (Malenka & Bear,

2004; Huang & Kandel, 2005). In particular, enhanced dendritic translation of CaMKIIα

and GluA1 and GluA2 AMPA receptor subunits may occur during L-LTP (Steward &

Schuman, 2001; Miller et al., 2002; Ju et al., 2004). Induction L-LTP is also dependent

on Ca2+ influx through NMDA receptors (Huang & Kandel, 2005; Bengtson et al., 2010).

In contrast to induction mechanisms of CA1 LTP, systemic injection of FZP-treated rats

with MK-801 did not prevent the appearance of anxiety or up-regulation of AMPA

receptor function after 1 day of withdrawal (Xiang & Tietz, 2007), suggesting that a

source of Ca2+ other than NMDA receptors may mediate this function.

2.10 Molecular substrates of drug dependence: Role of L-type voltage gated calcium

channels

VGCCs are Ca2+ permeable channels that open in response to membrane

depolarization. They are composed of a large (~190 - 250 kDa) transmembrane pore-

forming α1 subunit (10 subtypes) and 2 - 3 auxiliary subunits. An intracellular β subunit

(4 subtypes) acts as a chaperone and regulates gating properties. An extracellular α2 subunit disulfide-linked to a transmembrane δ subunit (4 α2δ subtypes) regulates trafficking and voltage-dependent channel properties (Catterall, 2000). A transmembrane

γ1 subunit was originally purified with skeletal muscle and cardiac Ca2+ channels, but not

with neuronal VGCCs. Additional γ homologs (γ2-8) are also not considered VGCC subunits, but may play a role in AMPA receptor trafficking (Dolphin, 2009). The α1 subunit consists of intracellular N- and C-termini and four repeated domains connected by intracellular loops, each domain containing six transmembrane segments (S1 - S6), with S4 serving as the voltage-sensor (Catterall, 2000). The different classes of VGCCs are characterized by their α1 pore-forming subunit, as the associated β and α2δ subunits are common to all VGCCs. VGCCs are divided into five classes based on pharmacology, as well as kinetic and conductance properties: high voltage-activated (HVA) channels contain L- (Cav1.1-1.4), P/Q- (Cav2.1), N- (Cav2.2), or R-type (Cav2.3) subunits, and low

voltage-activated channels contain T-type (Cav3.1-3.3) subunits. L-VGCCs are

selectively inhibited by DHPs, BTZs, and PAAs. P/Q-VGCCs are inhibited by ω-

agatoxin IVA, N-VGCCs by ω-conotoxin-GVIA, and R-VGCCs by SNX-482. T-VGCCs

are somewhat more sensitive to inhibition by mibefradil (Catterall et al., 2005).

The α1 subunits found in neuronal L-VGCCs are Cav1.2 and Cav1.3. Cav1.2 is four times more abundant than Cav1.3 in the rat cortex and hippocampus (Hell et al., 1993a)

and is localized in neuronal soma, as well as proximal and distal dendrites near excitatory

asymmetric synapses both in vitro (Obermair et al., 2004) and in vivo (Tippens et al.,

2008; Leitch et al., 2009). Although recent evidence suggested expression of Cav1.3 at

postsynaptic sites within CA1 neuron dendritic spines (Leitch et al., 2009), this study did

not confirm antibody specificity for immunohistochemical labeling. However, other

studies using a Cav1.3 antibody characterized as specific for immunohistochemistry using

knockout tissues (Olson et al., 2005) confirmed Cav1.3 localization within dendritic

spines in other brain regions, including the striatum (Day et al., 2006) and ventral horn

(Zhang et al., 2008). In addition, studies in hippocampal cultures indicated that Cav1.3 binds to and co-localizes with clusters of the postsynaptic adaptor protein, Shank (Zhang et al., 2005). Thus, there is substantial evidence that both Cav1.2 and Cav1.3 L-VGCCs

are situated near excitatory postsynapses where they can respond to neuronal activity and

mediate Ca2+-dependent synaptic plasticity and changes in gene expression (Dolmetsch et

al., 2001; Striessnig et al., 2006; Barbado et al., 2009). Moreover, L-VGCCs are located

in glial cells (Tippens et al., 2008), GABAergic interneurons (Vinet & Sik, 2006; Zhang

et al., 2008), and in axons and nerve terminals (Tippens et al., 2008), suggesting that L-

VGCCs can modulate neuronal plasticity via a variety of mechanisms.

In particular, both the early and late phases of LTP described above can also be

induced by Ca2+ through L-VGCCs during high frequency (100 - 200 Hz) or relatively

long-lasting theta-burst stimulation (Grover & Teyler, 1990; Morgan & Teyler, 2001;

Moosmang et al., 2005). Estimates of the contribution of L-VGCCs to these forms of

LTP may be underestimated for two reasons. First, application of NMDA receptor

antagonists often completely blocks LTP suggesting no role for other sources of Ca2+.

However, L-VGCCs may contribute to LTP but require the added NMDA receptor- mediated depolarization (Morgan & Teyler, 2001). Second, ineffectiveness of DHPs to block LTP may be due to how slowly they block L-VGCCs when activated by action potential stimuli even at high frequencies (Helton et al., 2005).

A role for L-VGCCs in contributing to withdrawal pathology from GABAA receptor

modulators has been proposed in numerous studies. Long-term treatment with BZs

enhances Ca2+ influx through L-VGCCs (Katsura et al., 2007; Xiang et al., 2008) and BZ

withdrawal symptoms were attenuated when L-VGCC antagonists were administered during the BZ withdrawal phase (Dolin et al., 1990; Gupta et al., 1996; Xiang & Tietz,

2007). However, reports differ on the effectiveness of L-VGCC antagonists when they are co-administered with BZs, with at least one study showing a reduction in withdrawal

symptoms (Hitchcott et al., 1992) and others showing no significant effects (Dolin et al.,

1990; Mizoguchi et al., 1993). Concurrent L-VGCC antagonist administration during

long-term barbiturate treatment, but not during the withdrawal phase protected against the

withdrawal syndrome (Rabbani et al., 1994). Barbiturate withdrawal was associated with an increase in the number of DHP binding sites in cerebral cortex and increased DHP- sensitive K+-stimulated Ca2+ influx in hippocampal slices (Rabbani & Little, 1999).

Concurrent administration of an L-VGCC antagonist with long-term ethanol treatment in

mice attenuated an up-regulation of brain DHP binding sites, convulsions, and

hippocampal slice hyperexcitability seen during withdrawal (Whittington et al., 1991;

Whittington & Little, 1991). Moreover, addition of an L-VGCC antagonist to perfusion

medium also acutely attenuated hippocampal slice hyperexcitability during ethanol

withdrawal (Whittington & Little, 1991). A role for L-VGCCs in mediating the

neurosteroid withdrawal phenomenon has not yet been reported.

The combination of data support a role for increased L-VGCC-mediated Ca2+ influx

in contributing to the GABAA receptor modulator withdrawal syndrome. In the presence

of a hyperexcitable state due to GABAA receptor dysfunction, a dramatic increase in

postsynaptic L-VGCC-mediated Ca2+ influx would ensue, contributing to withdrawal

phenomena via localized Ca2+ signal transduction cascades. At least for BZs, L-VGCC-

mediated Ca2+ signaling cascades may enhance AMPA receptor function (Xiang & Tietz,

2007), maintaining CA1 neuron hyperexcitability during withdrawal.

2.11 Regulation of L-VGCCs

Prolonged depolarization and activation of L-VGCCs leads to both voltage-dependent

and Ca2+-dependent inactivation, forms of negative feedback regulation. Unlike other

Ca2+ channels, L-VGCCs inactivate slowly leading to long-lasting Ca2+ currents.

2+ Voltage- and Ca -dependent inactivation are regulated by distinct sites within the α1 subunit. In particular, Ca2+-dependent inactivation is mediated by LA and IQ motifs

2+ located in the Cav1.2 C-terminus, which bind CaM in different Ca -bound states. The

2+ 2+ 2+ LA motif binds apo-CaM (Ca free) at low [Ca ]i (50 - 100 nM), as well as when [Ca ]i is high (>1 µM). In contrast, the IQ motif binds CaM when Ca2+ is conducted through the

channel. The transition from the LA to the IQ motif mediates Ca2+-dependent

inactivation, and may prime Ca2+-bound CaM for downstream signal transduction

cascades, linking L-VGCC inactivation with Ca2+ signaling (Morad & Soldatov, 2005).

Voltage-dependent rearrangements in channel confirmation also affect inhibition of L-

VGCCs by several drug classes, which generally show preferential interaction with open

and inactive channel states (Hockerman et al., 1997).

In addition to inactivation, L-VGCC function can be modulated by changes in plasma

membrane expression (Jarvis & Zamponi, 2007), proteolytic cleavage (Hell et al., 1996),

or phosphorylation (Kepplinger et al., 2000). As mentioned above both the β and α2δ

subunits are involved in trafficking Cav1.2 and Cav1.3 subunits to the membrane, as well

as modulating activation and inactivation kinetics and open probability of the channels

2+ (Lacinova, 2005). Proteolytic cleavage of Cav1.2 C-terminus by the Ca -dependent

protease, calpain, has been shown to increase Ca2+ influx through the channel in

hippocampal neurons (Hell et al., 1996). This cleavage also results in removal of S1928, a critical phosphorylation site on Cav1.2 (De Jongh et al., 1996; Yang et al., 2005; Hall et

al., 2006; Oliveria et al., 2007; Yang et al., 2007). Some of the kinases and phosphatases

involved in regulating L-VGCC function include PKA (Hell et al., 1993b; Gao et al.,

1997; Hall et al., 2007), PKC (Yang et al., 2005; Baroudi et al., 2006), PKG (Yang et al.,

2007), CaMKII (Lee et al., 2006; Grueter et al., 2008), PP2A (Davare et al., 2000; Hall et

al., 2006), and PP2B (Oliveria et al., 2007), also known as calcineurin. Many of these

kinases and phosphatases have been shown to act on Cav1.2, but also regulate other α1

(Liang & Tavalin, 2007) and β (Gao et al., 1997; Yang et al., 2007; Grueter et al., 2008) subunits. In particular, CaMKII is known to mediate Ca2+-dependent facilitation of both

Cav1.2 (Lee et al., 2006) and Cav1.3 (Calin-Jageman et al., 2007) channel function. G

protein-coupled receptors also regulate L-VGCCs, mainly via signaling cascades that

alter activity of kinases or phosphatases (Olson et al., 2005; Hulme et al., 2006).

Ca2+ influx is rapidly buffered by numerous cytosolic Ca2+-binding proteins, resulting

in localized elevation of Ca2+ concentrations mainly within the microdomain created by

2+ channel clusters. Thus, Cav1.2 and Cav1.3 Ca microdomains are capable of selectively

activating distinct localized signaling cascades leading to different downstream targets. In

particular, although both L-VGCCs contain class I PDZ (postsynaptic density-95/Discs

large/zona occludens-1)-interaction motifs in their C-termini, the Cav1.2 motif, VSNL,

interacts with different scaffolding and adaptor proteins than the Cav1.3 motif, ITTL

(Calin-Jageman & Lee, 2008). In addition, because of the different voltage-dependent

activation and inactivation properties of L-VGCC subtypes (Koschak et al., 2001; Xu &

Lipscombe, 2001), they are selectively activated at different phases of the action potential

and by different frequencies of neuronal activity (Zhang et al., 2006). Thus, Cav1.2 and

Cav1.3 L-VGCCs can mediate distinct neuronal functions. In particular, Cav1.2 channels

are involved in memory formation related to their role in an NMDA-independent form of

L-LTP (Moosmang et al., 2005), which may be stimulated solely by back-propagating

action potentials (Dudek & Fields, 2002). Cav1.3 channels on the other hand, stimulate

Ca2+-activated K+ channels enhancing the afterhyperpolarization and reducing further neuronal activity (Bowden et al., 2001), promote spontaneous firing activity in certain neurons (Putzier et al., 2009), and may promote age-related neuronal degeneration and

cognitive dysfunction (Thibault & Landfield, 1996; Porter et al., 1997; Chan et al., 2010).

Thus, differential regulation of Cav1.2 and Cav1.3 L-VGCCs could selectively target their distinct functions mediated by localized signaling cascades.

CHAPTER 3

INHIBITION OF RECOMBINANT L-TYPE VOLTAGE-GATED CALCIUM

CHANNELS BY POSITIVE ALLOSTERIC MODULATORS OF GABA-A

RECEPTORS

Damien E. Earl and Elizabeth I. Tietz

Department of Physiology and Pharmacology (D.E.E. and E.I.T.) and Department of

Neurosciences (E.I.T.), University of Toledo College of Medicine, Health Science

Campus, Toledo, Ohio

Running Title: GABAAR modulators inhibit recombinant L-VGCCs

Abbreviations: ANOVA, analysis of variance; BDZ, benzodiazepine; BTZ, benzothiazepine; DMSO, dimethyl sulfoxide; DHP, dihydropyridine; L-VGCC, L-type

voltage-gated calcium channel; PAA, phenylalkylamine; TEA, tetraethylammonium;

WT, wild type; LB, Luria-Bertani; HEK, human embryonic kidney

Keywords: barbiturate; benzodiazepine; calcium channel mutation; electrophysiology;

ethanol; neurosteroid

[The Journal of Pharmacology and Experimental Therapeutics 337 (2011) 301-11]

3.1 Abstract

Benzodiazepines (BDZs) depress neuronal excitability via positive allosteric

modulation of inhibitory GABAA receptors (GABAAR). BDZs and other positive

GABAAR modulators, including barbiturates, ethanol, and neurosteroids, can also inhibit

L-type voltage-gated calcium channels (L-VGCCs), which could contribute to reduced neuronal excitability. Because neuronal L-VGCC function is up-regulated after long-term

GABAAR modulator exposure, an interaction with L-VGCCs may also play a role in physical dependence. The current studies assessed the effects of BDZs (diazepam, flurazepam, and desalkylflurazepam), allopregnanolone, pentobarbital, and ethanol on

2+ whole-cell Ba currents through recombinant neuronal Cav1.2 and Cav1.3 L-VGCCs

expressed with β3 and α2δ-1 in HEK293T cells. Allopregnanolone was the most potent

inhibitor (IC50, ~10 µM), followed by BDZs (IC50, ~50 µM), pentobarbital (IC50, 0.3–1

mM), and ethanol (IC50, ~300 mM). Cav1.3 channels were less sensitive to pentobarbital

inhibition than Cav1.2 channels, similar to dihydropyridine (DHP) L-VGCC antagonists.

All GABAAR modulators induced a negative shift in the steady-state inactivation curve

of Cav1.3 channels, but only BDZs and pentobarbital induced a negative shift in Cav1.2 channel inactivation. Mutation of the high-affinity DHP binding site (T1039Y and

Q1043M) in Cav1.2 channels reduced pentobarbital potency. Despite the structural

similarity between benzothiazepines and BDZs, mutation of an amino acid important for

diltiazem potency (I1150A) did not affect diazepam potency. Although L-VGCC

inhibition by BDZs occurred at concentrations that are possibly too high to be clinically

relevant and is not likely to play a role in the up-regulation of L-VGCCs during long-

45 term treatment, pentobarbital and ethanol inhibited L-VGCCs at clinically relevant concentrations.

3.2 Introduction

Benzodiazepines (BDZs) are clinically useful anxiolytics, sedatives, and

anticonvulsants. The clinical effects of BDZs are attributed to their ability to allosterically enhance the inhibitory action of γ-aminobutyric acid type A receptors

(GABAARs). However, there is also evidence that BDZs directly inhibit L-type voltage-

gated calcium channels (L-VGCCs) in muscle and nerve cells at low micromolar concentrations (Yamakage et al., 1999; Xiang et al., 2008). Other GABAAR modulators,

such as the neurosteroid, allopregnanolone (3α-hydroxy-5α-pregnan-20-one), the

barbiturate, pentobarbital, and ethanol can also inhibit neuronal L-VGCCs (Messing et

al., 1986; ffrench-Mullen et al., 1993; Hu et al., 2007).

The interaction of GABAAR modulators with L-VGCCs could play a role in their

acute effects to reduce neuronal excitability and may also contribute to the neuronal hyperexcitability seen following withdrawal from chronic drug exposure. Notably, chronic treatment with BDZs, barbiturates, and ethanol increases neuronal Ca2+ influx in vitro and in vivo, possibly through L-VGCCs (Messing et al., 1986; Rabbani & Little,

1999; Katsura et al., 2006; Katsura et al., 2007; Xiang et al., 2008). An upregulation of L-

VGCC function during chronic treatment with the various GABAAR modulators is

implicated in contributing to withdrawal hyperexcitability (Dolin et al., 1987; Rabbani &

Little, 1999; Xiang & Tietz, 2007; Xiang et al., 2008).

Although it is known that GABAAR modulators inhibit L-VGCCs, there have been no

studies to assess the selective effects of these drugs on the neuronal L-VGCC α1 subtypes, Cav1.2 and Cav1.3. Using the whole-cell voltage-clamp technique, the current studies investigated the effects of BDZs (diazepam, flurazepam, and desalkylflurazepam),

allopregnanolone, pentobarbital, and ethanol on recombinant L-VGCCs containing the neuronal L-type α1 subunit, Cav1.2 or Cav1.3, along with β3 and α2δ-1 subunits. We found that positive allosteric GABAAR modulators reversibly inhibited both Cav1.2- and

Cav1.3-containing L-VGCCs in a state-dependent manner. The order of potency was

allopregnanolone > BDZs > pentobarbital > ethanol, with BDZs displaying 2- to 6-fold

less potency than the benzothiazepine (BTZ), diltiazem. Previous studies reported that

Cav1.3 channels were less sensitive to inhibition by dihydropyridines (DHPs),

phenylalkylamines (PAAs), and BTZs than Cav1.2 channels (Koschak et al., 2001; Xu &

Lipscombe, 2001; Tarabova et al., 2007). The structural similarities among BDZs and

BTZs (see Fig. 1) suggested the possibility that L-VGCCs might also display differential

sensitivity to GABAAR modulators. Compared to Cav1.2, recombinant Cav1.3 channels

were 1.5-fold less sensitive to inhibition by flurazepam, 3-fold less sensitive to inhibition

by pentobarbital, and 3-fold more sensitive to inhibition by diltiazem. Studies using

Cav1.2 mutants revealed that pentobarbital potency was reduced 3.5-fold by mutation of the high-affinity DHP binding site (T1039Y and Q1043M). Despite the aforementioned structural similarities, diltiazem did not compete with diazepam inhibition, and mutation of an amino acid within the BTZ binding site (I1150A) had no effect on diazepam potency. Based on their concentration-dependent effects, inhibition of L-VGCCs by pentobarbital and ethanol may contribute to their acute clinical actions. A direct interaction of pentobarbital and ethanol with L-VGCCs may also contribute to the adaptive upregulation of L-VGCC function following chronic use of these non-selective

CNS depressants.

3.3 Materials and Methods

3.3.1 Cell culture and transient transfection

SV40 large T-antigen stably transfected HEK293 (HEK293T) cells were utilized for

transient expression of L-VGCCs. Cells were maintained at 37ºC and 5% CO2 in

Dulbecco’s Modified Eagle’s Medium with GlutaMAX-I (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Sigma, St. Louis, MO), 100 U/mL penicillin, and 100 µg/mL streptomycin. Cells were grown to ~80% confluency and transfected with

α1 (Cav1.2 or Cav1.3), β3, α2δ-1, and monomeric red fluorescent protein (mRFP) plasmids

(1:1:1:0.5 μg, respectively) using Lipofectamine™ 2000 reagent (Invitrogen). One day after transfection, cells were trypsinized and replated on 35 mm culture dishes prior to

electrophysiological recording 2 – 3 days post-transfection. Except for experiments with

mutated Cav1.2, all experiments utilized mouse neuronal Cav1.2 (Helton et al., 2005), rat

neuronal Cav1.3 (+exon11, Δexon32, +exon42a, Xu & Lipscombe, 2001), rat neuronal β3, and rat neuronal α2δ-1 (Lin et al., 2004) subunit plasmids, which were generously provided by Dr. Diane Lipscombe (Brown Univ., Providence, RI). The mRFP plasmid was generously provided by Dr. Zi-Jian Xie (Univ. of Toledo College of Medicine,

Toledo, OH). A DHP binding site (T1039Y and Q1043M) mutant Cav1.2 (Hockerman et

al., 2000) and its wild-type form from rat brain (Snutch et al., 1991) were generously

provided by Dr. Gregory Hockerman (Purdue Univ., West Lafayette, IN).

3.3.2 Site-Directed Mutagenesis

A mutation previously shown to be important for diltiazem potency, I1150A

(Hockerman et al., 2000), was inserted into rat brain wild-type Cav1.2 using the

QuikChange II XL Site-Directed Mutagenesis Kit (Stratagene Cloning Systems, La Jolla,

CA) and the following primers:

5’-GTGGAGATCTCCATCTTCTTCGCGATCTACATCATCATCATTGCC-3’

5’-GGCAATGATGATGATGTAGATCGCGAAGAAGATGGAGATCTC-3’

(I1150A mutation in bold). The manufacturer’s protocol was followed with a few

modifications. Briefly, 50 ng of Cav1.2 plasmid template was used in PCR with the

following parameters: 1 cycle: 95°C, 30 s; 18 cycles: 95°C, 50 s, 60°C, 50 s, 68°C, 18

min (1.5 min/kb); 1 cycle: 68°C, 7 min. Following Dpn I treatment, the entire PCR

product was ethanol precipitated, resuspended in 5 μL ddH2O, and transformed into

XL10-Gold® ultracompetent cells. Transformed cells were plated on LB agar dishes

containing 100 μg/mL ampicillin for >16 hrs at 37°C. Single colonies were grown for

>16 hrs in LB broth containing 100 μg/mL ampicillin, then plasmid DNA was extracted

using phenol/chloroform/isoamyl alcohol (25:24:1). Mutation was confirmed by

sequencing (Eurofins MWG Operon, Huntsville, AL). A clone positive for the mutation

was grown in LB broth containing 100 μg/mL ampicillin, and the plasmid DNA was

purified using QIAGEN Plasmid Maxi Kit (QIAGEN Inc., Valencia, CA).

3.3.3 Recording solutions

The external solution (300 mOsm) contained 135 mM cholineCl, 1 mM MgCl2, 5

mM BaCl2, and 10 mM HEPES, adjusted to pH 7.4 with TEAOH. As indicated in the

results, a few experiments used 2 mM Ca2+ as the charge carrier instead of 5 mM Ba2+.

Glass recording pipettes were pulled from micro-hematocrit capillary tubes (Fisher

Scientific, Pittsburgh, PA) to a resistance of 2-5 MΩ. To facilitate seal formation, recording pipette tips were filled with a lower osmolarity internal solution (270 mOsm)

lacking ATP. Pipettes were then back-filled with internal solution (285 mOsm)

containing: 135 mM CsCl, 4 mM MgCl2, 4 mM ATP, 10 mM HEPES, 10 mM EGTA,

and 1 mM EDTA, adjusted to pH 7.2 with TEAOH.

3.3.4 Whole-cell voltage-clamp electrophysiology

Red-fluorescing cells were visualized on an Olympus IX51 fluorescent microscope.

Whole-cell currents were recorded with an Axopatch 200A amplifier (Molecular

Devices, Sunnyvale, CA). Data was sampled at 2 – 10 kHz, low pass-filtered at 1 kHz,

and analyzed using pClamp v9.2 (Molecular Devices). Following pipette and membrane

capacitance compensation the series resistance was corrected by 80-90% with a 20-100

µs lag time. Cells with a series resistance greater than 20 MΩ were not used for analysis.

After whole-cell break-in, Ba2+ currents were elicited in voltage-clamp mode by stepping

from a holding potential (Vh) of -80 mV to test potentials (Vt) of 0 mV (Cav1.2) or -25

mV (Cav1.3) for 200 ms. Current rundown, plotted as total charge transfer elicited at 10 s

intervals (or higher frequencies, as indicated), was fitted with a one- or two-phase

exponential decay curve. Recordings using this protocol were not leak subtracted.

Drugs were applied by gravity flow and washed out rapidly by external solution

applied with a separate pipette. Percent inhibition was calculated with the equation: %

inhibition = (1 – [Iactual/Iexpected]) × 100, where Iactual was the actual current remaining

during steady-state inhibition by the drug, and Iexpected was the expected current remaining

at the same time-point based on the exponential decay fit of the current rundown.

Concentration-response curves were fitted with a four-parameter logistic equation: %

inhibition = % min + (% max – % min)/(1 + 10(log IC50 – log [drug]) × nH)), where % min

represented percent inhibition by vehicle, % max represented maximal percent inhibition

by the drug, IC50 was the drug concentration which yielded half-maximal inhibition, and

nH was the Hill slope. % min was constrained to zero, since average inhibition by vehicle

was not significantly different than zero. Current decay was measured as the ratio of

current remaining after a 200 ms depolarization relative to peak current, which was

defined as the r200 value, similar to previous studies (Cai et al., 1997).

For voltage-dependent activation and inactivation protocols, data obtained from each

voltage step was leak-subtracted online by removing the sum of the current obtained from

four consecutive waveforms of 1/4 amplitude and opposite polarity to the test potentials

(a P/4 protocol). Voltage-dependence of activation was determined using either 5 mM

Ba2+ or 2 mM Ca2+ as charge carriers. Channels were activated with 200 ms voltage steps

from Vh = -80 mV to various test potentials, Vt = -75 mV to +40 mV (Cav1.3) or Vt = -50

mV to +65 mV (Cav1.2) in 5 mV steps. Peak current plotted against the test potential was

fitted with the equation: I = Gmax(Vt - Vrev)/(1 + exp[(V50 - Vt)/k]), where Gmax was the maximal conductance of the cell, Vrev was the reversal potential, V50 was the potential for

half-maximal activation, and k was the slope factor of activation. As the test potential

approached Vrev, currents deviated from the linear relationship predicted by this equation.

Therefore, currents obtained at potentials more positive than 25 mV were excluded from

fitting analysis.

The voltage-dependence of L-VGCC steady-state inactivation was determined by

stepping to various conditioning pulse potentials for 1.5 s, preceded by a control pulse to

-25 mV (Cav1.3) or 0 mV (Cav1.2) and followed by a test pulse to -25 mV (Cav1.3) or 0 mV (Cav1.2). The current elicited by the control pulse was used to control for current rundown. Inactivation curves were plotted as normalized peak currents (test peak/control

peak) versus the conditioning pulse potential and fit with the Boltzmann equation: In =

In(min) + (In(max) – In(min))/(1 + exp[(V50 – Vt)/k]), where In was the normalized peak current,

In(min) was minimal normalized current, In(max) was maximal normalized current, V50 was the potential for half-maximal inactivation, and k was the slope factor of inactivation.

Drug effects on steady-inactivation were tested by directly incubating cells in external solution containing the drug for at least 5 min prior to running the inactivation protocol.

3.3.5 Drugs

The three BDZs tested were the prototype diazepam, the relatively water soluble flurazepam, and its major bioactive metabolite in rat and man, desalkylflurazepam (Lau et al., 1987). Flumazenil, a competitive BDZ antagonist, was also tested. Stocks of nimodipine, diazepam, desalkylflurazepam, flumazenil, and allopregnanolone were made in dimethylsulfoxide (DMSO). Stocks of (+)-cis-diltiazem, flurazepam (pH 5.8), and racemic pentobarbital were made in dH2O. Drug stocks were dissolved into external solution by vortexing. Drugs dissolved in DMSO were diluted at least 1:1000, except 300 mM diazepam and desalkylflurazepam stocks were diluted 1:300 to achieve the final concentration of 1 mM. DMSO diluted 1:1000 or 1:300 in external solution was used as a vehicle control for some experiments and resulted in negligible inhibition of currents

(mean inhibition typically < 5% in each experiment). Absolute ethanol (empirical density of 0.75 g/mL) was dissolved directly into external solution. Flurazepam was generously provided by the NIDA Drug Supply Program. Flumazenil and desalkylflurazepam were obtained from Roche, Nutley, NJ. Allopregnanolone was obtained from Tocris

Bioscience, Ellisville, MO. Ethanol was obtained from Pharmco-AAPER, Shelbyville,

KY. All other drugs were obtained from Sigma-Aldrich, St. Louis, MO.

3.3.6 Statistical analyses

All curve fits were made using GraphPad Prism v5 (GraphPad Software, Inc., La

Jolla, CA). Except for curve fit parameters, all data are reported as mean ± S.E.M. Curve fit parameters (i.e., IC50, Hill slope, V50, and slope factor k) are reported as the best fit value to the experimental data set with the associated 95% confidence interval from a

Levenberg-Marquardt least squares fitting algorithm. Non-overlapping confidence intervals between values were accepted as significant differences (p < 0.05). A two-tailed

Student’s t-test was used to compare percent inhibition by select drug concentrations between two experimental conditions. Threshold drug concentrations were defined as the lowest concentration to yield significant inhibition measured by repeated measures one- way ANOVA with post-hoc comparison by Dunnett’s test. Mean r200 values were analyzed by repeated measures one-way ANOVA with post-hoc comparison by

Dunnett’s test or by a paired t-test. A p-value of 0.05 or less was considered significant.

3.4 Results

3.4.1 Functional Characterization of Recombinant L-VGCCs

As a confirmation that the transient transfection of HEK293T cells resulted in

functional L-VGCCs, the voltage-dependence of activation was evaluated for both

Cav1.2- and Cav1.3-containing L-VGCCs and was similar to previous reports for these

channels (Koschak et al., 2001; Xu & Lipscombe, 2001). Fig. 2A shows Cav1.3 and

2+ 2+ Cav1.2 L-VGCC current-voltage responses, with peak 5 mM Ba or 2 mM Ca currents

(pA) normalized to cell capacitance (pF). Notably, activation of Cav1.3 channels occurred

at more negative potentials than Cav1.2 channels. Half-maximal inactivation also

occurred at more negative potentials in Cav1.3 compared to Cav1.2 channels (see Fig. 6

and Table 3). As an additional test of channel integrity and appropriate pharmacologic

response, channels were inhibited by the DHP, nimodipine in a concentration-dependent manner. As shown in Fig. 2B, Cav1.3 channels were less sensitive to inhibition by

nimodipine than Cav1.2 channels, consistent with previous reports of recombinant L-

VGCC inhibition by DHPs (Koschak et al., 2001; Xu & Lipscombe, 2001).

3.4.2 GABAAR Modulators Reversibly Inhibit Recombinant L-VGCCs

In control cells transfected with Cav1.3 L-VGCCs (n = 4) in which no drug was

applied, the average percent current remaining 2, 5, and 10 min from the start of

recording was 71 ± 11%, 57 ± 13%, and 42 ± 9%, respectively. This extent of rundown

was similar to that of Cav1.2 L-VGCCs in this study and as reported previously

(Kepplinger et al., 2000). There was no apparent correlation between the extent of rundown in different cells and drug concentration-response, suggesting that the mechanisms causing the rundown phenomenon were not a significant factor in modifying

2+ L-VGCC inhibition by GABAAR modulators. As seen in Fig. 3A, 5 mM Ba currents through Cav1.3 L-VGCCs were inhibited in a concentration-dependent manner by diazepam. Inhibition by all drugs tested showed at least partial current recovery upon

washout. Figs. 3B and 3C illustrate that inhibition of Cav1.2 and Cav1.3 L-VGCCs,

respectively, was rapid for most GABAAR modulators tested. For BDZs, pentobarbital,

and ethanol, a maximal response was typically obtained by the 3rd activating pulse after

drug application. Inhibition was slower for allopregnanolone and diltiazem, generally

requiring at least 4 activation pulses to reach maximal inhibition. For allopregnanolone

inhibition was slower in Cav1.2 channels, and for diltiazem inhibition was slower in

Cav1.3 channels. Inhibition of Cav1.3 channels by desalkylflurazepam was similar when 2

mM Ca2+ was used as the charge carrier (data not shown).

Figs. 4B and C show the concentration-response relationship of diltiazem and all

GABAAR modulators tested in Cav1.2 and Cav1.3 L-VGCCs, respectively. Threshold

concentrations, IC50 values, Hill slopes, and numbers of cells tested are reported in Table

1. BDZs had relatively similar potencies, except Cav1.3 channels were 1.5-fold less

sensitive to flurazepam than Cav1.2 channels (p < 0.05). The BDZs were about 2- to 6-

fold less potent than diltiazem, with Cav1.3 channels displaying 3-fold greater sensitivity

to diltiazem than Cav1.2 channels (p < 0.05). The BDZ receptor competitive antagonist,

flumazenil had no effect to inhibit Cav1.2 or Cav1.3 channels at concentrations up to 300

µM (data not shown). Flumazenil (100 – 300 µM) was unable to antagonize diazepam

inhibition of Cav1.2 channels (Table 1). Interestingly, co-application with 100 µM

flumazenil resulted in a 1.8-fold reduction in desalkylflurazepam potency in Cav1.3 channels (Table 1). Although allopregnanolone was the most potent GABAAR modulator

tested (about 6-fold more potent than BDZs), it incompletely inhibited L-VGCCs and

was less efficacious at inhibiting Cav1.3 than Cav1.2 channels (29% versus 63% maximal

response, respectively). Pentobarbital was about 7- to 21-fold less potent than BDZs.

Cav1.3 channels were 3-fold less sensitive to pentobarbital than Cav1.2 channels (p <

0.05). Ethanol was the least potent drug tested; about 4,500-fold less potent than BDZs.

3.4.3 GABAAR Receptor Modulators Enhance L-VGCC Current Decay

Inhibition by various L-VGCC antagonists is known to depend on the state of the

channel (Lee & Tsien, 1983; Bean, 1984; Uehara & Hume, 1985; Cai et al., 1997;

Hockerman et al., 1997). In particular, antagonists can stabilize the inactive state or display enhanced binding during channel opening (Hockerman et al., 1997), resulting in

increased current decay during prolonged depolarization. Fig. 5A illustrates that the

2+ decay of Cav1.3 Ba current is enhanced by diazepam. Fig. 5B shows that the ratio of

residual current at 200 ms to peak current (r200) was reduced in a concentration-

dependent manner by diazepam. Table 2 summarizes L-VGCC r200 values in the

presence and absence of diltiazem and GABAAR modulators at approximately equipotent

concentrations. A significant decrease in the r200 value of both L-VGCCs was observed

for all GABAAR modulators, except for flurazepam at Cav1.3 channels and ethanol at

Cav1.2 channels, due to variability and the low numbers of cells tested. By comparison, diltiazem at its IC50 concentration resulted in substantially less enhancement of L-VGCC

current decay.

3.4.4 GABAAR Modulators Induce a Negative Shift in L-VGCC Steady-State

Inactivation

The voltage-dependence of L-VGCC steady-state inactivation was measured as described in the methods in the presence and absence of drug. As illustrated in Fig. 6A, there was a significant shift in the voltage-dependence of Cav1.2 L-VGCC steady-state

inactivation towards more negative potentials in the presence of approximately

equipotent concentrations of diazepam (60 μM) and pentobarbital (300 μM), but not

allopregnanolone (30 μM) or ethanol (300 mM). The potentials of half-maximal

inactivation (V50) in the presence and absence of diltiazem and GABAAR modulators are

reported in Table 3. Notably, there was a concentration dependent effect of diazepam to

shift Cav1.2 inactivation towards more negative potentials, which was significant at 60

and 100 μM. 100 μM desalkylflurazepam caused a significant shift in the Cav1.2 inactivation curve, but was smaller in magnitude than the shift by 100 μM diazepam. A similar result was obtained with 30 μM diltiazem. Consistent with data obtained from 0.1

Hz activation, flumazenil (300 μM) did not shift Cav1.2 inactivation and was unable to

antagonize the shift by 100 μM diazepam.

Fig. 6B shows a drug-induced shift of Cav1.3 steady-state inactivation towards more negative potentials in the presence of approximately equipotent concentrations of diazepam (60 μM), allopregnanolone (30 μM), pentobarbital (1 mM), and ethanol (300 mM), although ethanol was weakly effective relative to the other drugs. As shown in

Table 3, 100 μM desalkylflurazepam caused a significant shift in the Cav1.3 inactivation curve, but was of smaller magnitude than the shift by 100 μM diazepam. Diltiazem at its

IC50 concentration of 10 μM was completely ineffective to shift the Cav1.3 inactivation

curve.

3.4.5 Inhibition of Cav1.2 Channels by Desalkylflurazepam is State- and Frequency-

Dependent

To test the hypothesis that BDZ potency is enhanced by channel inactivation, the

inhibitory action of desalkylflurazepam was measured on channels activated from a more

depolarized holding potential resulting in a greater extent of channels in the inactive state.

As illustrated in Fig. 7A, desalkylflurazepam inhibited Cav1.2 channels 2-fold more

potently when channels were activated from a more depolarized holding potential, with a

significantly greater degree of inhibition occurring at 30 and 100 µM desalkylflurazepam.

For Cav1.3 channels, which inactivate at more negative membrane potentials, a holding

potential of -60 mV was used. As illustrated in Fig. 7B, desalkylflurazepam was similarly

potent when Cav1.3 channels were activated from a more depolarized holding potential,

and the degree of inhibition was not significantly different at any concentration at or

above threshold.

To test whether BDZ potency might be similarly enhanced by the frequency of

channel activation, desalkylflurazepam potency was tested on L-VGCCs activated at 1

Hz. As illustrated in Fig. 7C, desalkylflurazepam inhibited Cav1.2 L-VGCCs 2-fold more

potently when channels were activated at 1 Hz compared to 0.1 Hz, with a significantly

greater degree of inhibition occurring at 10, 30, and 100 µM desalkylflurazepam. As

illustrated in Fig. 7D, inhibition of Cav1.3 L-VGCCs by desalkylflurazepam was not significantly different when channels were activated at 1 Hz compared to 0.1 Hz and showed no significant difference in inhibition at any concentration at or above threshold.

3.4.6 Two Amino Acids Important for DHP Potency Reduce Pentobarbital Potency

While the various classes of L-VGCC antagonists have unique binding sites, they also have overlapping amino acids that contribute to binding. Thus, it was of interest to see if single- and double-amino acid mutations already characterized to affect binding of specific L-VGCC antagonists, might also affect binding of GABAAR modulators.

Previous studies have found two amino acids in domain IIIS5 of Cav1.2, T1039 and

Q1043, which are critical for DHP affinity (Mitterdorfer et al., 1996; Hockerman et al.,

2000). Mutation of these two amino acids thus creates a DHP insensitive Cav1.2 (DHPI).

As can be seen in Fig. 8A, half maximal inhibition of wild-type (WT) Cav1.2 L-VGCCs occurred at about 0.1 μM nimodipine and maximal block at 1 μM (data represent inhibition at 0 mV test potential shown in Fig. 2B). In two cells transfected with DHPI L-

VGCCs, application of 1 μM nimodipine resulted in negligible inhibition (5 ± 14%).

Application of 10 μM nimodipine resulted in substantial inhibition (58 ± 3%), but was about 100-fold less potent than in WT, confirming that DHPI channels are resistant to

DHPs.

As shown in Fig. 8B, DHPI L-VGCCs were significantly less sensitive to inhibition by pentobarbital compared to WT. Although this suggests that T1039 and Q1043 may play a role in pentobarbital binding to Cav1.2, the 3.5-fold reduction in potency is quite small compared to the estimated 100-fold reduction in nimodipine potency. As shown in

Figs. 8C and E, inhibition of Cav1.2 channels by diazepam and ethanol, respectively was unaffected by mutation of the DHP binding site. Fig. 8D illustrates that DHPI channels were slightly more sensitive to allopregnanolone than WT Cav1.2 channels, but only 10

μM allopregnanolone resulted in significantly more inhibition.

It should be noted that the WT Cav1.2 used for comparison of nimodipine,

allopregnanolone, and ethanol inhibition was a mouse brain clone, whereas the DHPI

mutant was created from a rat brain Cav1.2 clone. Thus, the small difference in inhibition

by allopregnanolone might be due to species differences between the two channels.

Additional controls in rat WT Cav1.2 were obtained for diazepam, pentobarbital, and

diltiazem (see below).

3.4.7 A Single Amino Mutation that Affects Diltiazem Potency does not Affect

Diazepam Potency

Based on the structural similarity between BDZs and BTZs, it was hypothesized that

they might share a similar binding site at L-VGCCs. A single amino acid mutation,

I1150A, was previously shown to affect diltiazem potency, but not other classes of L-

VGCC antagonists (Hockerman et al., 2000). As shown in Fig. 9A, mutation of I1150 in rat Cav1.2 resulted in a significant 2-fold decrease in diltiazem potency, with a

significantly lower degree of inhibition by 10, 30, and 300 μM diltiazem. However, as

shown in Fig. 9B, diazepam potency was not significantly affected by the mutation, with

no difference in the degree of inhibition by any concentration tested. In order to confirm

that diazepam likely does not share a binding site with diltiazem, inhibition of WT Cav1.2 channels by diazepam was tested in the presence of an IC50 concentration of diltiazem (30

μM). As seen in Fig. 9C, the presence of diltiazem did not significantly affect inhibition at any diazepam concentration tested.

3.5 Discussion

3.5.1 GABAAR Modulator Pharmacology at L-VGCCs: Comparison to GABAARs

Prior studies investigated inhibition of native L-VGCCs by GABAAR modulators

either in non-neuronal or neuronal cells that also contain GABAARs and multiple types of

calcium channels, making it difficult to ascertain the selective effects of these drugs on

the neuronal L-VGCC subtypes, Cav1.2 and Cav1.3. The current studies analyzed the

inhibitory action of GABAAR modulators on recombinant neuronal Cav1.2 and Cav1.3 L-

VGCCs assembled with β3 and α2δ subunits. Inhibition by modulators was not due to an indirect action at the GABAAR, since HEK293T cells do not express functional

GABAARs (Davies et al., 2000).

The BDZs were about 3 orders of magnitude less potent (Thomas et al., 1997),

allopregnanolone about 1.5 orders of magnitude less potent (Maitra & Reynolds, 1998),

pentobarbital about 1 order of magnitude less potent (Thomas et al., 1997), and ethanol

slightly less than 1 order of magnitude less potent (Wallner et al., 2003) at L-VGCCs

than at GABAARs. Though diazepam was several-fold more potent at GABAARs than

flurazepam (Thomas et al., 1997), they displayed similar potency at L-VGCCs. The

competitive BDZ antagonist, flumazenil did not inhibit L-VGCCs, yet it antagonized

BDZ inhibition of Cav1.3, but not Cav1.2 channels. This suggests that the Cav1.3 α1 subunit may have a flumazenil binding site analogous to GABAARs.

3.5.2 GABAAR Modulator Manner of L-VGCC Inhibition: Comparison to L-VGCC

Antagonists

Maximal inhibition by L-VGCC antagonists is dependent upon channel use, with

PAAs displaying high levels of use-dependence, followed by BTZs, then DHPs, which

display little use-dependence (Lee & Tsien, 1983; Uehara & Hume, 1985). Although the

current studies were not designed to assess use-dependence, maximal inhibition by

BDZs, pentobarbital, and ethanol consistently required fewer activating pulses compared to inhibition by allopregnanolone and diltiazem (Fig. 3B and C). While these time-course differences may be due to differential kinetics of drug binding and/or drug action, it could

also suggest that the former compounds may not be as dependent on channel opening to

bind and can better access their L-VGCC binding sites when channels are in a resting

confirmation. Alternatively, antagonists that slow recovery from inactivation may result

in an accumulation of channels in the inactive state during repeated activation and have

greater effect at higher frequencies of activation (Uehara & Hume, 1985). Although

inhibition of Cav1.2 channels by desalkylflurazepam was slightly enhanced in a

frequency-dependent manner (Fig. 7), it is possible that these modulators have a limited

ability to slow recovery from inactivation relative to BTZs and PAAs (Uehara & Hume,

1985).

GABAAR modulators enhance L-VGCC current decay, similar to other L-VGCC

antagonists (Lee & Tsien, 1983; Cai et al., 1997). This could represent open channel

block and/or stabilization of the inactive state. In support of the latter possibility,

desalkylflurazepam potency was increased at Cav1.2 channels by recording paradigms

that enhance channel inactivation (Fig. 7). Additionally, GABAAR modulators induced a significant negative shift in the Cav1.3 steady-state inactivation curve, and BDZs and pentobarbital also induced a significant negative shift in the Cav1.2 steady-state

inactivation curves (Fig. 6 and Table 3). Altogether, the data provide evidence for state-

dependent block by GABAAR modulators, preferentially interacting with inactive and

possibly open states.

3.5.3 Cav1.2 and Cav1.3 L-VGCC Differential Sensitivity to GABAAR Modulators

Recombinant Cav1.3 channels have been shown to be less sensitive to inhibition by

DHPs than Cav1.2 channels (Koschak et al., 2001; Xu & Lipscombe, 2001). Native

Cav1.3 channels in auditory hair cells were also found to be less sensitive to inhibition by

BTZs and PAAs, though this effect may be cell-type specific (Tarabova et al., 2007).

Indeed, recombinant Cav1.3 channels were 3-fold more sensitive to diltiazem inhibition than Cav1.2 channels. For the GABAAR modulators, Cav1.3 channels were 1.5-fold less

sensitive to flurazepam and 3-fold less sensitive to pentobarbital than Cav1.2 channels.

Diazepam, desalkylflurazepam, allopregnanolone, and ethanol displayed no significant

differential potency at L-VGCCs. However, since desalkylflurazepam inhibition of

Cav1.2, but not Cav1.3 channels was increased by protocols that enhance inactivation

(Fig. 7), it is possible that Cav1.2 L-VGCCs may display greater sensitivity to the latter

compounds in a state-dependent manner as proposed previously for DHPs (Koschak et

al., 2001). Whether this differential state-dependent effect may be related to the greater

extent of inactivation observed for Cav1.2 than Cav1.3 channels (Table 2) or a more

specific BDZ interaction with the channels is currently unknown. The differential

potency of these drugs at Cav1.2 and Cav1.3 channels may allow them to selectively

modify distinct L-VGCC subunit-mediated neuronal functions. In particular, reduced

Cav1.2 channel function impairs hippocampal spatial memory, whereas reduced Cav1.3 channel function affects neuronal firing patterns and may be protective in reducing age-

related neuronal degeneration, as occurs in Parkinson’s disease (Striessnig & Koschak,

2008).

3.5.4 GABAAR Modulator Site of Action

Organic L-VGCC antagonists have unique binding sites on the Cav1.2 subunit that

share a number of overlapping amino acids (Hockerman et al., 1997). Thus, it was of interest to see if amino acid mutations that are known to affect binding of specific L-

VGCC antagonists could similarly reduce inhibition by GABAAR modulators. Two

Cav1.2 mutants were used in the current studies: a double amino acid mutation, T1039Y

and Q1043M, that reduces DHP potency (Mitterdorfer et al., 1996; Hockerman et al.,

2000), and a single mutation, I1150A, that reduces diltiazem potency (Hockerman et al.,

2000). Neither mutation affected diazepam potency, suggesting that BDZs may have an

L-VGCC binding site distinct from DHPs and BTZs. Additionally, diltiazem did not

competitively interfere with diazepam inhibition of Cav1.2 channels and these

compounds were somewhat dissimilar in their manner of block in terms of state- and use-

dependence. Thus, despite the structural similarity between BDZs and BTZs and their

relatively similar potencies, the evidence suggests that these drugs act via distinct sites.

Interestingly, pentobarbital potency was significantly reduced 3.5-fold by the DHP

mutant, suggesting that the pentobarbital binding site on Cav1.2 might overlap that of

DHPs.

3.5.5 Clinical Relevance of L-VGCC Inhibition by GABAAR Modulators and

Implications for Physical Dependence

Calcium influx through postsynaptic L-VGCCs is linked to neuronal activity and

gene transcription, and L-VGCC inhibition has anxiolytic, antidepressant, and

anticonvulsant actions (Striessnig et al., 2006). Thus, inhibition of neuronal L-VGCCs

could play a role in the acute clinical actions of GABAAR modulators. Further, chronic

inhibition of L-VGCCs may lead to an adaptive upregulation of L-VGCC function during

prolonged GABAAR modulator treatment and contribute to physical dependence (Dolin

et al., 1987; Rabbani & Little, 1999; Xiang & Tietz, 2007; Xiang et al., 2008).

Free plasma and cerebrospinal fluid concentrations of BDZs, barbiturates, and ethanol exist in a ~1:1 ratio (Richards, 1972; Hallstrom et al., 1980; Harris et al., 2008). Taking into account extensive binding (>97%) of diazepam and its active metabolite, desmethyldiazepam to plasma proteins (Hallstrom et al., 1980; Divoll & Greenblatt,

1981), free plasma concentrations of about 20 – 60 nM are necessary for acute relief of convulsant activity (Rey et al., 1999) and about 50 – 100 nM to produce sedation

(Lundgren, 1987). Due to rapid tolerance to the sedative effects of diazepam, higher concentrations up to 200 nM are achieved during chronic treatment for psychiatric therapy (Rutherford et al., 1978; Hallstrom et al., 1980; Greenblatt et al., 1981). Since the threshold concentration of BDZs to significantly inhibit recombinant L-VGCCs was about 10 μM, inhibition of neuronal L-VGCCs is unlikely to contribute to BDZ clinical

actions. However, studies in hippocampal cultures revealed that 1 µM flurazepam

significantly inhibited VGCC current in a use-dependent manner (Xiang et al., 2008),

suggesting that greater potency can be observed in neuronal systems. The threshold concentration of allopregnanolone to inhibit L-VGCCs was 3 µM, which is much greater than the 5 nM concentration estimated to be produced from progesterone in the brain

(Uzunova et al., 1998). The threshold concentration of pentobarbital to inhibit L-VGCCs was 100 μM, within the 200 μM range required to induce anesthesia (Richards, 1972).

Unlike pentobarbital, phenobarbital has anticonvulsant actions with minimal sedative

effects at 20 to 90 μM (Schulz & Macdonald, 1981). It would be of interest to see if

phenobarbital inhibits L-VGCCs at concentrations required for anticonvulsant activity, or

if L-VGCC inhibition is more likely to pertain to the anesthetic effects of barbiturates

(ffrench-Mullen et al., 1993). The threshold concentration of ethanol to inhibit L-VGCCs

was 30 mM, within range of intoxicating ethanol concentrations, 10 to 100 mM (Harris et

al., 2008). Thus, inhibition of L-VGCCs may attribute to ethanol’s intoxicating effects,

including mood alteration and memory impairment.

The enhancement of neuronal L-VGCC-mediated Ca2+ influx observed following

chronic BDZ exposure is unlikely to be due to a direct effect at L-VGCCs, since this

upregulation occurs with BDZ concentrations as low as 0.3 μM in vitro (Katsura et al.,

2007) and ~1 μM measured in rat brain homogenates during chronic BDZ treatment in

vivo (Xiang et al., 2008). Thus, persistent allosteric enhancement of GABAARs seems the

most plausible mechanism associated with L-VGCC regulation during chronic BDZ

administration, since L-VGCC upregulation in cortical cultures can be prevented by

nanomolar concentrations of the BDZ competitive antagonist, flumazenil (Katsura et al.,

2007). In addition, a bicarbonate-dependent GABAAR-mediated depolarization that arises during BDZ withdrawal (Zeng & Tietz, 2000) may contribute to the hyperexcitable state

(Van Sickle et al., 2004), which could exacerbate the downstream Ca2+-mediated effects

of L-VGCC upregulation. As with other GABAAR modulators, withdrawal from chronic

exposure to allopregnanolone is associated with a withdrawal syndrome characterized by

anxiety and increased seizure susceptibility (Smith, 2002). However, a role for L-VGCC upregulation in this phenomenon has not been established. Inhibition of L-VGCCs may

67 contribute to the upregulation of L-VGCC function following chronic exposure to barbiturates (Rabbani & Little, 1999), as well as ethanol (Messing et al., 1986; Katsura et al., 2006). Collectively, these findings may provide a molecular basis for the adaptive changes that contribute to drug withdrawal symptoms and physical dependence on

GABAAR modulators.

3.6 Footnotes

This work was supported by the National Institutes of Health National Institute of Drug

Abuse [Grant RO1-DA184342 (EIT) and individual NRSA predoctoral fellowship F30-

DA026675 (DEE)].

Portions of this work were previously presented: Earl DE, Franklin JD, Boulineau N, and

Tietz EI, Direct inhibition of recombinant L-type voltage-gated calcium channel currents by benzodiazepines, in 2008 Abstract Viewer, 2008 Nov 15–19; Washington, DC. Vol

133, pp 13, Society for Neuroscience, Washington, DC; Earl DE and Tietz EI, Direct, concentration-dependent inhibition of recombinant neuronal L-type voltage-gated calcium channel currents by GABA-A receptor modulators, in 2009 Abstract Viewer,

2009 Oct 17–21; Chicago, IL. Vol 416 pp 17, Society for Neuroscience, Washington,

DC.

3.7 Acknowledgements

We thank Drs. L. John Greenfield, Jr., David Giovannucci, Paromita Das, and Nathalie

Boulineau for technical assistance and advice with plasmid preparation, transient transfection procedures, and experimental design. We thank Dr. Scott Molitor for critical review of the data analyses and manuscript and Drs. Diane Lipscombe and Gregory

Hockermann for the generous contribution of L-VGCC subunit cDNA plasmids.

3.8 References

Bean BP (1984) Nitrendipine block of cardiac calcium channels: high-affinity binding to

the inactivated state. Proc Natl Acad Sci U S A 81:6388-6392.

Cai D, Mulle JG, and Yue DT (1997) Inhibition of recombinant Ca2+ channels by

benzothiazepines and phenylalkylamines: class-specific pharmacology and underlying

molecular determinants. Mol Pharmacol 51:872-881.

Davies PA, Hoffmann EB, Carlisle HJ, Tyndale RF, and Hales TG (2000) The influence

of an endogenous beta3 subunit on recombinant GABA(A) receptor assembly and

pharmacology in WSS-1 cells and transiently transfected HEK293 cells.

Neuropharmacology 39:611-620.

Divoll M and Greenblatt DJ (1981) Binding of diazepam and desmethyldiazepam to

plasma protein: concentration-dependence and interactions. Psychopharmacology

(Berl) 75:380-382.

Dolin S, Little H, Hudspith M, Pagonis C, and Littleton J (1987) Increased

dihydropyridine-sensitive calcium channels in rat brain may underlie ethanol physical

dependence. Neuropharmacology 26:275-279.

ffrench-Mullen JM, Barker JL, and Rogawski MA (1993) Calcium current block by (-)-

pentobarbital, phenobarbital, and CHEB but not (+)-pentobarbital in acutely isolated

hippocampal CA1 neurons: comparison with effects on GABA-activated Cl- current. J

Neurosci 13:3211-3221.

Greenblatt DJ, Laughren TP, Allen MD, Harmatz JS, and Shader RI (1981) Plasma

diazepam and desmethyldiazepam concentrations during long-term diazepam therapy.

Br J Clin Pharmacol 11:35-40.

Hallstrom C, Lader MH, and Curry SH (1980) Diazepam and N-desmethyldiazepam

concentrations in saliva, plasma and CSF. Br J Clin Pharmacol 9:333-339.

Harris RA, Trudell JR, and Mihic SJ (2008) Ethanol's molecular targets. Sci Signal 1:re7.

Helton TD, Xu W, and Lipscombe D (2005) Neuronal L-type calcium channels open

quickly and are inhibited slowly. J Neurosci 25:10247–10251.

Hockerman GH, Dilmac N, Scheuer T, and Catterall WA (2000) Molecular determinants

of diltiazem block in domains IIIS6 and IVS6 of L-type Ca(2+) channels. Mol

Pharmacol 58:1264-1270.

Hockerman GH, Peterson BZ, Johnson BD, and Catterall WA (1997) Molecular

determinants of drug binding and action on L-type calcium channels. Annu Rev

Pharmacol Toxicol 37:361-396.

Hu AQ, Wang ZM, Lan DM, Fu YM, Zhu YH, Dong Y, and Zheng P (2007) Inhibition

of evoked glutamate release by neurosteroid allopregnanolone via inhibition of L-type

calcium channels in rat medial prefrontal cortex. Neuropsychopharmacology 32:1477-

1489.

Katsura M, Shibasaki M, Hayashida S, Torigoe F, Tsujimura A, and Ohkuma S (2006)

Increase in expression of alpha1 and alpha2/delta1 subunits of L-type high voltage-

gated calcium channels after sustained ethanol exposure in cerebral cortical neurons. J

Pharmacol Sci 102:221-230.

Katsura M, Shibasaki M, Kurokawa K, Tsujimura A, and Ohkuma S (2007) Up-

regulation of L-type high voltage-gated calcium channel subunits by sustained exposure

to 1,4- and 1,5-benzodiazepines in cerebrocortical neurons. J Neurochem 103:2518-

2528.

Kepplinger KJ, Forstner G, Kahr H, Leitner K, Pammer P, Groschner K, Soldatov NM,

and Romanin C (2000) Molecular determinant for run-down of L-type Ca2+ channels

localized in the carboxyl terminus of the 1C subunit. J Physiol 529 Pt 1:119-130.

Koschak A, Reimer D, Huber I, Grabner M, Glossmann H, Engel J, and Striessnig J

(2001) alpha 1D (Cav1.3) subunits can form l-type Ca2+ channels activating at

negative voltages. J Biol Chem 276:22100-22106.

Lau CE, Falk JL, Dolan S, and Tang M (1987) Simultaneous determination of flurazepam

and five metabolites in serum by high-performance liquid chromatography and its

application to pharmacokinetic studies in rats. J Chromatogr 423:251-259.

Lee KS and Tsien RW (1983) Mechanism of calcium channel blockade by verapamil,

D600, diltiazem and nitrendipine in single dialysed heart cells. Nature 302:790-794.

Lin Y, McDonough SI, and Lipscombe D (2004) Alternative splicing in the

voltagesensing region of N-type Cav2.2 channels modulates channel kinetics. J

Neurophysiol 92:2820–2830.

Lundgren S (1987) Serum concentration and drug effect after intravenous and rectal

administration of diazepam. Anesth Prog 34:128-133.

Maitra R and Reynolds JN (1998) Modulation of GABA(A) receptor function by

neuroactive steroids: evidence for heterogeneity of steroid sensitivity of recombinant

GABA(A) receptor isoforms. Can J Physiol Pharmacol 76:909-920.

Messing RO, Carpenter CL, Diamond I, and Greenberg DA (1986) Ethanol regulates

calcium channels in clonal neural cells. Proc Natl Acad Sci U S A 83:6213-6215.

Mitterdorfer J, Wang Z, Sinnegger MJ, Hering S, Striessnig J, Grabner M, and

Glossmann H (1996) Two amino acid residues in the IIIS5 segment of L-type calcium

channels differentially contribute to 1,4-dihydropyridine sensitivity. J Biol Chem

271:30330-30335.

Rabbani M and Little HJ (1999) Increases in neuronal Ca2+ flux after withdrawal from

chronic barbiturate treatment. Eur J Pharmacol 364:221-227.

Rey E, Treluyer JM, and Pons G (1999) Pharmacokinetic optimization of benzodiazepine

therapy for acute seizures. Focus on delivery routes. Clin Pharmacokinet 36:409-424.

Richards CD (1972) On the mechanism of barbiturate anaesthesia. J Physiol 227:749-

767.

Rutherford DM, Okoko A, and Tyrer PJ (1978) Plasma concentrations of diazepam and

desmethyldiazepam during chronic diazepam therapy. Br J Clin Pharmacol 6:69-73.

Schulz DW and Macdonald RL (1981) Barbiturate enhancement of GABA-mediated

inhibition and activation of chloride ion conductance: correlation with anticonvulsant

and anesthetic actions. Brain Res 209:177-188.

Smith SS (2002) Withdrawal properties of a neuroactive steroid: implications for

GABA(A) receptor gene regulation in the brain and anxiety behavior. Steroids 67:519-

528.

Snutch TP, Tomlinson WJ, Leonard JP, and Gilbert MM (1991) Distinct calcium

channels are generated by alternative splicing and are differentially expressed in the

mammalian CNS. Neuron 7:45-57.

Striessnig J and Koschak A (2008) Exploring the function and pharmacotherapeutic

potential of voltage-gated Ca2+ channels with gene knockout models. Channels

(Austin) 2:233-251.

Striessnig J, Koschak A, Sinnegger-Brauns MJ, Hetzenauer A, Nguyen NK, Busquet P,

Pelster G, and Singewald N (2006) Role of voltage-gated L-type Ca2+ channel

isoforms for brain function. Biochem Soc Trans 34:903-909.

Tarabova B, Lacinova L, and Engel J (2007) Effects of phenylalkylamines and

benzothiazepines on Ca(v)1.3-mediated Ca2+ currents in neonatal mouse inner hair

cells. Eur J Pharmacol 573:39-48.

Thomas P, Sundaram H, Krishek BJ, Chazot P, Xie X, Bevan P, Brocchini SJ, Latham

CJ, Charlton P, Moore M, Lewis SJ, Thornton DM, Stephenson FA, and Smart TG

(1997) Regulation of neuronal and recombinant GABA(A) receptor ion channels by

xenovulene A, a natural product isolated from Acremonium strictum. J Pharmacol Exp

Ther 282:513-520.

Uehara A and Hume JR (1985) Interactions of organic calcium channel antagonists with

calcium channels in single frog atrial cells. J Gen Physiol 85:621-647.

Uzunova V, Sheline Y, Davis JM, Rasmusson A, Uzunov DP, Costa E, and Guidotti A

(1998) Increase in the cerebrospinal fluid content of neurosteroids in patients with

unipolar major depression who are receiving fluoxetine or fluvoxamine. Proc Natl

Acad Sci U S A 95:3239-3244.

Van Sickle BJ, Xiang K, and Tietz EI (2004) Transient plasticity of hippocampal CA1

neuron glutamate receptors contributes to benzodiazepine withdrawal-anxiety.

Neuropsychopharmacology 29:1994-2006.

Wallner M, Hanchar HJ, and Olsen RW (2003) Ethanol enhances alpha 4 beta 3 delta and

alpha 6 beta 3 delta gamma-aminobutyric acid type A receptors at low concentrations

known to affect humans. Proc Natl Acad Sci U S A 100:15218-15223.

Xiang K, Earl DE, Davis KM, Giovannucci DR, Greenfield LJ, Jr., and Tietz EI (2008)

Chronic benzodiazepine administration potentiates high voltage-activated calcium

currents in hippocampal CA1 neurons. J Pharmacol Exp Ther 327:872-883.

Xiang K and Tietz EI (2007) Benzodiazepine-induced hippocampal CA1 neuron alpha-

amino-3-hydroxy-5-methylisoxasole-4-propionic acid (AMPA) receptor plasticity

linked to severity of withdrawal anxiety: differential role of voltage-gated calcium

channels and N-methyl-D-aspartic acid receptors. Behav Pharmacol 18:447-460.

Xu W and Lipscombe D (2001) Neuronal Ca(V)1.3alpha(1) L-type channels activate at

relatively hyperpolarized membrane potentials and are incompletely inhibited by

dihydropyridines. J Neurosci 21:5944-5951.

Yamakage M, Matsuzaki T, Tsujiguchi N, Honma Y, and Namiki A (1999) Inhibitory

effects of diazepam and midazolam on Ca2+ and K+ channels in canine tracheal

smooth muscle cells. Anesthesiology 90:197-207.

Zeng XJ and Tietz EI (2000) Role of bicarbonate ion in mediating decreased synaptic

conductance in benzodiazepine tolerant hippocampal CA1 pyramidal neurons. Brain

Res 868:202-214.

3.9 Figures and Tables

Figure 3.1

Chemical structures of the compounds used in the current studies (with the exception of ethanol). Note the similarity in the core structure of the BTZ, diltiazem to the BDZs.

Structures were created using CS ChemDraw software, version 5.0 (CambridgeSoft

Corporation, Cambridge, MA).

Figure 3.2

Functional expression of recombinant L-VGCCs in HEK293T cells. A, the average

current density (picoamperes/picofarads, pA/pF) of recombinant Cav1.3 (squares) and

Cav1.2 (circles) L-VGCCs expressed with β3 and α2δ-1 subunits in HEK293T cells is shown plotted against the membrane test potential using either 5 mM Ba2+ (open

symbols) or 2 mM Ca2+ (filled symbols) as the charge carrier. Using Ba2+ as the charge

carrier, potentials of half-maximal activation (V50) were -41.3 ± 2.1 mV (n = 15) and -

2+ 12.9 ± 3.8 mV (n = 6) for Cav1.3 and Cav1.2 channels, respectively. Using Ca as the charge carrier, activation V50 values were -35.3 ± 4.3 mV (n = 6) and -12.5 ± 5.1 mV (n =

2+ 8) for Cav1.3 and Cav1.2 channels, respectively. B, average peak Ba currents through

Cav1.3 channels in the absence (open squares, n = 3) and presence of 1 µM (half-filled

squares, n = 3) or 10 µM nimodipine (filled squares, n = 3) and Cav1.2 channels in the

absence (open circles, n = 2) and presence of 0.1 µM (half-filled circles, n = 2) or 1 µM

nimodipine (filled circles, n = 2).

Figure 3.3

2+ A, concentration-dependent inhibition of Cav1.3 Ba currents by diazepam. Cav1.3 L-

VGCCs were activated by 200 ms test pulses to −25 mV every 10 s from a holding

potential of −80 mV. The total charge transfer is normalized to its initial value and

plotted as a function of time post break-in. Diazepam inhibited Cav1.3 currents in a

concentration-dependent manner and was reversible upon washout. VEH represents

1:1000 DMSO. Exemplar current traces in the presence and absence of diazepam are

shown above. Normalized, rundown-corrected time-courses of Cav1.2 (B) and Cav1.3 (C)

L-VGCC inhibition by diltiazem (filled circles, 30 and 10 µM for Cav1.2 and Cav1.3, respectively), flurazepam (open triangles, 30 µM), pentobarbital (half-filled diamonds,

0.3 and 1 mM for Cav1.2 and Cav1.3, respectively), ethanol (half-filled circles, 300 mM),

and allopregnanolone (half-filled squares, 10 µM). Time point zero represents current just

prior to drug application. Because the time-courses were taken from the middle of a

concentration-response, current at time point zero had not in all cases completely

recovered from the prior concentration of drug applied. Thus, inhibition that occurs

during the first pulse after drug application (10 sec) does not accurately represent closed-

channel block. Numbers of cells tested are the same as shown in Table 1.

Figure 3.4

Concentration-response curves representing inhibition of Cav1.2 (A) and Cav1.3 (B) L-

VGCCs by GABAAR modulators. Diltiazem (filled circles) is shown for comparison. The

rank order of potency amongst GABAAR modulators was allopregnanolone (half-filled

squares) > BDZs > pentobarbital (half-filled diamonds) > ethanol (half-filled circles).

The BDZs tested, diazepam (open circles), flurazepam (open triangles), and

desalkylflurazepam (open squares), were about 2- to 6-fold less potent than diltiazem.

Cav1.2 and Cav1.3 L-VGCCs had similar sensitivities to inhibition by diazepam, desalkylflurazepam, allopregnanolone, and ethanol. Cav1.3 channels were 1.5-fold less

sensitive to flurazepam, 3-fold less sensitive to pentobarbital, and 3-fold more sensitive to

diltiazem than Cav1.2 channels. VEH represents inhibition by vehicle. IC50 values, Hill

slopes, and numbers of cells tested are reported in Table 1.

Figure 3.5

Diazepam enhances L-VGCC current decay. A, current traces from the same cell shown

2+ in Fig. 3A are normalized to peak current. Diazepam enhanced Cav1.3 Ba current decay during prolonged depolarization in a concentration-dependent manner. B, the ratio of residual current at 200 ms normalized to peak current (r200 value) is shown for various diazepam concentrations. 30 and 100 μM diazepam significantly reduced the r200 value compared to vehicle (n = 3). This effect was reversed upon washout. *, p < 0.05.

Figure 3.6

L-VGCC voltage-dependent steady-state inactivation curves in the presence and absence of GABAAR modulators. Normalized peak currents are plotted against the membrane potential of a 1.5 s conditioning pulse. A, the Cav1.2 steady-state inactivation curve was significantly shifted towards more negative potentials by approximately equipotent concentrations of diazepam (open circles, 60 μM) and pentobarbital (half-filled diamonds, 300 μM), but not by allopregnanolone (half-filled squares, 30 μM) or ethanol

(half-filled circles, 300 mM). The V50 values (potential of half-maximal inactivation) in the presence and absence (filled circles) of each drug are plotted in the inset. Error bars were omitted for clarity in both A and B. *, p < 0.05. B, the Cav1.3 steady-state inactivation curve was significantly shifted towards more negative potentials by approximately equipotent concentrations of diazepam (60 μM), allopregnanolone (30

μM), pentobarbital (1 mM), and ethanol (300 mM). The inactivation V50 values are plotted in the inset. *, p < 0.05 relative to control V50. Inactivation V50 values, slope factors, and numbers of cells tested are reported in Table 3.

Figure 3.7

Cav1.2 L-VGCCs are inhibited by desalkylflurazepam in a state- (A and B) and frequency-dependent (C and D) manner. A, desalkylflurazepam (30 and 100 μM) inhibited significantly more current when Cav1.2 channels were activated from -45 mV

(open circles) compared to activation from -80 mV (filled circles). The inset illustrates a corresponding significant 2-fold decrease in the desalkylflurazepam IC50 value at Vh = -

45 mV (28 µM, 23 – 34 µM, n = 4) compared to Vh = -80 mV (55 µM, 45 – 68 µM, n =

3). VEH represents inhibition by vehicle. *, p < 0.05, **, p < 0.01. B, inhibition by desalkylflurazepam was not significantly different when Cav1.3 channels were activated from -60 mV (open circles) compared to -80 mV (filled circles). The inset illustrates the similarity in desalkylflurazepam IC50 values at Vh = -60 mV (32 µM, 13 – 80 µM, n = 4) and Vh = -80 mV (37 µM, 29 – 48 µM, n = 6). C, desalkylflurazepam (10, 30, and 100

μM) inhibited significantly more current when Cav1.2 channels were activated at 1 Hz

(open circles) compared to activation at 0.1 Hz (filled circles). The inset illustrates a corresponding significant 2-fold decrease in the desalkylflurazepam IC50 value at 1 Hz

(28 µM, 21 – 37 µM, n = 4) compared to 0.1 Hz activation (55 µM, 45 – 68 µM, n = 3).

*, p < 0.05. D, inhibition by desalkylflurazepam was not significantly different when

Cav1.3 L-VGCCs were activated at 1 Hz (open circles) compared to 0.1 Hz (filled circles). The inset illustrates the similarity in desalkylflurazepam IC50 values at 1 Hz (35

µM, 17 – 70 µM, n = 3) and 0.1 Hz activation (37 µM, 29 – 48 µM, n = 6).

Figure 3.8

DHPI contains mutations of two amino acids important for high-affinity DHP binding,

T1039Y and Q1043M. The sensitivity of DHPI L-VGCCs to GABAAR modulators was

tested. A, DHPI channels were about 100-fold less sensitive to inhibition by nimodipine

compared to WT Cav1.2 channels. B, DHPI channels were significantly less sensitive to

inhibition by pentobarbital compared to WT Cav1.2 channels. The inset illustrates a

significant 3.5-fold increase the pentobarbital IC50 value in DHPI (758 µM, 579 – 992

µM, n = 4) compared to WT Cav1.2 channels (215 µM, 167 – 276 µM, n = 4). VEH

represents inhibition by vehicle. *, p < 0.05, **, p < 0.01, ***, p < 0.001. C, no difference

in inhibition by diazepam was observed between WT and DHPI channels. Diazepam IC50 values for WT (41 µM, 31 – 55 µM, n = 4) and DHPI channels (48 µM, 32 – 73 µM, n =

3) are shown in the inset. D, DHPI channels were slightly more sensitive to inhibition by allopregnanolone than WT Cav1.2 channels, with 10 µM allopregnanolone inhibiting significantly more current through DHPI channels. As shown in the inset, there was no significant difference in allopregnanolone potency to inhibit in DHPI (3 µM, 2 – 5 µM, n

= 3) compared to WT channels (7 µM, 3 – 16 µM, n = 5). E, no difference in inhibition

by ethanol was observed between WT and DHPI channels. Ethanol IC50 values for WT

(183 mM, 46 – 723 mM, n = 3) and DHPI channels (157 mM, 117 – 216 mM, n = 3) are

shown in the inset.

Figure 3.9

A single amino acid mutation in Cav1.2, I1150A reduces diltiazem, but not diazepam

potency. A, as reportedly previously (Hockerman et al., 2000), the I1150A mutation

reduced diltiazem potency, with significantly less inhibition by 10, 30 and 300 µM

diltiazem. The inset illustrates a significant 2-fold increase the diltiazem IC50 value in

I1150A (53 µM, 40 – 70 µM, n = 6) compared to WT channels (26 µM, 21 – 32 µM, n =

4). VEH represents inhibition by vehicle. *, p < 0.05, **, p < 0.01. B, the I1150A mutation had no effect on diazepam potency. Diazepam IC50 values for WT (41 µM, 31 –

55 µM, n = 4) and I1150A channels (50 µM, 32 – 77 µM, n = 4) are shown in the inset.

C, as a confirmation that diazepam likely does not share a binding site with diltiazem,

inhibition of WT Cav1.2 channels by diazepam was assessed in the presence of an IC50 concentration of diltiazem (30 µM). There was no significant difference in inhibition by diazepam at any concentration tested when compared to inhibition in the absence of diltiazem. The inset illustrates that there was no significant difference in diazepam potency in the presence (38 µM, 21 – 68 µM, n = 3) compared to the absence of diltiazem

(49 µM, 38 – 64 µM, n = 4).

Table 3.1 Parameters for concentration-dependent inhibition of L-VGCCs by diltiazem and GABAAR modulators

Cav1.2 Cav1.3 a a Drug Threshold IC50 IC50 95% CI Hill slope n Threshold IC50 IC50 95% CI Hill slope n µM µM diltiazem 1 26* 21 – 32 1.0 ± 0.2 3 1 8 6 – 12 1.1 ± 0.3 4 diazepam 30 49 38 – 64 1.5 ± 0.4 4 10 53 20 – 138 1.3 ± 0.7 3 flurazepam 10 48* 40 – 57 1.2 ± 0.2 4 10 72 59 – 88 1.2 ± 0.2 3 desalkylflurazepam 10 55 45 – 68 1.3 ± 0.3 3 10 37 29 – 48 1.1 ± 0.3 6 diazepam N.D. 66 56 – 77 1.5 ± 0.3 2 N.D. N.D. N.D. N.D. +100 µM flumazenil diazepam 10 56 39 – 79 1.1 ± 0.4 5 N.D. N.D. N.D. N.D. +300 µM flumazenil desalkylflurazepam N.D. N.D. N.D. N.D. 10 65† 51 – 82 1.3 ± 0.3 8

+100 µM flumazenil 95 allopregnanolone 3 7 3 – 16 0.8 ± 0.4 5 10 11 4 – 30 1.0 ± 0.6 3 pentobarbital 100 336* 266 – 424 1.0 ± 0.2 3 100 1053 860 – 1291 1.5 ± 0.4 4 ethanol 30000 182523 46091 – 722802 1.4 ± 0.9 3 30000 276452 35612 – 2146000 1.0 ± 0.5 3 a IC50 values can be considered absolute for all drugs except allopregnanolone, which has a relative IC50 since maximal L-VGCC inhibition by allopregnanolone was less than 100%.

N.D., not determined

*, p < 0.05, compared to respective Cav1.3 IC50 value.

† , p < 0.05, compared to Cav1.3 desalkylflurazepam IC50 value in the absence of flumazenil.

Table 3.2 GABAAR modulators enhance L-VGCC current decay

Ca 1.2 Ca 1.3 Drug v v CON r200c Drug r200 % Dec.d n CON r200c Drug r200 % Dec.d n 30/10 μM diltiazema 0.53 ± 0.03 0.45 ± 0.06 15 ± 7% 3 0.73 ± 0.06 0.76 ± 0.05 -5 ± 4% 4 30 μM diazepam 0.49 ± 0.09 0.26 ± 0.08** 50 ± 7% 4 0.68 ± 0.07 0.49 ± 0.07* 28 ± 5% 3 30 μM flurazepam 0.35 ± 0.09 0.25 ± 0.07* 31 ± 4% 4 0.79 ± 0.07 0.64 ± 0.10 19 ± 6% 3 30 μM desalkylflurazepam 0.36 ± 0.08 0.26 ± 0.07* 30 ± 5% 3 0.49 ± 0.07 0.35 ± 0.08** 32 ± 7% 6 30 μM allopregnanolone 0.50 ± 0.08 0.24 ± 0.06*** 56 ± 5% 5 0.66 ± 0.11 0.57 ± 0.11* 15 ± 3% 3 0.3/1 mM pentobarbitalb 0.44 ± 0.10 0.19 ± 0.06* 58 ± 5% 3 0.69 ± 0.09 0.37 ± 0.11** 49 ± 8% 4 300 mM ethanol 0.29 ± 0.12 0.12 ± 0.05 62 ± 4% 3 0.54 ± 0.04 0.45 ± 0.04** 16 ± 2% 3 a

30 and 10 μM diltiazem were analyzed for Cav1.2 and Cav1.3, respectively. 96 b 0.3 and 1 mM pentobarbital were analyzed for Cav1.2 and Cav1.3, respectively. c r200 is the ratio of current remaining after a 200 ms depolarization relative to the peak current. Control r200 values were obtained in the same cells in the absence of drug. d Average percent drug-induced decrease in the r200 value.

*, p < 0.05, **, p < 0.01, ***, p < 0.001, Drug versus CON r200, paired t-test.

Table 3.3 Negative shift in L-VGCC steady-state inactivation by GABAAR modulators

Ca 1.2 Ca 1.3 Drug(s) v v V50 k n V50 k n control -25.7 ± 1.7 7.6 ± 1.6 8 -46.1 ± 1.6 5.4 ± 1.5 10 30/10 μM diltiazema -29.9 ± 1.9* 7.7 ± 1.8 3 -45.4 ± 3.3 6.8 ± 3.1 3 30 μM diazepam -28.0 ± 1.6 6.9 ± 1.5 5 -50.2 ± 3.2 5.2 ± 2.9 4 60 μM diazepam -33.5 ± 1.1* 7.4 ± 1.0 6 -59.2 ± 2.7* 7.1 ± 2.4 6 100 μM diazepam -36.9 ± 1.3* 6.7 ± 1.1 3 -55.1 ± 0.9* 4.8 ± 0.8 6 100 μM desalkylflurazepam -30.9 ± 2.0* 9.0 ± 2.0 8 -50.9 ± 1.1* 5.6 ± 1.1 10 300 μM flumazenil -27.2 ± 2.9 9.6 ± 3.0 3 N.D. N.D. 100 µM diazepam + 300 µM flumazenil -37.3 ± 1.2* 6.6 ± 1.1 3 N.D. N.D.

30 μM allopregnanolone -25.1 ± 4.2 12.0 ± 4.6 4 -55.6 ± 2.4* 6.5 ± 2.3 5 97 0.3/1 mM pentobarbitalb -31.2 ± 1.5* 7.5 ± 1.4 6 -54.7 ± 1.3* 5.6 ± 1.2 4 300 mM ethanol -25.2 ± 1.5 6.7 ± 1.4 5 -48.8 ± 0.6* 4.3 ± 0.5 4 a 30 and 10 μM diltiazem were used for Cav1.2 and Cav1.3, respectively.

b 0.3 and 1 mM pentobarbital were used for Cav1.2 and Cav1.3, respectively.

N.D., not determined.

* = p < 0.05 compared to respective control inactivation V50 in the absence of drug

CHAPTER 4

CA2+/CALMODULIN-DEPENDENT PROTEIN KINASE II LOCALIZATION

AND AUTOPHOSPHORYLATION WITHIN HIPPOCAMPAL CA1

EXCITATORY POSTSYNAPSES DURING FLURAZEPAM WITHDRAWAL

Damien E. Earla, Paromita Dasa, Francisco J. Alvarezb,1, William T. Gunningc,

Elizabeth I. Tietza,d

aDepartment of Physiology and Pharmacology, University of Toledo College of

Medicine, Health Science Campus, Toledo, OH 43614, USA,

[email protected], [email protected]

bDepartment of Neuroscience, Cell Biology and Physiology, Wright State University,

Dayton, OH 45435, USA

cDepartment of Pathology, University of Toledo College of Medicine, Health Science

Campus, Toledo, OH 43614, USA, [email protected]

dDepartment of Neurosciences, University of Toledo College of Medicine, Health

Science Campus, Toledo, OH 43614, USA

Present address:

1Department of Physiology, Emory University School of Medicine, Atlanta, GA 30322,

USA, [email protected]

Abbreviations: AMPAR, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid

receptor; BZ, benzodiazepine; CaM, calmodulin; CaMKII, Ca2+/calmodulin-dependent

protein kinase II; EDTA, Ethylenediaminetetraacetic acid; EGTA, ethylene glycol

tetraacetic acid; FZP, flurazepam; GABAAR, γ-aminobutyric acid type A receptor; L-

VGCC, L-type voltage-gated Ca2+ channel; NMDAR, N-methyl-D-aspartate receptor;

PEG, poly(ethylene) glycol; PSD, postsynaptic density; TBST, Tris-buffered saline with

0.1% Tween 20

4.1 Abstract

Benzodiazepines (BZs) are clinically useful anxiolytics, sedatives, and anticonvulsants

attributable to their positive allosteric modulation of GABAA receptors. Long-term use

can lead to development of physical dependence manifested by withdrawal symptoms.

One week oral administration of rats with the benzodiazepine flurazepam (FZP) results in

withdrawal anxiety, correlated with hippocampal CA1 neuron hyperexcitability mediated

by enhancement of GluA1-containing AMPARs. A CaMKII-mediated increase in

AMPAR conductance after 2 days of FZP withdrawal may result from potentiation of L-

type voltage-gated Ca2+ channel (L-VGCC) currents that precedes the GluA1-AMPAR

enhancement and continues through 2 days of withdrawal. The mechanistic basis of L-

VGCC potentiation and CaMKII activation remains unknown. The current studies

assessed L-VGCC Cav1.2 and Cav1.3 subunit expression by immunoblot and Cav1.2 immunohistochemistry. Total and autophosphorylated (Thr286) CaMKII expression and

distribution were examined in CA1 asymmetric synapses by postembedding immunogold

electron microscopy. The findings suggested that up-regulated L-VGCC function was not

due to increased subunit expression, rather may result from trafficking from intracellular

stores or post-translational modifications such as phosphorylation. Interestingly,

postsynaptic CaMKIIα expression was decreased with no change in autophosphorylated

CaMKII expression. Decreased CaMKII expression may be due to the corresponding removal of GluN1/GluN2B-containing N-methyl-D-aspartate receptors from CA1 synapses after 2 days of FZP withdrawal, suggesting CaMKII binding to GluN2B during

FZP withdrawal. An enhanced CaMKII-GluN2B interaction may reflect autonomous

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CaMKII activation stimulated by L-VGCC-mediated Ca2+ influx in this model of BZ withdrawal-induced glutamatergic plasticity.

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4.2 Introduction

Benzodiazepines (BZs) are clinically useful anxiolytics, sedative-hypnotics, and

anticonvulsants attributable to their enhancement of Cl− flux through γ-aminobutyric acid

type A receptors (GABAARs), the main inhibitory neurotransmitter receptors in the

mammalian central nervous system. However, long-term treatment can result in the

development of tolerance to BZ effects and physical dependence manifested by

withdrawal symptoms such as anxiety and insomnia (Griffiths & Johnson, 2005). Current

evidence suggests that while tolerance may result from impaired GABAergic function, withdrawal symptoms such as anxiety and in some cases increased seizure susceptibility

may relate to neuronal hyperexcitability mediated by enhanced function of the

glutamateric system (Izzo et al., 2001; Van Sickle et al., 2004; Song et al., 2007; Xiang &

Tietz, 2007). Specifically, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid

receptors (AMPARs) and N-methyl-D-aspartate receptors (NMDARs) are the excitatory

ionotropic receptors that respond to glutamate neurotransmission, and AMPAR

potentiation in particular may mediate withdrawal hyperexcitability. For example, long-

term treatment of rats with the BZ diazepam lead to withdrawal anxiety and increased

seizure susceptibility after 96 hrs of withdrawal, which correlated with increased

AMPAR GluA1 subunit mRNA and protein in the cortex and hippocampus, with no

alteration in AMPAR GluA2 or NMDAR GluN1 subunits (Izzo et al., 2001).

In flurazepam (FZP)-treated rats, withdrawal anxiety significantly correlated with

potentiation of AMPAR currents in hippocampal CA1 pyramidal neurons (Van Sickle et

al., 2004; Xiang & Tietz, 2007). The initial increase in AMPAR current amplitude

resulted from incorporation of GluA1 homomeric receptors into the postsynatpic density

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(PSD) during the first day of withdrawal (Song et al., 2007; Das et al., 2008; Shen et al.,

2010). Increased AMPAR conductance after 2 days of withdrawal was attributable to

Ca2+/calmodulin-dependent protein kinase II (CaMKII)-mediated phosphorylation of

GluA1 subunits at Ser831 (Shen et al., 2009; Shen et al., 2010).

Although FZP withdrawal-induced glutamatergic plasticity is similar to that of long-

term potentiation (LTP), several differences are notable. First, following AMPAR

potentiation, a compensatory removal of GluN1 and GluN2B NMDAR subunits and

reduced NMDAR current amplitude was observed 2 days after FZP withdrawal. Down-

regulation of GluN2B-containing NMDARs normalized excitatory total charge transfer in

CA1 neurons and precluded expression of anxiety (Van Sickle et al., 2004; Shen et al.,

2009; Das et al., 2010; Shen & Tietz, 2011). Anxiety was unmasked after 2 days of

withdrawal by preventing the NMDAR down-regulation via systemic pre-injection of an

NMDAR antagonist (Van Sickle et al., 2004). Second, while LTP is induced by Ca2+ influx through NMDARs during high frequency stimulation (Malenka & Nicoll, 1999), a near doubling of high-voltage-activated (HVA) Ca2+ currents was observed in CA1 neurons of FZP-treated rats, preceding the AMPAR enhancement and continuing through

2 days of withdrawal (Xiang et al., 2008). Moreover, blockade of L-type voltage-gated calcium channels (L-VGCCs), but not NMDARs reversed the withdrawal anxiety and

AMPAR potentiation at 1 and 2 days of FZP withdrawal (Van Sickle et al., 2004; Xiang

& Tietz, 2007; Xiang et al., 2008). Taken together, the data suggest that Ca2+ influx through L-VGCCs contributes to the increased number and function of synaptic homomeric GluA1 AMPARs during FZP withdrawal via a CaMKII-mediated mechanism.

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Although we reported that CaMKII mediates the phosphorylation of GluA1 homomers during the second day of FZP withdrawal, the mechanism of CaMKII activation remains uncertain. During LTP, persistent Ca2+ influx through NMDARs leads

to activation and autophosphorylation of CaMKII at Thr286 resulting in partially

autonomous (Ca2+-independent) activity (Coultrap et al., 2010). During FZP withdrawal,

total CaMKIIα, but not β expression was increased in PSD-enriched CA1 homogenates

from FZP-withdrawn rats without a concomitant increase in autophosphorylated Thr286-

CaMKII (pCaMKII) (Shen et al., 2010), suggesting that alternate mechanisms of CaMKII

autonomous activation and/or PSD localization may occur, perhaps through binding to

membrane-incorporated GluN2B subunits (Bayer et al., 2001; Bayer et al., 2006). It is

interesting to speculate that CaMKII activation and/or translocation may be differentially

mediated by the near doubling of Ca2+ influx through L-VGCCs during FZP withdrawal.

One mechanism by which this might occur is through increased expression of the L-

VGCC α1 pore-forming subunits, Cav1.2 and Cav1.3, near CA1 PSDs of FZP-withdrawn

rats. A hyperpolarizing shift in the L-VGCC activation curve (Xiang et al., 2008) suggested that Cav1.3, which activates at relatively negative membrane potentials (Xu &

Lipscombe, 2001; Earl & Tietz, 2011), might be selectively enhanced in CA1 neurons as

a consequence of long-term FZP treatment.

The current studies first assessed Cav1.2 and Cav1.3 L-VGCC subunit expression in

FZP-withdrawn and control tissues by immunoblot of PSD-enriched CA1 homogenates

and confocal immunofluorescent analysis of Cav1.2-labeled hippocampal slices. Next,

post-embedding immunogold labeling was used to assess CaMKIIα and

autophosphorylated (Thr286/Thr287) α/β-CaMKII expression and localization within

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asymmetric synapses located in stratum radiatum (SR) of CA1 neurons from 2-day FZP- withdrawn and matched control rats.

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4.3 Materials and Methods.

4.3.1 Long-term FZP treatment

All procedures involving the use of animals were performed in compliance with The

University of Toledo College of Medicine Institutional Animal Care and Use Committee

(IACUC) and National Institutes of Health guidelines. One-week oral treatment of rats

with the relatively water-soluble benzodiazepine, FZP was as described previously (Van

Sickle et al., 2004). Briefly, male Sprague-Dawley rats (P22-25, Harlan, Indianapolis, IN)

were acclimated 2 - 4 days to 0.02% saccharin water. Rats were then offered saccharin

water containing FZP (pH 5.8) as the sole drinking source. The FZP concentration was

periodically adjusted based on the body weight and volume consumed to yield doses of

100 mg/kg/day for 3 days, then 150 mg/kg/day for 4 days. The final average daily FZP

dose was always greater than 100 mg/kg, generally 125 - 130 mg/kg, with a goal of

achieving a minimum average daily dose of 120 mg/kg (Das et al., 2010). Related to the

half-life (< 12 hrs) of FZP and its major bioactive metabolites in rats (Lau et al., 1987),

this treatment paradigm results in BZ levels of about 1.2 µM measured in rat brain

homogenates by radioreceptor assay (Xie & Tietz, 1992). After 1-week FZP treatment,

rats were offered saccharin water for 1 or 2 days. FZP withdrawal consistently results in

anxiety-like behavior after 1 day of withdrawal (Van Sickle et al., 2004; Xiang & Tietz,

2007), which can be masked or expressed on day 2 of withdrawal as a function of

NMDAR down-regulation (Van Sickle et al., 2004; Shen & Tietz, 2011). Matched

control rats were offered saccharin water for the same experimental period.

4.3.2 Antibodies and specificity

4.3.2.1 Antibodies used for immunoblot and immunofluorescent staining

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A polyclonal anti-Cav1.3 antibody (2.49 mg/mL stock) which recognizes the N-

terminal sequence, MQHQRQQQEDHANEANYARGTRKC was generously provided

by Dr. Amy Lee (Univertsity of Iowa, Iowa City, Iowa). This antibody was previously

characterized as recognizing two bands consistent in size with Cav1.3 (>170 kDa and

>250 kDa) in hippocampal lysates from wild-type, but not Cav1.3 knockout mice.

Immunoblots of rat brain lysates revealed a 250 kDa band (Jenkins et al., 2010). A

polyclonal anti-Cav1.2 antibody (0.85 mg/mL stock) which recognizes an epitope located

in the intracellular loop between domains II and III, TTKINMDDLQPSENEDKS was

from Alomone Labs (Jerusalem, Israel, Cat. # ACC-003). Antibodies generated against this epitope were previously characterized as recognizing a 250 kDa band consistent with

Cav1.2, which was absent from cortical and hippocampal lysates of Cav1.2 conditional

knockout mice (Tippens et al., 2008). Immunoblot of rat hippocampal lysates revealed a

smeared band with a molecular weight >170 kDa (Tippens et al., 2008). Control

experiments used in the current studies included omitting the primary antibody and pre-

incubation with the antigenic peptide, both of which abolished bands on immunoblot (see

Fig. 1B) and immunofluorescent labeling (see Fig. 3B). A monoclonal anti-Cav1.2 antibody (clone N263/31) generated using a peptide fragment containing amino acids

808-874 (intracellular loop between domains I and II) was from NeuroMab (UC Davis,

Davis, CA). In the current studies, this antibody recognized full-length and truncated

Cav1.2 size-forms with identical apparent molecular weights as those recognized by the

1928 Alomone antibody (data not shown). Polyclonal anti-pS -Cav1.2 (0.34 mg/mL stock)

was generously provided by Dr. William Catterall (University of Washington, Seattle,

WA). This antibody was previously characterized as specifically recognizing Cav1.2

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1928 phosphorylated at Ser (Hulme et al., 2006). Polyclonal anti-Cavβ3 antibody (0.3

mg/mL stock) was from Alomone Labs (Cat. # ACC-008) and recognized a prominent 57 kDa band by immunoblot of PSD-enriched protein consistent with the Cavβ3 subunit

(Ludwig et al., 1997; Catterall, 2000), but also recognized fainter 60 and 69 kDa bands that likely represent Cavβ3 subunit splice variants (Hullin et al., 1992; Ludwig et al.,

1997), the latter of which was enriched in the cytosolic and Triton-soluble membrane

subcellular fractions (data not shown, see subcellular fractionation below).

4.3.2.2 Antibodies used for post-embedding immunogold electron microscopic

analysis

A monoclonal anti-CaMKIIα antibody (clone 6G9-2, 1 mg/mL stock) generated using

purified CaMKIIα protein (Kennedy et al., 1983a; Kennedy et al., 1983b) was from

Chemicon-Millipore (Billerica, MA, Cat. # MAB8699). This antibody recognized a

specific band at ~50 kDa, whereas no bands were observed in hippocampal lysates from

knockout mice (Silva et al., 1992). Additional controls for post-embedding immunogold

labeling in the current studies included omitting the primary antibody, which yielded 0.01

15-nm particles/bouton, 0.10 particles/spine, and 0.01 particles/PSD in 69 asymmetric

synaptic profiles (compare to CaMKIIα immunogold labeling in these same

compartments in Table 1), and replacing the monocloncal anti-CaMKIIα with the

polyclonal anti-pCaMKII antibody, which yielded no particles pre- or postsynaptically in

60 asymmetric synaptic profiles. A polyclonal anti-pCaMKII antibody which recognizes

the epitope, MHRQET(PO4)VDCLKKFN was from Promega (Madison, WI, Cat. #

V1111). This antibody recognizes phosphorylated Thr286/287 likely on all CaMKII isoforms (Lorenz et al., 2002; Larsson & Broman, 2006). Labeling using this antibody

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was substantially reduced when used in immunocytochemistry of visual cortex from mice

with CaMKIIα Thr286 mutated to alanine (Taha et al., 2002). Pre-embedding immunogold pCaMKII labeling using this antibody revealed increased postsynaptic labeling in

hippocampal cultures exposed to NMDA (Dosemeci et al., 2002). Additional controls for

post-embedding immunogold labeling in the current studies included omitting the

primary antibody which yielded 0.30 10-nm particles/bouton, 0.24 particles/spine, and

0.06 particles/PSD in 63 asymmetric synaptic profiles (compare to pCaMKII immunogold labeling in these same compartments in Table 2), and replacing the polyclonal anti-pCaMKII with monoclonal anti-CaMKII, which yielded no particles pre- or postsynaptically in 60 asymmetric synaptic profiles.

4.3.3 CA1 minislice subcellular fractionation

Subcellular fractionation to obtain a PSD-enriched membrane fraction was performed as described previously (Song et al., 2007; Shen et al., 2010). Briefly, following decapitation the CA1 region of the dorsal hippocampus was rapidly dissected on ice and stored at -80ºC for future use (2-3 CA1 minislices/group) or homogenized immediately, then stored at -80ºC. Homogenates contained phosphatase and protease inhibitors (P8340,

Sigma, St. Louis, MO). After removing nuclei and debris, membranes were separated from cytosol with a 10,000 g spin. Subcellular fractions used in the current studies included a cytosolic fraction (S2), a 0.5% Triton X-100-soluble membrane fraction (S3), and a Triton X-100-insoluble membrane fraction (P3). The P3 fraction was previously shown to be ~40% enriched in PSDs (Song et al., 2007). Moreover, changes in AMPAR and NMDAR subunits detected in the P3 fraction (Song et al., 2007; Shen et al., 2010;

Shen & Tietz, 2011) were also detected at CA1 synapses using immunogold techniques

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(Das et al., 2008; Das et al., 2010). Protein content was determined using either the

Bradford or bicinchoninic acid (BCA) protein assays using bovine serum albumin as a standard. Proteins were mixed with Laemmli sample buffer and heated to 70ºC for 10 min prior to blotting.

4.3.4 Western blot

Protein samples were electrophoretically separated in 4-12% Bis-Tris gradient gels

(Invitrogen, Carlsbad, CA) along with a Novex Sharp pre-stained protein standard

(Invitrogen) in SDS-MOPS running buffer (Invitrogen), then wet transferred to polyvinylidene fluoride membrane (Immobilon-FL, Millipore) in NuPAGE methanol-free transfer buffer (Invitrogen) at 90 V for 4 hr on ice. Transferred protein bands were detected by 5 min incubation with 0.5% Ponceau S (w/v) in 1% acetic acid (v/v). As indicated in the results, in some cases Ponceau S-stained bands were scanned and used as loading controls in lieu of actin. Blots were blocked with 5% non-fat milk in Tris- buffered saline (50 mM Tris, pH 7.4, 150 mM NaCl, 2.3 mM CaCl2), probed with primary antibodies as indicated in the results, then secondary antibodies conjugated to infrared probes (700 or 800 nm, LiCOR, Lincoln, NE). Reacted blots were scanned using the Odyssey Infrared Imaging System (LiCOR) at an intensity that yielded band densities in a linear range. Integrated band densities (ID) were quantified using Image J software

(NIH, Bethesda, MD), and signals were normalized to β-actin ID as a loading control.

Data from each blot were expressed as a percent of the control average.

4.3.5 Hippocampal slice confocal immunofluorescence

Hippocampal tissue preparation, immunofluorescent staining, and confocal analysis were performed as described previously (Song et al., 2007). Briefly, 1 cm hippocampal

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blocks were post-fixed in 0.1 M phosphate buffer (pH 7.4) containing 4%

paraformaldehyde for 1 hr at room temperature, then fixed overnight at 4°C. Fixed brains

were incubated in 15% sucrose overnight at 4°C. Coronal dorsal hippocampal slices (50

μm) were quenched in 0.5 M NH4Cl, blocked 1 hr in 10% normal goat serum (NGS, from

here forward solutions contained 1% fish gelatin and 0.1% Tween-20), incubated overnight at 4°C in primary antibody (anti-Cav1.2, 4.25 µg/mL, Alomone Labs), blocked

3 x 10 min in 5% NGS, then incubated 2 hr in secondary antibody (goat anti-rabbit IgG

conjugated to Alexa488, 1:500). Confocal images of each slice were captured at 5 µm

intervals on a Leica TCS SP5 system (Leica Microsystems Inc., Bannockburn, IL). Leica

Application Suite Advanced Fluorescence software was used to create two-dimensional

projections for each slice using only the maximal intensity pixels from each slice’s

collection of confocal images. Regions of interest were drawn in several hippocampal

regions (as noted in the results) and the mean gray value measured using Image J

software. Each region’s mean gray value was averaged from 2 - 5 slices for each rat. A

total of 4 separate immunofluorescent experiments were performed with 1 - 2 rats per

treatment group in each experiment (n = 6 rats/group). Data from each experiment were

expressed a percent of the mean control gray value in order to reduce interexperimental

variability resulting from differences in confocal laser intensity, gain, and offset.

4.3.6 Post-embedding immunogold electron microscopy

Transcardial fixation, cryosubstitution, and post-embedding immunogold labeling

procedures were as described previously, and the ultrathin cut sections used in the current

studies were cut from the same tissues as those used in a previous study of GluN1 and

GluN2A/B subunits at CA1 neuron asymmetric postsynapses (Das et al., 2010). Briefly,

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isoflurane anesthetized rats were transcardially perfused with an oxygenated vascular

rinse followed by 4% paraformaldehyde and 0.5% glutaraldehyde. Hippocampal slices

(200 or 500 μm) were slam-frozen (-190°C, Leica EM CPC, Bannockburn, IL),

cryosubstituted, and flat-embedded in lowicryl resin. Ultrathin CA1 sections (80 nm)

were collected on nickel grids, equilibrated in Tris-buffered saline with 0.1% Triton X-

100 (TBST, pH 7.6), quenched in 1% NaBH4 and 50 mM glycine, blocked in 10%

normal goat serum (NGS), then incubated in primary antibodies (1:20 anti-CaMKIIα

and/or 1:10 anti-pCaMKII with 1% NGS) 2 hrs at room temperature, then overnight at

4°C. Tissues were switched to pH 8.2 TBST, incubated in 0.5% poly(ethylene) glycol

(PEG), then in secondary antibody containing 0.5% PEG (1:25 goat anti-rabbit IgG

conjugated to 10-nm gold and/or 1:25 goat anti-mouse IgG conjugated to 15-nm gold,

BBInternational, UK) for 1.5 hr. Sections were counterstained with 5% uranyl acetate

and Reynold’s lead citrate. The CA1 proximal SR region of reacted tissues was scanned

using a Phillips CM10 PW6020 transmission electron microscope to randomly identify

profiles of asymmetric synapses. Images were captured at 52,000X magnification. The

negatives were developed and scanned, then immunogold labeling was measured using

Image Pro Plus software (Media Cybernetics, Inc., Bethesda, MD) as previously

described (Das et al., 2010). Pre- and postsynaptic immunogold particles were binned

into distances perpendicular to the cleft surface of the PSD out to 300 nm. Immunogold

particles were also assigned to PSD, active zone, perisynaptic, membrane, or intracellular

compartments: particles within 20 nm of the PSD were counted as PSD labeling as

described previously (Das et al., 2008); particles within 20 nm of the presynaptic membrane, but not within 20 nm of the PSD membrane were considered within the active

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zone; particles within 20 nm of the membrane were considered membrane-bound; and postsynaptic membrane-bound particles within 100 nm lateral to the PSD were considered perisynaptic (Das et al., 2008). All procedures and measurements were performed with the experimenter blinded to the experimental group.

4.3.7 Statistical analyses

Data from immunoblot and immunofluorescent analyses were normalized to mean control values within each experiment so that data could be pooled from multiple experiments for statistical analysis. Immunoblot and immunofluorescent means were compared between control and FZP-withdrawn groups by Student’s unpaired t-test.

Presynaptically and postsynaptically binned immunogold particles were analyzed by

Two-way Repeated Measures ANOVA with post-hoc comparisons between control and

FZP-withdrawn groups within each bin using Bonferroni’s Multiple Comparison test.

Percent labeling and the mean number of immunogold particles were compared between control and FZP-withdrawn groups by Student’s t-test. Relative frequencies of the numbers of immunogold particles per PSD between control FZP-withdrawn groups (0 to

5) were compared by Mann-Whitney U-test. Data were statistically analyzed and graphs generated using Prism 4.0 software (GraphPad Software Inc., San Diego, CA). Values are reported as mean ± SEM and were considered significantly different if the p-value was less than or equal to 0.05.

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4.4 Results

4.4.1 Unaltered L-VGCC subunit expression in CA1 PSD-enriched fraction during

FZP-withdrawal

To investigate the mechanistic basis of the voltage-gated Ca2+ current enhancement in

CA1 neurons during FZP withdrawal (Xiang et al., 2008), L-VGCC subunit expression

was assessed by immunoblot of CA1 homogenates from rats withdrawn 1 or 2 days from

1-week FZP treatment. Figs. 1A and B show the staining pattern of Cav1.2 and Cav1.3 immunoblots, respectively in different subcellular fractions. Importantly, signals with a molecular weight consistent with that of Cav1.2 and Cav1.3 were primarily seen in the

PSD-enriched, Triton-insoluble membrane fraction. Long and short size-forms of both

Cav1.2 and Cav1.3 were observed with apparent molecular weights of 220 and 180 kDa.

The different Cav1.2 size-forms arise from calpain-mediated cleavage of the C-terminus resulting in full-length and truncated peptides (Hell et al., 1996). For Cav1.3, these size forms likely result from neuronal splice variation, which yields full-length and C-terminal truncated peptides (Safa et al., 2001; Xu & Lipscombe, 2001; Singh et al., 2008), similar to Cav1.2. In the Triton-soluble membrane fraction, a weakly staining diffuse band with an approximate molecular weight between 180 and 220 kDa was observed for both

Cav1.2 and Cav1.3 subunits. The cytosolic fraction revealed no signal in the range of 180

– 220 kDa. Other signals above (for Cav1.3) and below the 180 – 200 kDa range were

observed. Based on previous characterizations using knockout tissue, the low molecular

weight signals may not be calcium channel α1 subunits, including the prominent 120 kDa

band observed in Cav1.2 immunoblots that was removed by peptide pre-incubation (Fig.

1B).

114

Fig. 2A illustrates that there was no change in Cav1.2 or Cav1.3 subunit expression in

the PSD-enriched fraction after 1 day of FZP withdrawal. Although increased expression of the auxiliary Cavβ3 subunit could enhance trafficking of L-VGCCs to the plasma

membrane from the endoplasmic reticulum (Bichet et al., 2000), no alteration in Cavβ3 subunit expression was detected (Fig. 2A). Interestingly, each Cav1.2 size form was

expressed as a doublet of bands separated by about 10 kDa. These doublets could

represent splice variants (Tang et al., 2004), each of which undergoes the same

proteolytic cleavage by calpain. Separate measurements of the integrated densities of

each doublet band also revealed no change in the expression of Cav1.2 (data not shown).

2+ Calpain-mediated cleavage of the Cav1.2 C-terminus increases Ca currents through the

channel (Klockner et al., 1995), which could be a factor in L-VGCC subunit regulation

during FZP withdrawal. However, there was also no difference in the ratio of the short to

long size-form by measuring integrated density of the doublet bands together (CON ratio:

1.16 ± 0.10, FZP ratio: 1.30 ± 0.08, p = 0.30, n = 4/group) or separately (top band: CON ratio: 0.96 ± 0.08, FZP ratio: 1.07 ± 0.11, p = 0.42; bottom band: CON ratio: 1.86 ± 0.22,

FZP ratio: 1.88 ± 0.10, p = 0.96, n = 4/group). Fig. 2B illustrates that Cav1.2, Cav1.3, and

Cavβ3 subunit expression was similarly unaltered after 2 days of FZP withdrawal. The

Cav1.2 doublet bands were not easily distinguishable on this immunoblot, and were not

separately analyzed. The Cav1.2 short to long size-form ratio was also unaltered after 2

days of FZP withdrawal (CON ratio: 0.60 ± 0.09, n = 3; FZP ratio: 0.57 ± 0.03, n = 4; p =

0.81).

1928 2+ Phosphorylation of Cav1.2 at Ser enhances Ca current density (Oliveria et al.,

1928 2007). Using an antibody specific for pS -Cav1.2, immunoblot analysis of CA1 PSD-

115

enriched proteins revealed no change in the Cav1.2 phosphorylation state when

normalized to Ponceau S-stained protein bands (CON: 1.00 ± 0.06, n = 8, FZP: 1.09 ±

0.16, n = 6, p = 0.56, pooled data from two blots) or when normalized to total Cav1.2 expression measured using the NeuroMab monoclonal antibody (CON: 1.00 ± 0.06, n =

8, FZP: 1.36 ± 0.16, n = 6, p = 0.08 with Welch’s correction, pooled data from two blots).

1928 The anti-pS -Cav1.2 antibody recognized doublet bands with apparent molecular

weights of about 220-240 kDa, similar to full-length Cav1.2. This is consistent with the

1928 fact that the C-terminal truncated short-form of Cav1.2 does not contain Ser .

Consistent with immunoblot data, experiments in which fixed hippocampal slices were immunostained with the Alomone anti-Cav1.2 antibody also revealed no change in expression after 2 days of FZP withdrawal (Fig. 3A and C). Control experiments in which the primary antibody was omitted or pre-incubated with the Cav1.2-specific antigenic

peptide abolished the labeling (Fig. 3B). Immunohistochemical experiments could not be

conducted for Cav1.3, since the antibody used for immunoblot is not specific for

hippocampal immunohistochemical staining (Amy Lee, unpublished observations).

4.4.2 CaMKIIα expression, but not Thr286 autophosphorylation is reduced in FZP-

withdrawn CA1 asymmetric synapses

Prior evidence suggested that Ca2+ influx through L-VGCCs might activate CaMKII,

which then phosphorylates GluA1 subunits at Ser831 increasing AMPAR conductance

after 2 days of FZP withdrawal (Xiang & Tietz, 2007; Xiang et al., 2008; Shen et al.,

2009; Shen et al., 2010). To further evaluate this hypothesis, ultrathin cut sections of the

CA1 region were co-labeled with anti-CaMKIIα and anti-autophosphorylated Thr286/287

CaMKIIα/β (anti-pCaMKII) antibodies recognized using secondary antibodies

116

conjugated to 15- or 10-nm gold, respectively. Dual labeling was assessed in asymmetric synapses in the proximal dendritic SR region, where decreased GluN1 and GluN2B subunit postsynaptic expression was previously detected in these same tissues (Das et al.,

2010). Increased postsynaptic expression of homomeric GluA1 AMPA receptors was also detected in this region after 2 days of FZP withdrawal (Das et al., 2008). Similar levels of labeling were observed in tissues reacted with CaMKII or pCaMKII antibodies alone (data not shown) rather than as a cocktail, suggesting that the antibodies did not sterically interfere with each other during dual reaction.

Fig. 4 illustrates dual labeling observed in asymmetric synapses from control (A,B) and FZP-withdrawn (C,D) rats. Immunogold particles were counted within 300 nm pre- and postsynaptic to the cleft surface of the PSD. There was no significant change in the percentage of boutons, spines, or PSDs labeled with CaMKII or pCaMKII antibodies

(Fig. 4E-G, Tables 1 and 2, respectively). Similarly, no significant change in the mean number of CaMKII or pCaMKII immugold particles was observed in boutons, spines, or

PSDs (Fig. 4H-J, Tables 1 and 2, respectively). Interestingly, there was a trend towards an increased ratio of pCaMKII/CaMKII expression in PSDs (Fig. 4J). A significant decrease in the number of CaMKII immunogold particles was observed in spines and

PSDs, but not boutons when only positively labeled (≥1 immunogold particle) boutons, spines, or PSDs were considered (Table 1). No alteration in pCaMKII labeling was observed in any of these synaptic compartments (Table 2). A significant decrease in the percent labeling and mean number of CaMKII immunogold particles was also observed in FZP-withdrawn active zones, but no alteration was observed in pre- or postsynaptic membrane or intracellular CaMKII or pCaMKII immunogold labeling (Table 3).

117

Fig. 5 illustrates CaMKII and pCaMKII expression within binned distances pre- and postsynaptic to the PSD membrane for control and FZP-withdrawn rats. Analysis of binned CaMKII expression revealed a significant interaction between experimental group and binned distance with a significant decrease 60 nm postsynaptic to the PSD membrane

(see Fig. 5A). There was no significant interaction between experimental group and binned distance for presynaptic CaMKII expression or pCaMKII either pre- or postsynaptically. Decreased CaMKII expression within the 60 nm postsynaptic bin without a change in pCaMKII expression indicates an alteration in PSD labeling.

Moreover, as illustrated in Fig. 6, comparing the relative frequency of numbers of 15-

(CaMKII) and 10-nm (pCaMKII) particles within PSDs (0 to 5 immunogold particles) between control and FZP-withdrawn groups revealed a significant decrease in the frequency of PSDs labeled with two 15-nm particles in FZP-withdrawn tissues. Taken together the data support decreased CaMKIIα, but not absolute pCaMKII expression in

PSDs within the CA1 region of 2-day FZP-withdrawn rats.

118

4.5. Discussion

4.5.1 Molecular basis of FZP-induced enhancement of CA1 VGCC function

The current results suggest that a mechanism other than increased expression of L-

VGCC subunits mediates the increased HVA Ca2+ current density in CA1 neurons of

FZP-treated and withdrawn rats (Xiang et al., 2008). It is possible that L-VGCC surface

expression is enhanced via trafficking from intracellular compartments, which went

undetected by the methods used in the current studies. This could be assessed in surface

protein crosslinking experiments. Although it was reported that the number of

postsynaptic profiles with plasma membrane-bound Cav1.2 immunogold labeling was

relatively low (Tippens et al., 2008), it may also be possible to detect increased surface

expression of Cav1.2 or Cav1.3 L-VGCCs using immunogold EM methods. The specificity of anti-Cav1.2 and Cav1.3 antibodies for pre- or post-embedding immunogold

labeling will need to be confirmed using knockout tissues to determine if the low level of

surface labeling represents actual Cav1.2 and Cav1.3-containing surface-expressed L-

VGCCs near CA1 PSDs.

Another possible L-VGCC regulatory mechanism involves phosphorylation at sites that enhance channel currents and may also result in channel activation at more negative

membrane potentials (Gao et al., 2006). Although the phosphorylation state of Cav1.2

Ser1928 was unchanged during FZP withdrawal, several other phosphorylation sites

2+ regulate channel gating. In particular, Ca -dependent facilitation of both Cav1.2 and

Cav1.3 is mediated by CaMKII (Hudmon et al., 2005; Lee et al., 2006; Jenkins et al.,

2010). This facilitation occurs during increased channel activity (Hudmon et al., 2005),

though BZs would likely decrease channel activity via enhancement of GABAAR-

119

mediated neuronal inhibition. Nevertheless, a depolarizing shift in the GABAAR reversal

potential and a GABAAR-mediated depolarization was observed during FZP-withdrawal

(Zeng et al., 1995; Zeng & Tietz, 1997), which could contribute to CA1 neuron

hyperexcitability and activate L-VGCCs during FZP withdrawal. The precise mechanism

of the observed GABA-mediated depolarization and whether it exists during drug

treatment to enhance L-VGCC activity remains unknown.

4.5.2 Molecular basis of CaMKII activation during FZP withdrawal

L-VGCC-mediated Ca2+ influx may related to CaMKII activation and the observed

phosphorylation of GluA1 homomers and AMPAR potentiation during FZP withdrawal

(Shen et al., 2010). CaMKII forms oligomeric holoenzymes containing up to 12 subunits

(Bennett et al., 1983). The β-CaMKII isoform mediates binding of CaMKII holoenzymes

to the actin cytoskeleton within dendritic arbors (Shen et al., 1998). Upon elevation of

2+ 2+ 2+ 2+ intracellular Ca concentration ([Ca ]i), Ca binds to all four Ca -binding sites on calmodulin (CaM), resulting in association with and activation of CaMKII via

2+ displacement of an autoinhibitory domain. Elevations in [Ca ]i also result in dissociation

2+ of CaMKII holoenzymes from the actin cytoskeleton. If [Ca ]i remains elevated for a

sufficient length of time, CaMKII enzymes phosphorylate neighboring CaMKII molecules within the holoenzyme at Thr286 on α and Thr287 on β isoforms. This

autophosphorylation results in Ca2+/CaM trapping and autonomous kinase activity

2+ without the need for continued elevation of [Ca ]i (reviewed in Fink & Meyer, 2002).

The mechanism of drug withdrawal-induced CaMKII activation prior to the

phosphorylation of GluA1 homomeric AMPARs observed after 2 days of FZP

withdrawal is unclear. The trend towards an increased ratio of pCaMKII/CaMKII

120

immunogold PSD labeling suggests that a greater proportion of CaMKII molecules

within the PSD are autophosphorylated at Thr286 in FZP-withdrawn rats. However, since the absolute amount of pCaMKII was unaltered, this suggests that an additional

mechanism of CaMKII activation may occur during FZP withdrawal. A possible hint to

an alternate activation mechanism was the loss of CaMKIIα from CA1 asymmetric

synapses, which may be related to the corresponding loss of GluN1/GluN2B NMDA

receptors observed in a prior EM study using tissue sections from the same experimental

groups (Das et al., 2010). Thus, there may be an increased interaction between CaMKII and GluN2B prior to the second day of FZP withdrawal. Direct binding of CaMKII to

GluN2B resulted in autonomous CaMKII activation independent of autophosphorylation

(Bayer et al., 2001). A loss of GluN2B-containing NMDARs after 2 days of withdrawal

would serve to homeostatically reset the CA1 glutamatergic system mediating recovery

from FZP withdrawal-induced anxiety (Shen & Tietz, 2011). Since a correlation in the

loss of both CaMKII and GluN2B from PSDs does not in itself indicate an enhanced

interaction between these molecules, future experiments will address this hypothesis directly using co-immunoprecipitation studies.

The amino acid sequence surrounding the Thr286/287 autophosphorylation site is nearly

identical in each CaMKII isoform and phospho-specific antibodies will recognize this

epitope on all isoforms. In particular, β-CaMKII is the second highest expressed isoform

in the brain (Bennett et al., 1983; Erondu & Kennedy, 1985). Autophosphorylation of β-

CaMKII results in increased affinity for Ca2+/CaM, leading to dissociation from the actin

cytoskeleton promoting association of CaMKII holoenzymes with other proteins.

Although, there was no change in β-CaMKII expression observed in CA1 PSD-enriched

121

homogenates from 2-day FZP-withdrawn rats (Shen et al., 2010), autophosphorylation of

β-CaMKII cannot be ruled out.

4.5.3 Discrepancies with prior studies

The decrease in postsynaptic expression of total CaMKIIα was surprising given that prior studies detected increased CaMKIIα expression in the PSD-enriched fraction after 2 days of FZP withdrawal (Shen et al., 2010). One possibility to explain this discrepancy is that the Triton-insoluble PSD-enriched fraction contains other subcellular compartments in which CaMKII expression may be increased. For example, CaMKII is expressed in a heavy microsomal cytoskeletal compartment insoluble in 0.5% Triton X-100 (Dosemeci et al., 2000). It is possible that immunoblot analysis revealed increased CaMKII expression due to translocation to this cytoskeletal compartment, rather than to PSDs.

One caveat is that the heavy microsomal cytoskeleton was pelleted from the cytosolic, rather than the membrane fraction. Yet, CaMKII expression may also be enriched in other non-PSD, Triton-insoluble compartments (Song et al., 2007) after 2 days of FZP withdrawal.

Another discrepancy is that CaMKII inhibitors were able to reverse the enhanced

AMPAR currents and conductance in CA1 neurons recorded in hippocampal slices from

2-day FZP-withdrawn rats (Shen et al., 2009; Shen et al., 2010). If CaMKII is already removed from dendritc spines at this time-point, it seems unlikely that CaMKII inhibitors would be effective. This may be explained by the fact that the level of pCaMKII within the PSD remained unchanged in the current studies. Thus, although total CaMKII is being removed from postsynaptic sites along with the GluN2B subunit, the level of autophosphorylated CaMKII remains constant to phosphorylate the significantly greater

122

number of homomeric GluA1 AMPARs in 2-day FZP-withdrawn rats (Song et al., 2007;

Das et al., 2008). Phosphatase activity may also be reduced in FZP-withdrawn tissues, so that the same level of kinase activity is unopposed by a corresponding amount of phosphatase activity, as may occur during LTP in CA1 synapses (Blitzer et al., 1998).

This possibility remains to be tested in this model of drug withdrawal-induced plasticity.

123

4.6 Footnotes

This work was supported by the National Institutes of Health National Institute of Drug

Abuse [Grant RO1-DA184342 (EIT) and Fellowship F30-DA026675 (DEE)].

Parts of this work were previously presented at the following meetings: Earl DE and

Tietz EI. (2010). Increased Cav1.3, but not Cav1.2 L-VGCC subunit expression in the rat

hippocampus during flurazepam withdrawal, in 2010 Abstract Viewer, 2010 Nov 13–17;

San Diego, CA. Society for Neuroscience, Washington, DC.

124

4.7 Acknowledgements

The authors would like to thank Yana Fedotova and Paula Kramer for their technical assistance with electron microscopic image capture and processing negatives, Krista Pette and Nikki Wenzlaff for assistance with dosing procedures, and Krista Pettee, Dr. Nathalie

Boulineau, and Dr. David Giovannucci for their assistance with the immunofluorescent staining procedure, confocal imaging, and image analysis, respectively.

125

4.8 References

Bayer, K. U., De Koninck, P., Leonard, A. S., Hell, J. W., & Schulman, H. (2001).

Interaction with the NMDA receptor locks CaMKII in an active conformation.

Nature, 411(6839), 801-805.

Bayer, K. U., LeBel, E., McDonald, G. L., O'Leary, H., Schulman, H., & De

Koninck, P. (2006). Transition from reversible to persistent binding of

CaMKII to postsynaptic sites and NR2B. J Neurosci, 26(4), 1164-1174.

Bennett, M. K., Erondu, N. E., & Kennedy, M. B. (1983). Purification and

characterization of a calmodulin-dependent protein kinase that is highly

concentrated in brain. J Biol Chem, 258(20), 12735-12744.

Bichet, D., Cornet, V., Geib, S., Carlier, E., Volsen, S., Hoshi, T., et al. (2000). The I-

II loop of the Ca2+ channel alpha1 subunit contains an endoplasmic reticulum

retention signal antagonized by the beta subunit. Neuron, 25(1), 177-190.

Blitzer, R. D., Connor, J. H., Brown, G. P., Wong, T., Shenolikar, S., Iyengar, R., et

al. (1998). Gating of CaMKII by cAMP-regulated protein phosphatase activity

during LTP. Science, 280(5371), 1940-1942.

Catterall, W. A. (2000). Structure and regulation of voltage-gated Ca2+ channels.

Annu Rev Cell Dev Biol, 16, 521-555.

Das, P., Lilly, S. M., Zerda, R., Gunning, W. T., 3rd, Alvarez, F. J., & Tietz, E. I.

(2008). Increased AMPA receptor GluR1 subunit incorporation in rat

hippocampal CA1 synapses during benzodiazepine withdrawal. J Comp

Neurol, 511(6), 832-846.

126

Das, P., Zerda, R., Alvarez, F. J., & Tietz, E. I. (2010). Immunogold electron

microscopic evidence of differential regulation of GluN1, GluN2A, and

GluN2B, NMDA-type glutamate receptor subunits in rat hippocampal CA1

synapses during benzodiazepine withdrawal. J Comp Neurol, 518(21), 4311-

4328.

Dosemeci, A., Reese, T. S., Petersen, J., & Tao-Cheng, J. H. (2000). A novel

particulate form of Ca(2+)/calmodulin-dependent [correction of

Ca(2+)/CaMKII-dependent] protein kinase II in neurons. J Neurosci, 20(9),

3076-3084.

Dosemeci, A., Vinade, L., Winters, C. A., Reese, T. S., & Tao-Cheng, J. H. (2002).

Inhibition of phosphatase activity prolongs NMDA-induced modification of

the postsynaptic density. J Neurocytol, 31(8-9), 605-612.

Earl, D. E., & Tietz, E. I. (2011). Inhibition of Recombinant L-Type Voltage-Gated

Calcium Channels by Positive Allosteric Modulators of GABAA Receptors. J

Pharmacol Exp Ther, 337(1), 301-311.

Erondu, N. E., & Kennedy, M. B. (1985). Regional distribution of type II

Ca2+/calmodulin-dependent protein kinase in rat brain. J Neurosci, 5(12),

3270-3277.

Fink, C. C., & Meyer, T. (2002). Molecular mechanisms of CaMKII activation in

neuronal plasticity. Curr Opin Neurobiol, 12(3), 293-299.

Gao, L., Blair, L. A., Salinas, G. D., Needleman, L. A., & Marshall, J. (2006).

Insulin-like growth factor-1 modulation of CaV1.3 calcium channels depends

on Ca2+ release from IP3-sensitive stores and calcium/calmodulin kinase II

127

phosphorylation of the alpha1 subunit EF hand. J Neurosci, 26(23), 6259-

6268.

Griffiths, R. R., & Johnson, M. W. (2005). Relative abuse liability of hypnotic drugs:

a conceptual framework and algorithm for differentiating among compounds.

J Clin Psychiatry, 66 Suppl 9, 31-41.

Hell, J. W., Westenbroek, R. E., Breeze, L. J., Wang, K. K., Chavkin, C., & Catterall,

W. A. (1996). N-methyl-D-aspartate receptor-induced proteolytic conversion

of postsynaptic class C L-type calcium channels in hippocampal neurons.

Proc Natl Acad Sci U S A, 93(8), 3362-3367.

Hudmon, A., Schulman, H., Kim, J., Maltez, J. M., Tsien, R. W., & Pitt, G. S. (2005).

CaMKII tethers to L-type Ca2+ channels, establishing a local and dedicated

integrator of Ca2+ signals for facilitation. J Cell Biol, 171(3), 537-547.

Hulme, J. T., Westenbroek, R. E., Scheuer, T., & Catterall, W. A. (2006).

Phosphorylation of serine 1928 in the distal C-terminal domain of cardiac

CaV1.2 channels during beta1-adrenergic regulation. Proc Natl Acad Sci U S

A, 103(44), 16574-16579.

Izzo, E., Auta, J., Impagnatiello, F., Pesold, C., Guidotti, A., & Costa, E. (2001).

Glutamic acid decarboxylase and glutamate receptor changes during tolerance

and dependence to benzodiazepines. Proc Natl Acad Sci U S A, 98(6), 3483-

3488.

Jenkins, M. A., Christel, C. J., Jiao, Y., Abiria, S., Kim, K. Y., Usachev, Y. M., et al.

(2010). Ca2+-dependent facilitation of Cav1.3 Ca2+ channels by densin and

Ca2+/calmodulin-dependent protein kinase II. J Neurosci, 30(15), 5125-5135.

128

Kennedy, M. B., Bennett, M. K., & Erondu, N. E. (1983a). Biochemical and

immunochemical evidence that the "major postsynaptic density protein" is a

subunit of a calmodulin-dependent protein kinase. Proc Natl Acad Sci U S A,

80(23), 7357-7361.

Kennedy, M. B., McGuinness, T., & Greengard, P. (1983b). A calcium/calmodulin-

dependent protein kinase from mammalian brain that phosphorylates Synapsin

I: partial purification and characterization. J Neurosci, 3(4), 818-831.

Klockner, U., Mikala, G., Varadi, M., Varadi, G., & Schwartz, A. (1995).

Involvement of the carboxyl-terminal region of the alpha 1 subunit in voltage-

dependent inactivation of cardiac calcium channels. J Biol Chem, 270(29),

17306-17310.

Larsson, M., & Broman, J. (2006). Pathway-specific bidirectional regulation of

Ca2+/calmodulin-dependent protein kinase II at spinal nociceptive synapses

after acute noxious stimulation. J Neurosci, 26(16), 4198-4205.

Lau, C. E., Falk, J. L., Dolan, S., & Tang, M. (1987). Simultaneous determination of

flurazepam and five metabolites in serum by high-performance liquid

chromatography and its application to pharmacokinetic studies in rats. J

Chromatogr, 423, 251-259.

Lee, T. S., Karl, R., Moosmang, S., Lenhardt, P., Klugbauer, N., Hofmann, F., et al.

(2006). Calmodulin kinase II is involved in voltage-dependent facilitation of

the L-type Cav1.2 calcium channel: Identification of the phosphorylation

sites. J Biol Chem, 281(35), 25560-25567.

129

Lorenz, J. M., Riddervold, M. H., Beckett, E. A., Baker, S. A., & Perrino, B. A.

(2002). Differential autophosphorylation of CaM kinase II from phasic and

tonic smooth muscle tissues. Am J Physiol Cell Physiol, 283(5), C1399-1413.

Ludwig, A., Flockerzi, V., & Hofmann, F. (1997). Regional expression and cellular

localization of the alpha1 and beta subunit of high voltage-activated calcium

channels in rat brain. J Neurosci, 17(4), 1339-1349.

Malenka, R. C., & Nicoll, R. A. (1999). Long-term potentiation--a decade of

progress? Science, 285(5435), 1870-1874.

Oliveria, S. F., Dell'Acqua, M. L., & Sather, W. A. (2007). AKAP79/150 anchoring

of calcineurin controls neuronal L-type Ca2+ channel activity and nuclear

signaling. Neuron, 55(2), 261-275.

Safa, P., Boulter, J., & Hales, T. G. (2001). Functional properties of Cav1.3

(alpha1D) L-type Ca2+ channel splice variants expressed by rat brain and

neuroendocrine GH3 cells. J Biol Chem, 276(42), 38727-38737.

Shen, G., Mohamed, M. S., Das, P., & Tietz, E. I. (2009). Positive allosteric

activation of GABAA receptors bi-directionally modulates hippocampal

glutamate plasticity and behaviour. Biochem Soc Trans, 37(Pt 6), 1394-1398.

Shen, G., & Tietz, E. I. (2011). Down-regulation of synaptic GluN2B subunit-

containing N-methyl-D-aspartate receptors: a physiological brake on CA1

neuron alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid

hyperexcitability during benzodiazepine withdrawal. J Pharmacol Exp Ther,

336(1), 265-273.

130

Shen, G., Van Sickle, B. J., & Tietz, E. I. (2010). Calcium/calmodulin-dependent

protein kinase II mediates hippocampal glutamatergic plasticity during

benzodiazepine withdrawal. Neuropsychopharmacology, 35(9), 1897-1909.

Shen, K., Teruel, M. N., Subramanian, K., & Meyer, T. (1998). CaMKIIbeta

functions as an F-actin targeting module that localizes CaMKIIalpha/beta

heterooligomers to dendritic spines. Neuron, 21(3), 593-606.

Silva, A. J., Stevens, C. F., Tonegawa, S., & Wang, Y. (1992). Deficient hippocampal

long-term potentiation in alpha-calcium-calmodulin kinase II mutant mice.

Science, 257(5067), 201-206.

Singh, A., Gebhart, M., Fritsch, R., Sinnegger-Brauns, M. J., Poggiani, C., Hoda, J.

C., et al. (2008). Modulation of voltage- and Ca2+-dependent gating of

CaV1.3 L-type calcium channels by alternative splicing of a C-terminal

regulatory domain. J Biol Chem, 283(30), 20733-20744.

Song, J., Shen, G., Greenfield, L. J., Jr., & Tietz, E. I. (2007). Benzodiazepine

withdrawal-induced glutamatergic plasticity involves up-regulation of GluR1-

containing alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid

receptors in Hippocampal CA1 neurons. J Pharmacol Exp Ther, 322(2), 569-

581.

Taha, S., Hanover, J. L., Silva, A. J., & Stryker, M. P. (2002). Autophosphorylation

of alphaCaMKII is required for ocular dominance plasticity. Neuron, 36(3),

483-491.

Tang, Z. Z., Liang, M. C., Lu, S., Yu, D., Yu, C. Y., Yue, D. T., et al. (2004).

Transcript scanning reveals novel and extensive splice variations in human l-

131

type voltage-gated calcium channel, Cav1.2 alpha1 subunit. J Biol Chem,

279(43), 44335-44343.

Tippens, A. L., Pare, J. F., Langwieser, N., Moosmang, S., Milner, T. A., Smith, Y.,

et al. (2008). Ultrastructural evidence for pre- and postsynaptic localization of

Cav1.2 L-type Ca2+ channels in the rat hippocampus. J Comp Neurol, 506(4),

569-583.

Van Sickle, B. J., Xiang, K., & Tietz, E. I. (2004). Transient plasticity of

hippocampal CA1 neuron glutamate receptors contributes to benzodiazepine

withdrawal-anxiety. Neuropsychopharmacology, 29(11), 1994-2006.

Xiang, K., Earl, D. E., Davis, K. M., Giovannucci, D. R., Greenfield, L. J., Jr., &

Tietz, E. I. (2008). Chronic benzodiazepine administration potentiates high

voltage-activated calcium currents in hippocampal CA1 neurons. J Pharmacol

Exp Ther, 327(3), 872-883.

Xiang, K., & Tietz, E. I. (2007). Benzodiazepine-induced hippocampal CA1 neuron

alpha-amino-3-hydroxy-5-methylisoxasole-4-propionic acid (AMPA) receptor

plasticity linked to severity of withdrawal anxiety: differential role of voltage-

gated calcium channels and N-methyl-D-aspartic acid receptors. Behav

Pharmacol, 18(5-6), 447-460.

Xie, X. H., & Tietz, E. I. (1992). Reduction in potency of selective gamma-

aminobutyric acidA agonists and diazepam in CA1 region of in vitro

hippocampal slices from chronic flurazepam-treated rats. J Pharmacol Exp

Ther, 262(1), 204-211.

132

Xu, W., & Lipscombe, D. (2001). Neuronal Ca(V)1.3alpha(1) L-type channels

activate at relatively hyperpolarized membrane potentials and are

incompletely inhibited by dihydropyridines. J Neurosci, 21(16), 5944-5951.

Zeng, X., & Tietz, E. I. (1997). Depression of early and late monosynaptic inhibitory

postsynaptic potentials in hippocampal CA1 neurons following prolonged

benzodiazepine administration: role of a reduction in Cl- driving force.

Synapse, 25(2), 125-136.

Zeng, X., Xie, X. H., & Tietz, E. I. (1995). Reduction of GABA-mediated inhibitory

postsynaptic potentials in hippocampal CA1 pyramidal neurons following oral

flurazepam administration. Neuroscience, 66(1), 87-99.

133

4.9 Figures and Tables

Figure 4.1

134

Figure 4.1

Cav1.2 and Cav1.3 immunoblot analysis of subcellular fractions created from CA1 homogenates. (A) Immunoblot with the polyclonal anti-Cav1.2 antibody (4.25 µg/mL) revealed a differential staining pattern in various subcellular fractions (25 – 30 µg).

Notably, a long (220 kDa) and short (180 kDa) size-form was observed in the PSD- enriched, Triton-insoluble membrane fraction (P3), consistent with previous reports for

Cav1.2 (Hell et al., 1996). A smeared band was observed in the Triton-soluble membrane fraction (S3), which also contained an enriched ~170 kDa band. A 120 kDa band enriched in the cytosolic fraction (S2) was not seen following antigenic peptide preincubation. (B) Immunoblot with anti-Cav1.3 antibody (12.45 µg/mL, Jenkins et al.,

2010) also revealed different staining patterns in the various fractions. Notably, two size- forms were detected in the PSD-enriched fraction with apparent molecular weights of 220 and 180 kDa. A weakly staining smear of protein surrounding an ~200 kDa band was observed in the Triton-soluble membrane fraction, and signals with unclear specificity outside the 180 - 220 kDa range were detected in the cytosolic fraction.

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Figure 4.2

136

Figure 4.2

No change in L-VGCC subunit expression was detected in the PSD-enriched fraction after either 1 day (A) or 2 days (B) of FZP withdrawal. Blots were probed with the polyclonal anti-Cav1.2 (4.25 µg/mL, 25 - 30 µg protein/lane), anti-Cav1.3 (7.11 µg/mL,

50 µg protein/lane), or anti-Cavβ3 antibodies (1.5 µg/mL, 50 µg protein/lane). For the day

2 withdrawal point, data for Cav1.2 was averaged from 3 control (CON) and 4 FZP samples, and data for Cavβ3 represents an average of 7 CON and 8 FZP samples pooled from two blots. All other graphs represent data from a single blot in which 4 CON and 4

FZP samples were loaded. All statistical comparisons resulted in p-values > 0.05.

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Figure 4.3

138

Figure 4.3

No change in Cav1.2 subunit expression was detected by immunofluorescent staining of

hippocampal slices from control (CON) and 2-day FZP-withdrawn rats. (A)

Immunostaining with the polyclonal anti-Cav1.2 antibody (4.25 µg/mL) revealed

comparable fluorescent intensities between CON and FZP-withdrawn hippocampi in

every subregion, including the CA1 regions, stratum oriens (SO), s. pyramidale (SP), s.

radiatum (SR), and s. lacunosum-moleculare (SLM), and the dentate gyrus regions,

molecular layer (ML) and granule cell layer (GCL). (B) Omission of the primary

antibody abolished the immunofluorescent signal. Pre-incubation of anti-Cav1.2 with its antigenic peptide resulted in only sparse non-specific signal. (C) Averaged data from 4 separate experiments indicated no statistically significant difference in the Cav1.2 immunofluorescent intensity between CON and FZP-withdrawn tissues in any subregion

(p > 0.05 for each region, n = 6 rats/group).

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Figure 4.4

140

Figure 4.4

Images of asymmetric synapses in CA1 region of control (CON, A,B) and FZP- withdrawn (C,D) rats co-labeled with anti-CaMKIIα (1:20, recognized with 15-nm gold,

arrows) and anti-pCaMKII antibodies (1:10, recognized with 10-nm gold, arrowheads).

PSD-associated (black arrows), as well as membrane-bound and intracellular (white

arrows) CaMKII immunogold particles were observed. Compared to CaMKII, pCaMKII

presynaptic expression (white arrowheads) was higher relative to postsynaptic expression

(black arrowheads, see also Fig. 5). (E-G) There was no significant change in the percent

of boutons (E), spines (F), or PSDs (G) labeled with either CaMKII or pCaMKII (p >

0.05 in each case, Student’s t-test). (H-J) There was no significant change in the mean

number of either CaMKII or pCaMKII immunogold particles located within boutons (H),

spines (I), or PSDs (J, measured per PSD length in µm, p > 0.05 in each case, Student’s t-

test). There was a trend towards decreased CaMKII labeling in spines (61% of control, p

= 0.20, I), as well as a trend towards decreased CaMKII immunogold particles/PSD

length (38% of control, p = 0.12, G and J, respectively). Coupled with unaltered

pCaMKII labeling, this resulted in a trend towards an increased pCaMKII/CaMKII ratio

in PSDs of 2-day FZP-withdrawn rats (J, right panel, p = 0.12).

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Figure 4.5

142

Figure 4.5

Pre- and postsynaptic distribution of CaMKII and pCaMKII immunogold particles in

CA1 asymmetric synapses of control and FZP-withdrawn rats. (A) Analysis of CaMKII

postsynaptic labeling revealed a significant interaction between experimental group and

binned distance (p < 0.05, F(6,48) = 3.13, Two-way Repeated Measures ANOVA) with

significantly decreased CaMKII labeling 60 nm postsynaptic to the PSD membrane in

FZP-withdrawn relative to control synapses (38% of control, p < 0.01, Bonferroni’s

posttest). In contrast, no significant interaction between treatment and binned distance

was observed for presynaptic CaMKII (p > 0.05, Two-way Repeated Measures

ANOVA). (B) There was no significant interaction between experimental group and

binned distance for pre- or postsynaptic pCaMKII labeling (p > 0.05, Two-way Repeated

Measures ANOVA).

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Figure 4.6

144

Figure 4.6

Distribution histograms of postsynaptic profiles containing different numbers of CaMKII

(A) and pCaMKII (B) immunogold particles within the PSD. Histograms represent the average relative frequencies of synapses lacking (0 particles) CaMKII or pCaMKII immunogold particles or containing 1 to 5 immunogold particles. There was a significant decrease in the fraction of FZP-withdrawn synapses containing two CaMKII immunogold particles (p < 0.05, Mann-Whitney U-test), whereas no significant difference was detected in the distribution of pCaMKII immunogold particles (p > 0.05, Mann-Whitney

U-test).

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Table 4.1 CaMKIIα Immunogold Synaptic Labeling

All synapses (≥0 particles) ≥1 particle in respective compartment Bouton Spine PSD Bouton Spine PSD Labeled Mean Labeled Mean Labeled Particles/ PSD Mean Mean Particles/ PSD Synapses (%) ≥ 1 No. of (%) ≥1 No. of (%) ≥ 1 PSD Length No. of No. of PSD Length Rat Sampled Particle Particles Particle Particles Particle Length (µm) Particles Particles Length (µm) CON 1 43 14.0 0.19 20.9 0.60 11.6 1.02 0.184 1.33 2.89 8.77 0.206 2 56 28.6 0.61 46.4 0.89 28.6 2.79 0.156 2.13 1.92 9.76 0.162 3 43 25.6 0.58 58.1 1.74 27.9 1.93 0.196 2.27 3.00 6.90 0.224 4 44 15.9 0.27 40.9 1.18 18.2 2.07 0.170 1.71 2.89 11.37 0.182 5 53 58.5 1.47 84.9 2.66 54.7 6.61 0.170 2.52 3.13 12.07 0.175 Total 239 Mean 28.5 0.62 50.2 1.41 28.2 2.88 0.175 1.99 2.77 9.77 0.190 ± SEM ± 8.0 ± 0.23 ± 10.6 ± 0.36 ± 7.3 ± 0.97 ± 0.007 ± 0.21 ± 0.22 ± 0.92 ± 0.011 146 FZP 1 45 11.1 0.20 17.8 0.27 6.7 0.44 0.165 1.80 1.50 6.56 0.159 2 44 27.3 0.52 56.8 0.98 29.5 1.92 0.170 1.92 1.72 6.50 0.178 3 51 29.4 0.90 45.1 1.27 11.8 0.95 0.158 3.07 2.83 8.04 0.152 4 44 22.7 0.36 38.6 0.75 9.1 0.54 0.168 1.60 1.94 5.92 0.169 5 54 16.7 0.17 59.3 1.02 22.2 1.64 0.169 1.00 1.72 7.37 0.172 Total 238 Mean 21.4 0.43 43.5 0.86 15.9 1.10 0.166 1.88 1.94 6.88 0.166 ± SEM ± 3.4 ± 0.13 ± 7.5 ± 0.17 ± 4.3 ± 0.29 ± 0.002 ± 0.34 ± 0.23 ± 0.37 ± 0.005 p-value 0.44 0.48 0.62 0.20 0.19 0.12 0.25 0.78 0.03* 0.02* 0.08 *, p < 0.05, Student’s t-test

Table 4.2 pCaMKII Immunogold Synaptic Labeling

All synapses (≥0 particles) ≥1 particle in respective compartment Bouton Spine PSD Bouton Spine PSD Labeled Mean Labeled Mean Labeled Particles/ PSD Mean Mean Particles/ PSD Synapses (%) ≥ 1 No. of (%) ≥1 No. of (%) ≥ 1 PSD Length No. of No. of PSD Length Rat Sampled Particle Particles Particle Particles Particle Length (µm) Particles Particles Length (µm) Control 1 43 37.2 1.28 53.5 1.37 30.2 2.60 0.184 3.44 2.57 8.61 0.183 2 56 21.4 0.32 12.5 0.20 8.9 0.98 0.156 1.50 1.57 11.01 0.162 3 43 81.4 3.56 67.4 2.70 34.9 3.94 0.196 4.37 4.00 11.29 0.218 4 44 38.6 0.82 20.5 0.48 11.4 0.66 0.170 2.12 2.33 5.77 0.177 5 53 56.6 1.42 30.2 0.55 28.3 2.75 0.170 2.50 1.81 9.72 0.189 Total 239 Mean 47.0 1.48 36.8 1.06 22.7 2.19 0.175 2.79 2.46 9.28 0.186 ± SEM ± 10.2 ± 0.55 ± 10.3 ± 0.45 ± 5.3 ± 0.61 ± 0.007 ± 0.51 ± 0.43 ± 1.00 ± 0.009 147 FZP 1 45 44.4 0.91 33.3 0.73 17.8 1.87 0.165 2.05 2.20 10.51 0.167 2 44 47.7 1.11 13.6 0.20 11.4 0.86 0.170 2.33 1.50 7.59 0.199 3 51 74.5 2.27 54.9 1.08 23.5 2.68 0.158 3.05 1.96 11.37 0.174 4 44 79.5 3.30 63.6 1.86 25.0 1.70 0.168 4.14 2.93 6.80 0.196 5 54 61.1 1.59 44.4 0.91 29.6 3.12 0.169 2.61 2.04 10.52 0.192 Total 238 Mean 61.4 1.83 42.0 0.96 21.5 2.05 0.166 2.84 2.13 9.36 0.186 ± SEM ± 7.0 ± 0.44 ± 8.7 ± 0.27 ± 1.3 ± 0.39 ± 0.002 ± 0.37 ± 0.23 ± 0.91 ± 0.006 p-value 0.28 0.63 0.71 0.85 0.84 0.85 0.25 0.94 0.52 0.96 0.99

Table 4.3

CaMKIIα and pCaMKII Immunogold Non-PSD Synaptic Labeling

Presynaptic Postsynaptic Perisynaptic Extrasynaptic Active Zone Membrane Intracellular Intracellular Membrane Membrane Labeled Mean Labeled Mean Labeled Mean Labeled Mean Labeled Mean Labeled Mean (%) ≥ 1 No. of (%) ≥1 No. of (%) ≥ 1 No. of (%) ≥ 1 No. of (%) ≥ 1 No. of (%) ≥ 1 No. of Particle Particles Particle Particles Particle Particles Particle Particles Particle Particles Particle Particles CaMKII 4.0 0.04 3.0 0.04 26.2 0.54 0.8 0.02 3.9 0.07 38.8 0.86 CON ± 1.3 ± 0.01 ± 1.5 ± 0.02 ± 7.6 ± 0.20 ± 0.5 ± 0.01 ± 1.4 ± 0.04 ± 9.9 ± 0.22

0.8 0.008 2.6 0.03 19.8 0.39 2.6 0.03 4.8 0.06 32.2 0.59 FZP

± 0.5 ± 0.005 ± 1.3 ± 0.01 ± 3.0 ± 0.12 ± 1.6 ± 0.02 ± 2.6 ± 0.04 ± 5.7 ± 0.12 148 p-value 0.049* 0.047* 0.83 0.88 0.45 0.54 0.37 0.70 0.75 0.94 0.58 0.32 pCaMKII 7.2 0.09 4.5 0.10 43.7 1.29 0.9 0.01 2.3 0.02 21.9 0.62 CON ± 2.3 ± 0.03 ± 2.5 ± 0.08 ± 9.7 ± 0.46 ± 0.9 ± 0.01 ± 1.1 ± 0.01 ± 10.5 ± 0.34

7.3 0.08 6.7 0.09 58.6 1.67 3.1 0.04 5.1 0.07 26.9 0.49 FZP ± 2.2 ± 0.03 ± 3.0 ± 0.04 ± 7.6 ± 0.40 ± 2.2 ± 0.03 ± 2.8 ± 0.05 ± 7.8 ± 0.18 p-value 0.98 0.96 0.60 0.87 0.26 0.56 0.40 0.38 0.37 0.36 0.71 0.76 *, p < 0.05, Student’s t-test. Values represent mean ± SEM.

CHAPTER 5

CONCLUSIONS AND SUMMARY

5.1 Conclusions

The following novel findings were generated during the dissertation work outlined in

Chapters 3 and 4:

1. GABAA receptor modulators inhibited recombinant neuronal Cav1.2 and Cav1.3 L-

VGCCs in a concentration-dependent manner, yet only pentobarbital and ethanol, but not

BZs and allopregnanolone inhibited L-VGCCs at concentrations achieved in vivo.

2. L-VGCC inhibition by GABAA receptor modulators was state-dependent, with

preference for inactivate and possibly open channel states.

3. Pentobarbital and flurazepam were more potent inhibitors of Cav1.2 than Cav1.3 channels, and desalkylflurazepam was also more potent at Cav1.2 channels in a state-

dependent manner.

4. Diltiazem was a more potent inhibitor of Cav1.3 channels and inhibited Cav1.3 with greater apparent use-dependence than Cav1.2 channels.

5. In addition to a dissimilar manner of L-VGCC inhibition between diltiazem and BZs, mutation of a Cav1.2 amino acid important for diltiazem potency did not alter diazepam

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potency, and diltiazem did not antagonize diazepam-mediated inhibition of Cav1.2 channels, suggesting that despite their structural similarity, BZs and BTZs act at distinct

L-VGCC binding sites.

6. Mutation of the DHP binding site in Cav1.2 channels reduced pentobarbital potency,

suggesting that the barbiturate L-VGCC binding site may overlap that of DHPs.

7. Flumazenil, a competitive BZ antagonist at the GABAA receptor, did not inhibit L-

VGCCs, yet antagonized BZ-mediated inhibition of Cav1.3 but not Cav1.2 channels,

suggesting that a flumazenil binding site might exist on Cav1.3 channels analogous to the

GABAA receptor.

1928 8. Cav1.2, Cav1.3, Cavβ3, and pSer -Cav1.2 expression was not altered in PSD-enriched

CA1 homogenates from 1- and 2-day FZP-withdrawn rats, suggesting that an alternate

mechanism of up-regulated L-VGCC function occurs following long-term FZP treatment.

9. CaMKIIα, but not Thr286/287 autosphophorylated CaMKII expression was decreased in

PSDs of CA1 SR asymmetric synapses in 2-day FZP-withdrawn rats.

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5.2 Summary and inferences

Long-term administration of positive GABAA receptor modulators can lead to tolerance, especially to their sedative and anticonvulsant effects (File, 1985; Rosenberg &

Chiu, 1985; Rosenberg et al., 1991), and with higher doses also increases the risk of

developing physical dependence manifested by a characteristic withdrawal syndrome that is more severe for barbiturates and ethanol relative to BZs (Hodding et al., 1980; Griffiths

& Weerts, 1997). Differences in the manifestation and severity of withdrawal from the various GABAA modulators likely pertain to their differential effects at other molecular

targets. However, one common molecular mechanism proposed to contribute to

withdrawal hyperexcitability induced by long-term exposure to BZs (Katsura et al., 2007;

Xiang et al., 2008), barbiturates (Rabbani & Little, 1999), and ethanol (Messing et al.,

1986; Katsura et al., 2006) is enhanced Ca2+ influx through L-VGCCs. One aim of the

current studies was to assess whether various GABAA receptor modulators directly

inhibit L-VGCCs, both as a putative mechanism to mediate the enhanced L-VGCC function and a mechanism to prevent downstream affects of L-VGCCs until drug withdrawal.

Findings related to the possibility that GABAA receptor modulators directly inhibit

recombinantly expressed Cav1.2 and Cav1.3 L-VGCCs were published in the Journal of

Pharmacology and Experimental Therapeutics manuscript in Chapter 3 (Earl & Tietz,

2011). These findings supported a differential role of direct L-VGCC inhibition by

various GABAA receptor modulators in contributing to the neuronal adaptations that take place during long-term drug exposure. While pentobarbital and ethanol directly inhibit L-

VGCCs at clinically relevant concentrations, the concentrations of BZs and

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allopregnanolone required to inhibit L-VGCCs are likely too high to be clinically

relevant. These findings are consistent with the relative selectivity of these drugs for the

GABAA receptor, with ethanol having the greatest number of alternate molecular targets

at clinically relevant concentrations, followed by barbiturates, then neurosteroids and

BZs. Barbiturate and ethanol actions at alternate molecular targets, including L-VGCCs,

may also contribute to the toxic effects of these drugs, such as memory, cognitive, and

psychomotor impairment and potential for overdose toxicity, whereas BZs exhibit a

relatively low toxicity profile (Griffiths & Johnson, 2005). Nevertheless, acute drug effects measured using recombinantly expressed L-VGCCs in HEK cells may not represent that which occurs during long-term treatment at native channels expressed in cultured neurons or in vivo. Notably, a significant concentration and use-dependent inhibition of VGCCs was measured by preincubation of cultured hippocampal neurons with flurazepam at concentrations as low as 1 µM (Xiang et al., 2008). However, when working with neuronal preparations effects on non-L-VGCC subtypes cannot be ruled out. The contribution of different VGCCs to BZ effects could be addressed in future experiments by pharmacologically and/or electrophysiologically isolating L-VGCC currents.

Interestingly, Cav1.2 channels were more sensitive to inhibition by pentobarbital and

FZP and were less sensitive to inhibition by the L-VGCC benzothiazepine antagonist,

diltiazem, than Cav1.3 channels. Selective inhibition could independently block the

2+ different Ca signaling cascades mediated by Cav1.2 and Cav1.3 L-VGCCs. In particular,

Cav1.2 channels play a role in a protein synthesis-dependent form of L-LTP involved in memory formation (Moosmang et al., 2005), whereas Cav1.3 channels impact neuronal

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excitability and firing rates (Bowden et al., 2001; Putzier et al., 2009) and may promote age-related neuronal degeneration and cognitive dysfunction, as occurs in Alzheimer’s

and Parkinson’s diseases (Thibault & Landfield, 1996; Porter et al., 1997; Chan et al.,

2010). Thus, not only does selective inhibition provide a useful pharmacologic tool in

dissecting the differential functions of L-VGCC subtypes, but may also be valuable for treating age-related neuronal degeneration, without impairing Cav1.2-dependent memory.

However, other side-effects related to Cav1.3 inhibition may be an issue for therapeutic

treatment. For example, Cav1.3 knockout mice are congenitally deaf and display

arrhythmias due to the important role of Cav1.3 channels in the function of cochlear hair

cells and sinoatrial node cells, respectively (Platzer et al., 2000).

GABAA receptor modulators inhibited L-VGCCs in a state-dependent manner, which

appeared to involve a more selective interaction with the inactive and/or open state of

channels. This state-dependent interaction lead to more potent inhibition of Cav1.2 channels by desalkylflurazepam relative to Cav1.3 channels (see Fig. 3.7), suggesting that

differential potency dependent on the holding potential and frequency of activation can

also be observed. Interestingly, the BTZ, diltiazem appeared to inhibit L-VGCCs in a

manner distinct from BZs in terms of state- (Tables 3.2 and 3.3) and use-dependence

(Fig. 3.3B,C). Moreover, mutation of an amino acid (Ile1150) important for diltiazem

binding did not affect diazepam affinity, nor was diltiazem able to antagonize diazepam-

mediated inhibition (Fig. 3.9). Together the data suggested that BZs interact with L-

VGCCs at a site distinct from BTZs despite the structural similarity amongst these

compounds. In contrast, pentobarbital potency was reduced 3.5-fold by mutations that

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reduce Cav1.2 sensitivitity to DHPs (Fig. 3.8), suggesting that the barbiturate site on L-

VGCCs may overlap that of DHPs.

It is interesting that all the positive GABAA receptor modulators tested enhance

currents through GABAA receptors, but inhibit L-VGCCs. Thus, the current studies support a common finding amongst drugs that act at both anion and cation channels. In particular, drugs that enhance anion currents inhibit cation currents, and vice versa.

Additional examples include the neurosteroid pregnenolone sulfate, a negative modulator at GABAA receptors (Akk et al., 2001) but a positive modulator at NMDA receptors

(Malayev et al., 2002) and several general anesthetics such as etomidate, isoflurane,

propofol, sevoflurane, and nitrous oxide, which are generally positive modulators of

GABAA and glycine receptors but inhibitors of nicotinic acetylcholine, 5-HT3, AMPA, kainate, and NMDA receptors (Grasshoff et al., 2006), as well as voltage-gated Na+ channels (Ouyang et al., 2003). It is interesting to speculate that this effect may be due to intrinsic gating mechanisms that are opposite for anion and cation channels. However, this generalized principle is not true in all cases, and one exception includes DHPs, which inhibit L-VGCCs, as well as GABAA receptors (Das et al., 2004).

The mechanisms underlying the neuronal adaptations that occur during treatment and

the ensuing CA1 neuron hyperexcitability during withdrawal (Whittington & Little,

1991; Smith et al., 1998; Izzo et al., 2001; Van Sickle et al., 2004) remain unclear, but

inhibition of L-VGCCs may contribute. Decreased GABAA receptor function and

alterations in GABAA receptor subunit mRNA and protein expression during long-term

treatment may result of chronic enhancement of GABA-mediated inhibition. However,

other mechanisms are probably involved, since these changes are not identical between

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GABAA receptor modulators. For example, allosteric uncoupling of GABA and modulator binding sites is a common finding, but differences exist between GABAA receptor modulators in which sites are uncoupled and the extent of uncoupling (Friedman et al., 1996). These differences may be explained by the varied molecular targets of

GABAA receptor modulators. For example, since GABAA receptor function is affected by

Ca2+-mediated signaling cascades (Houston et al., 2009; Saliba et al., 2009), direct inhibition of L-VGCCs could promote GABAA receptor dysfunction both acutely and by

long-term treatment with barbiturates and ethanol. Moreover, the tight link between L-

VGCC-mediated Ca2+ influx and gene transcription in neurons (Barbado et al., 2009),

allows for the potential regulation of a wide variety of proteins, including GABAA receptor subunits following long-term L-VGCC inhibition. For example, long-term exposure to both barbiturates and ethanol led to increased α6 subunit expression in the cerebellum (Ito et al., 1996; Sanna et al., 2004a). However, it remains to be determined if long-term inhibition of L-VGCCs alone is sufficient to induce changes in GABAA receptor subunit expression, or if these changes might involve cross-talk amongst the other molecular targets of barbiturates and ethanol.

Direct inhibition of L-VGCCs by barbiturates and ethanol may also contribute to L-

VGCC up-regulation observed following long-term drug exposure (Messing et al., 1986;

Grant et al., 1993; Rabbani & Little, 1999; Katsura et al., 2006). In contrast, enhanced L-

VGCC function following long-term BZ exposure (Katsura et al., 2007; Xiang et al.,

2008) likely occurs via a different mechanism. One possibility is that BZs reduce L-

VGCC activity indirectly via enhancement of GABAA receptor-mediated

hyperpolarization. This hypothesis is supported by the fact that 0.1 µM flumazenil was

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able to antagonize up-regulated L-VGCC function in cortical cultures chronically

exposed to 0.3 µM of diazepam, 1 µM brotizolam, or 1 µM clobazam (Katsura et al.,

2007), suggesting that BZ binding to the GABAA receptor was required. Although the

current studies support antagonism of BZ-mediated inhibition of Cav1.3 channels by

flumazenil (see Table 3.1), only a small degree of antagonism was observed and required

a relatively high concentration (100 µM) of flumazenil. In addition, BZ antagonism was

not observed at Cav1.2 channels in the presence of up to 300 µM flumazenil. Thus,

flumazenil likely precluded L-VGCC functional enhancement by preventing BZ binding

to GABAA receptors in cultured neurons. It would be interesting to determine if

flumazenil administration could similarly prevent the enhanced HVA Ca2+ current

density observed in CA1 neurons following long-term FZP treatment in vivo (Xiang et

al., 2008).

Co-administration of a DHP with BZs in vitro (Katsura et al., 2007) or with ethanol in

vivo (Whittington et al., 1991) also prevented up-regulation of L-VGCCs. This may suggest that increased Ca2+ influx through L-VGCCs mediates the up-regulation. In

2+ hippocampal cultures Cav1.2 trafficking to distal dendrites was enhanced in a Ca /CaM- dependent fashion upon K+-stimulated depolarization (Wang et al., 2007). However, this

does not indicate incorporation into the plasma membrane as it was shown that neuronal

cells exposed to prolonged depolarizing conditions resulted in a down-regulation of L-

VGCC surface expression (DHP binding), which was dependent on Ca2+ influx and calmodulin and recovered rapidly (DeLorme et al., 1988; Ferrante et al., 1991; Liu et al.,

1994; Feron & Godfraind, 1995). The converse is therefore conceivable; that basal Ca2+ influx through L-VGCCs would serve to maintain their membrane expression at

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relatively constant levels, and reduced L-VGCC activity may result in a corresponding

increase in surface expression. This would require a Ca2+ sensor capable of responding to

2+ 2+ lowered [Ca ]i and/or changes in temporal Ca dynamics. For example, in addition to the Thr286 autophosphorylation site on CaMKII that mediates autonomous activity,

autophosphorylation of Thr305/306 prevents Ca2+/CaM binding and inhibits holoenzyme

activity (Hanson & Schulman, 1992). This dynamic regulation allows CaMKII to

maintain a short-term molecular memory of past Ca2+ signals, and its activity can

therefore be modulated by changes in the frequency and amplitude of Ca2+ oscillations

(De Koninck & Schulman, 1998). In addition, Cav1.2 and Cav1.3 L-VGCCs themselves

are coupled to different intracellular signaling complexes and respond to different

stimulation intensities (Zhang et al., 2006), suggesting that changes in neuronal activity

can differentially activate L-VGCC subtype signaling cascades. Thus, complete blockade

of L-VGCC-mediated Ca2+ influx by a DHP could also prevent a Ca2+-dependent

enhancement of L-VGCC surface expression resulting from reduced (but not abolished)

L-VGCC activity.

It is also possible that the BZ-GABAA receptor interaction paradoxically increases L-

VGCC-mediated Ca2+ influx during FZP treatment, evidenced by the possibly

extrasynaptic GABAA receptor-mediated depolarization observed during FZP withdrawal

(Zeng et al., 1995; Zeng & Tietz, 2000). However, if this same mechanism existed during

treatment, it would mediate increased CA1 neuron activity and NMDA-dependent and

potentially L-VGCC-dependent plasticity, which seems counter-intuitive. In order for

BZs to maintain anxiolytic effects, activity of the neuronal circuits mediating anxiety

must still be reduced during treatment. On the other hand, if functional inhibition is

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maintained in other parts of the circuit, this may allow for localized dendritic

hyperexcitability in CA1 neurons during treatment, while still reducing overall activity

through the circuit. Distinguishing these possibilities would require measurement of

− − 2+ GABA-mediated Cl , HCO3 , and Ca fluxes in CA1 neurons during FZP treatment.

There is a practical difficulty in doing this due to the continued presence of BZs in the

brain during treatment, which would interfere with these measurements. This could

potentially be avoided by competitively antagonizing BZs from their GABAA receptor

binding sites using flumazenil prior to experimental manipulations. Clearly, additional

studies are needed to decipher the complex regulation of ion channels by GABAA receptor modulators, keeping in mind the importance of interneuron networks on neuronal activity in vivo, which may be lost with in vitro studies.

In conjunction with enhanced voltage-dependent Ca2+ influx, numerous studies

detected an increased number of radiolabeled L-VGCC antagonist binding sites on

neuronal membranes following long-term exposure to BZs (Katsura et al., 2007),

barbiturates (Rabbani & Little, 1999), and ethanol (Messing et al., 1986; Whittington et

al., 1991; Katsura et al., 2006). Therefore, the second aim was to determine if expression

of L-VGCC subunits is altered during FZP withdrawal. In particular, Cav1.3 channels,

which activate at relatively negative membrane potentials, could mediate the negative

shift in the voltage-dependence of HVA Ca2+ channel activation observed following

long-term FZP treatment (Xiang et al., 2008). A number of changes in excitatory receptor

function also occur during FZP withdrawal. Notably, previous findings suggested that L-

VGCC-mediated Ca2+ influx may lead to insertion of homomeric GluA1 AMPA

receptors into CA1 neuron PSDs during the first day of FZP withdrawal (Van Sickle et

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al., 2004; Song et al., 2007; Xiang & Tietz, 2007; Das et al., 2008; Xiang et al., 2008).

Further, L-VGCCs maintain AMPA receptor potentiation (Xiang et al., 2008) and may

also induce the CaMKII-mediated enhancement of GluA1-containing AMPA receptor

conductance during the second day of withdrawal (Shen et al., 2009). However, the

extent of CaMKII Thr286 autophosphorylation detected in immunoblots was not changed during FZP withdrawal (Shen et al., 2010), suggesting that an alternative mechanism of

CaMKII autonomous activity and/or translocation of autophosphorylated CaMKII to the

PSD is induced by L-VGCCs. Thus, the final aim of the current studies was to assess the expression level and postsynaptic localization of total CaMKII and its Thr286 autophosphorylated form in asymmetric synapses located in the CA1 SR region during

FZP withdrawal.

Findings contained in the second manuscript (Chapter 4) indicated that enhanced L-

VGCC function during FZP withdrawal was not explained by a simple increase in L-

VGCC subunit expression. Other possibilities include enhanced trafficking of channels from intracellular compartments to the plasma membrane and/or post-translational modifications such as phosphorylation, which regulate channel gating. One well-

1928 characterized site on the Cav1.2 subunit, Ser , enhances time spent in the in open state

when phosphorylated by PKA, PKC, or PKG (De Jongh et al., 1996; Yang et al., 2005;

1928 Yang et al., 2007). However, there was no change in the Cav1.2 Ser phosphorylation

state after 2 days of FZP withdrawal. Numerous other phosphorylation sites have also

been characterized on both Cav1.2 and Cav1.3 subunits, including sites regulated by

CaMKII, which mediates Ca2+-dependent facilitation of channel function (Hudmon et al.,

159

2005; Jenkins et al., 2010). Creation of an antibody to measure phosphorylation at these

sites may reveal if L-VGCC function is enhanced in a Ca2+-dependent manner.

Studies of CaMKIIα and Thr286 autophosphorylated CaMKII expression and synaptic

localization at the EM level revealed that CaMKIIα, but not pCaMKII expression was

decreased in CA1 postsyanpses after 2 days of FZP withdrawal. This resulted in a trend

towards an increased ratio of pCaMKII/CaMKII within the PSD, suggesting that a greater

proportion of CaMKII molecules are autophosphorylated at Thr286 during FZP

withdrawal. The loss of CaMKIIα may relate to the significant decrease in GluN2B-

subunit containing NMDA receptors at the same withdrawal time point. While this

correlation alone does not indicate an increased interaction, it is possible that drug-

induced enhancement of L-VGCC-mediated Ca2+ influx may lead to an increased

interaction of CaMKII with GluN2B to increase CaMKII autonomous activity (Bayer et

al., 2001). Subsequent to CaMKII-mediated potentiation of AMPA receptor currents and

conductance (Shen et al., 2009; Shen et al., 2010), removal of GluN2B-CaMKII

complexes from the PSD would serve to homestatically reverse AMPA recptor-mediated

hyperexcitability, attenuating withdrawal anxiety. Since similar mechanisms are not observed during barbiturate or ethanol withdrawal, this may explain why the withdrawal syndrome for BZs is much less severe compared to the former non-selective GABAA receptor modulators.

160

REFERENCES

Aguayo, L. G., Peoples, R. W., Yeh, H. H., & Yevenes, G. E. (2002). GABA(A)

receptors as molecular sites of ethanol action. Direct or indirect actions? Curr

Top Med Chem, 2(8), 869-885.

Akk, G., Bracamontes, J., & Steinbach, J. H. (2001). Pregnenolone sulfate block of

GABA(A) receptors: mechanism and involvement of a residue in the M2

region of the alpha subunit. J Physiol, 532(Pt 3), 673-684.

Alger, B. E., & Nicoll, R. A. (1979). GABA-mediated biphasic inhibitory responses

in hippocampus. Nature, 281(5729), 315-317.

Alger, B. E., & Nicoll, R. A. (1982). Pharmacological evidence for two kinds of

GABA receptor on rat hippocampal pyramidal cells studied in vitro. J Physiol,

328, 125-141.

Allan, A. M., Zhang, X., & Baier, L. D. (1992). Barbiturate tolerance: effects on

GABA-operated chloride channel function. Brain Res, 588(2), 255-260.

Allison, C., & Pratt, J. A. (2003). Neuroadaptive processes in GABAergic and

glutamatergic systems in benzodiazepine dependence. Pharmacol Ther, 98(2),

171-195.

Allison, C., Pratt, J. A., Ripley, T. L., & Stephens, D. N. (2005). alpha-Amino-3-

hydroxy-5-methylisoxazole-4-propionate receptor autoradiography in mouse

161

brain after single and repeated withdrawal from diazepam. Eur J Neurosci,

21(4), 1045-1056.

Amaral, D. G., & Witter, M. P. (1989). The three-dimensional organization of the

hippocampal formation: a review of anatomical data. Neuroscience, 31(3),

571-591.

Amin, Z., Mason, G. F., Cavus, I., Krystal, J. H., Rothman, D. L., & Epperson, C. N.

(2006). The interaction of neuroactive steroids and GABA in the development

of neuropsychiatric disorders in women. Pharmacol Biochem Behav, 84(4),

635-643.

Barbado, M., Fablet, K., Ronjat, M., & De Waard, M. (2009). Gene regulation by

voltage-dependent calcium channels. Biochim Biophys Acta, 1793(6), 1096-

1104.

Barnard, E. A., Skolnick, P., Olsen, R. W., Mohler, H., Sieghart, W., Biggio, G., et al.

(1998). International Union of Pharmacology. XV. Subtypes of gamma-

aminobutyric acidA receptors: classification on the basis of subunit structure

and receptor function. Pharmacol Rev, 50(2), 291-313.

Baroudi, G., Qu, Y., Ramadan, O., Chahine, M., & Boutjdir, M. (2006). Protein

kinase C activation inhibits Cav1.3 calcium channel at NH2-terminal serine

81 phosphorylation site. Am J Physiol Heart Circ Physiol, 291(4), H1614-

1622.

Bayer, K. U., De Koninck, P., Leonard, A. S., Hell, J. W., & Schulman, H. (2001).

Interaction with the NMDA receptor locks CaMKII in an active conformation.

Nature, 411(6839), 801-805.

162

Bayer, K. U., LeBel, E., McDonald, G. L., O'Leary, H., Schulman, H., & De

Koninck, P. (2006). Transition from reversible to persistent binding of

CaMKII to postsynaptic sites and NR2B. J Neurosci, 26(4), 1164-1174.

Bean, B. P. (1984). Nitrendipine block of cardiac calcium channels: high-affinity

binding to the inactivated state. Proc Natl Acad Sci U S A, 81(20), 6388-6392.

Bengtson, C. P., Freitag, H. E., Weislogel, J. M., & Bading, H. (2010). Nuclear

calcium sensors reveal that repetition of trains of synaptic stimuli boosts

nuclear calcium signaling in CA1 pyramidal neurons. Biophys J, 99(12),

4066-4077.

Bennett, M. K., Erondu, N. E., & Kennedy, M. B. (1983). Purification and

characterization of a calmodulin-dependent protein kinase that is highly

concentrated in brain. J Biol Chem, 258(20), 12735-12744.

Benson, J. A., Low, K., Keist, R., Mohler, H., & Rudolph, U. (1998). Pharmacology

of recombinant gamma-aminobutyric acidA receptors rendered diazepam-

insensitive by point-mutated alpha-subunits. FEBS Lett, 431(3), 400-404.

Bichet, D., Cornet, V., Geib, S., Carlier, E., Volsen, S., Hoshi, T., et al. (2000). The I-

II loop of the Ca2+ channel alpha1 subunit contains an endoplasmic reticulum

retention signal antagonized by the beta subunit. Neuron, 25(1), 177-190.

Bitran, D., Dugan, M., Renda, P., Ellis, R., & Foley, M. (1999). Anxiolytic effects of

the neuroactive steroid pregnanolone (3 alpha-OH-5 beta-pregnan-20-one)

after microinjection in the dorsal hippocampus and lateral septum. Brain Res,

850(1-2), 217-224.

163

Blaesse, P., Airaksinen, M. S., Rivera, C., & Kaila, K. (2009). Cation-chloride

cotransporters and neuronal function. Neuron, 61(6), 820-838.

Blaustein, M. P., & Ector, A. C. (1975). Barbiturate inhibition of calcium uptake by

depolarized nerve terminals in vitro. Mol Pharmacol, 11(3), 369-378.

Blitzer, R. D., Connor, J. H., Brown, G. P., Wong, T., Shenolikar, S., Iyengar, R., et

al. (1998). Gating of CaMKII by cAMP-regulated protein phosphatase activity

during LTP. Science, 280(5371), 1940-1942.

Boehm, J., & Malinow, R. (2005). AMPA receptor phosphorylation during synaptic

plasticity. Biochem Soc Trans, 33(Pt 6), 1354-1356.

Borden, L. A., Czajkowski, C., Chan, C. Y., & Farb, D. H. (1984). Benzodiazepine

receptor synthesis and degradation by neurons in culture. Science, 226(4676),

857-860.

Borghese, C. M., & Harris, R. A. (2007). Studies of ethanol actions on recombinant

delta-containing gamma-aminobutyric acid type A receptors yield

contradictory results. Alcohol, 41(3), 155-162.

Bormann, J., Hamill, O. P., & Sakmann, B. (1987). Mechanism of anion permeation

through channels gated by glycine and gamma-aminobutyric acid in mouse

cultured spinal neurones. J Physiol, 385, 243-286.

Bowden, S. E., Fletcher, S., Loane, D. J., & Marrion, N. V. (2001). Somatic

colocalization of rat SK1 and D class (Ca(v)1.2) L-type calcium channels in

rat CA1 hippocampal pyramidal neurons. J Neurosci, 21(20), RC175.

Brandon, N. J., Delmas, P., Kittler, J. T., McDonald, B. J., Sieghart, W., Brown, D.

A., et al. (2000). GABAA receptor phosphorylation and functional modulation

164

in cortical neurons by a protein kinase C-dependent pathway. J Biol Chem,

275(49), 38856-38862.

Bray, J. G., & Mynlieff, M. (2009). Influx of calcium through L-type calcium

channels in early postnatal regulation of chloride transporters in the rat

hippocampus. Dev Neurobiol, 69(13), 885-896; erratum 897-912.

Brown, N., Kerby, J., Bonnert, T. P., Whiting, P. J., & Wafford, K. A. (2002).

Pharmacological characterization of a novel cell line expressing human

alpha(4)beta(3)delta GABA(A) receptors. Br J Pharmacol, 136(7), 965-974.

Buck, K. J., & Harris, R. A. (1990). Benzodiazepine agonist and inverse agonist

actions on GABAA receptor-operated chloride channels. II. Chronic effects of

ethanol. J Pharmacol Exp Ther, 253(2), 713-719.

Burnashev, N. (1998). Calcium permeability of ligand-gated channels. Cell Calcium,

24(5-6), 325-332.

Bussieres, N., & El Manira, A. (1999). GABA(B) receptor activation inhibits N- and

P/Q-type calcium channels in cultured lamprey sensory neurons. Brain Res,

847(2), 175-185.

Cagetti, E., Liang, J., Spigelman, I., & Olsen, R. W. (2003). Withdrawal from chronic

intermittent ethanol treatment changes subunit composition, reduces synaptic

function, and decreases behavioral responses to positive allosteric modulators

of GABAA receptors. Mol Pharmacol, 63(1), 53-64.

Cai, D., Mulle, J. G., & Yue, D. T. (1997). Inhibition of recombinant Ca2+ channels

by benzothiazepines and phenylalkylamines: class-specific pharmacology and

underlying molecular determinants. Mol Pharmacol, 51(5), 872-881.

165

Calin-Jageman, I., & Lee, A. (2008). Ca(v)1 L-type Ca2+ channel signaling

complexes in neurons. J Neurochem, 105(3), 573-583.

Calin-Jageman, I., Yu, K., Hall, R. A., Mei, L., & Lee, A. (2007). Erbin enhances

voltage-dependent facilitation of Ca(v)1.3 Ca2+ channels through relief of an

autoinhibitory domain in the Ca(v)1.3 alpha1 subunit. J Neurosci, 27(6),

1374-1385.

Catterall, W. A. (2000). Structure and regulation of voltage-gated Ca2+ channels.

Annu Rev Cell Dev Biol, 16, 521-555.

Catterall, W. A., Perez-Reyes, E., Snutch, T. P., & Striessnig, J. (2005). International

Union of Pharmacology. XLVIII. Nomenclature and structure-function

relationships of voltage-gated calcium channels. Pharmacol Rev, 57(4), 411-

425.

Cenquizca, L. A., & Swanson, L. W. (2007). Spatial organization of direct

hippocampal field CA1 axonal projections to the rest of the cerebral cortex.

Brain Res Rev, 56(1), 1-26.

Chaix, Y., Ferraro, T. N., Lapouble, E., & Martin, B. (2007). Chemoconvulsant-

induced seizure susceptibility: toward a common genetic basis? Epilepsia, 48

Suppl 5, 48-52.

Chan, C. S., Gertler, T. S., & Surmeier, D. J. (2010). A molecular basis for the

increased vulnerability of substantia nigra dopamine neurons in aging and

Parkinson's disease. Mov Disord, 25 Suppl 1, S63-70.

Chebib, M., & Johnston, G. A. (1999). The 'ABC' of GABA receptors: a brief review.

Clin Exp Pharmacol Physiol, 26(11), 937-940.

166

Chen, S., Huang, X., Zeng, X. J., Sieghart, W., & Tietz, E. I. (1999). Benzodiazepine-

mediated regulation of alpha1, alpha2, beta1-3 and gamma2 GABA(A)

receptor subunit proteins in the rat brain hippocampus and cortex.

Neuroscience, 93(1), 33-44.

Clements, J. D. (1996). Transmitter timecourse in the synaptic cleft: its role in central

synaptic function. Trends Neurosci, 19(5), 163-171.

Collingridge, G. L., Olsen, R. W., Peters, J., & Spedding, M. (2009). A nomenclature

for ligand-gated ion channels. Neuropharmacology, 56(1), 2-5.

Collinson, N., Kuenzi, F. M., Jarolimek, W., Maubach, K. A., Cothliff, R., Sur, C., et

al. (2002). Enhanced learning and memory and altered GABAergic synaptic

transmission in mice lacking the alpha 5 subunit of the GABAA receptor. J

Neurosci, 22(13), 5572-5580.

Conn, P. J., & Pin, J. P. (1997). Pharmacology and functions of metabotropic

glutamate receptors. Annu Rev Pharmacol Toxicol, 37, 205-237.

Coultrap, S. J., Buard, I., Kulbe, J. R., Dell'Acqua, M. L., & Bayer, K. U. (2010).

CaMKII autonomy is substrate-dependent and further stimulated by

Ca2+/calmodulin. J Biol Chem, 285(23), 17930-17937.

Crestani, F., Keist, R., Fritschy, J. M., Benke, D., Vogt, K., Prut, L., et al. (2002).

Trace fear conditioning involves hippocampal alpha5 GABA(A) receptors.

Proc Natl Acad Sci U S A, 99(13), 8980-8985.

Crestani, F., Low, K., Keist, R., Mandelli, M., Mohler, H., & Rudolph, U. (2001).

Molecular targets for the myorelaxant action of diazepam. Mol Pharmacol,

59(3), 442-445.

167

Crews, F. T., Morrow, A. L., Criswell, H., & Breese, G. (1996). Effects of ethanol on

ion channels. Int Rev Neurobiol, 39, 283-367.

Dailey, J. W. (1990). Sedative-hypnotic and anxiolytic agents. In C. R. Craig & R. E.

Stitzel (Eds.), Modern Pharmacology (3rd ed., pp. 440-451). Boston: Little,

Brown and Company.

Das, P., Bell-Horner, C. L., Huang, R. Q., Raut, A., Gonzales, E. B., Chen, Z. L., et

al. (2004). Inhibition of type A GABA receptors by L-type calcium channel

blockers. Neuroscience, 124(1), 195-206.

Das, P., Lilly, S. M., Zerda, R., Gunning, W. T., 3rd, Alvarez, F. J., & Tietz, E. I.

(2008). Increased AMPA receptor GluR1 subunit incorporation in rat

hippocampal CA1 synapses during benzodiazepine withdrawal. J Comp

Neurol, 511(6), 832-846.

Das, P., Zerda, R., Alvarez, F. J., & Tietz, E. I. (2010). Immunogold electron

microscopic evidence of differential regulation of GluN1, GluN2A, and

GluN2B, NMDA-type glutamate receptor subunits in rat hippocampal CA1

synapses during benzodiazepine withdrawal. J Comp Neurol, 518(21), 4311-

4328.

Davare, M. A., Horne, M. C., & Hell, J. W. (2000). Protein phosphatase 2A is

associated with class C L-type calcium channels (Cav1.2) and antagonizes

channel phosphorylation by cAMP-dependent protein kinase. J Biol Chem,

275(50), 39710-39717.

Davies, P. A., Hoffmann, E. B., Carlisle, H. J., Tyndale, R. F., & Hales, T. G. (2000).

The influence of an endogenous beta3 subunit on recombinant GABA(A)

168

receptor assembly and pharmacology in WSS-1 cells and transiently

transfected HEK293 cells. Neuropharmacology, 39(4), 611-620.

Day, M., Wang, Z., Ding, J., An, X., Ingham, C. A., Shering, A. F., et al. (2006).

Selective elimination of glutamatergic synapses on striatopallidal neurons in

Parkinson disease models. Nat Neurosci, 9(2), 251-259.

De Jongh, K. S., Murphy, B. J., Colvin, A. A., Hell, J. W., Takahashi, M., &

Catterall, W. A. (1996). Specific phosphorylation of a site in the full-length

form of the alpha 1 subunit of the cardiac L-type calcium channel by

adenosine 3',5'-cyclic monophosphate-dependent protein kinase.

Biochemistry, 35(32), 10392-10402.

De Koninck, P., & Schulman, H. (1998). Sensitivity of CaM kinase II to the

frequency of Ca2+ oscillations. Science, 279(5348), 227-230.

Degroot, A., & Treit, D. (2004). Anxiety is functionally segregated within the septo-

hippocampal system. Brain Res, 1001(1-2), 60-71.

Deisseroth, K., Mermelstein, P. G., Xia, H., & Tsien, R. W. (2003). Signaling from

synapse to nucleus: the logic behind the mechanisms. Curr Opin Neurobiol,

13(3), 354-365.

DeLorme, E. M., Rabe, C. S., & McGee, R., Jr. (1988). Regulation of the number of

functional voltage-sensitive Ca++ channels on PC12 cells by chronic changes

in membrane potential. J Pharmacol Exp Ther, 244(3), 838-843.

Devaud, L. L., Fritschy, J. M., Sieghart, W., & Morrow, A. L. (1997). Bidirectional

alterations of GABA(A) receptor subunit peptide levels in rat cortex during

chronic ethanol consumption and withdrawal. J Neurochem, 69(1), 126-130.

169

Diaz, M. R., Christian, D. T., Anderson, N. J., & McCool, B. A. (2011). Chronic

ethanol and withdrawal differentially modulate lateral/basolateral amygdala

paracapsular and local GABAergic synapses. J Pharmacol Exp Ther, 337(1),

162-170.

Divoll, M., & Greenblatt, D. J. (1981). Binding of diazepam and desmethyldiazepam

to plasma protein: concentration-dependence and interactions.

Psychopharmacology (Berl), 75(4), 380-382.

Dolin, S., Little, H., Hudspith, M., Pagonis, C., & Littleton, J. (1987). Increased

dihydropyridine-sensitive calcium channels in rat brain may underlie ethanol

physical dependence. Neuropharmacology, 26(2-3), 275-279.

Dolin, S. J., Patch, T. L., Rabbani, M., Siarey, R. J., Bowhay, A. R., & Little, H. J.

(1990). Nitrendipine decreases benzodiazepine withdrawal seizures but not

the development of benzodiazepine tolerance or withdrawal signs. Br J

Pharmacol, 101(3), 691-697.

Dolmetsch, R. E., Pajvani, U., Fife, K., Spotts, J. M., & Greenberg, M. E. (2001).

Signaling to the nucleus by an L-type calcium channel-calmodulin complex

through the MAP kinase pathway. Science, 294(5541), 333-339.

Dolphin, A. C. (2009). Calcium channel diversity: multiple roles of calcium channel

subunits. Curr Opin Neurobiol, 19(3), 237-244.

Dosemeci, A., Reese, T. S., Petersen, J., & Tao-Cheng, J. H. (2000). A novel

particulate form of Ca(2+)/calmodulin-dependent [correction of

Ca(2+)/CaMKII-dependent] protein kinase II in neurons. J Neurosci, 20(9),

3076-3084.

170

Dosemeci, A., Vinade, L., Winters, C. A., Reese, T. S., & Tao-Cheng, J. H. (2002).

Inhibition of phosphatase activity prolongs NMDA-induced modification of

the postsynaptic density. J Neurocytol, 31(8-9), 605-612.

Dudek, S. M., & Fields, R. D. (2002). Somatic action potentials are sufficient for late-

phase LTP-related cell signaling. Proc Natl Acad Sci U S A, 99(6), 3962-3967.

Dzhala, V. I., Talos, D. M., Sdrulla, D. A., Brumback, A. C., Mathews, G. C., Benke,

T. A., et al. (2005). NKCC1 transporter facilitates seizures in the developing

brain. Nat Med, 11(11), 1205-1213.

Earl, D. E., & Tietz, E. I. (2011). Inhibition of Recombinant L-Type Voltage-Gated

Calcium Channels by Positive Allosteric Modulators of GABAA Receptors. J

Pharmacol Exp Ther, 337(1), 301-311.

Ebert, B., Wafford, K. A., Whiting, P. J., Krogsgaard-Larsen, P., & Kemp, J. A.

(1994). Molecular pharmacology of gamma-aminobutyric acid type A

receptor agonists and partial agonists in oocytes injected with different alpha,

beta, and gamma receptor subunit combinations. Mol Pharmacol, 46(5), 957-

963.

Emmett-Oglesby, M. W., Mathis, D. A., Moon, R. T., & Lal, H. (1990). Animal

models of drug withdrawal symptoms. Psychopharmacology (Berl), 101(3),

292-309.

Ernst, M., Bruckner, S., Boresch, S., & Sieghart, W. (2005). Comparative models of

GABAA receptor extracellular and transmembrane domains: important

insights in pharmacology and function. Mol Pharmacol, 68(5), 1291-1300.

171

Erondu, N. E., & Kennedy, M. B. (1985). Regional distribution of type II

Ca2+/calmodulin-dependent protein kinase in rat brain. J Neurosci, 5(12),

3270-3277.

Fanselow, M. S., & Dong, H. W. (2010). Are the dorsal and ventral hippocampus

functionally distinct structures? Neuron, 65(1), 7-19.

Feng, H. J., Bianchi, M. T., & Macdonald, R. L. (2004). Pentobarbital differentially

modulates alpha1beta3delta and alpha1beta3gamma2L GABAA receptor

currents. Mol Pharmacol, 66(4), 988-1003.

Feron, O., & Godfraind, T. (1995). Regulation of the L-type calcium channel alpha-1

subunit by chronic depolarization in the neuron-like PC12 and aortic smooth

muscle A7r5 cell lines. Pflugers Arch, 430(3), 323-332.

Ferrante, J., Triggle, D. J., & Rutledge, A. (1991). The effects of chronic

depolarization on L-type 1,4-dihydropyridine-sensitive, voltage-dependent

Ca2+ channels in chick neural retina and rat cardiac cells. Can J Physiol

Pharmacol, 69(7), 914-920. ffrench-Mullen, J. M., Barker, J. L., & Rogawski, M. A. (1993). Calcium current

block by (-)-pentobarbital, phenobarbital, and CHEB but not (+)-pentobarbital

in acutely isolated hippocampal CA1 neurons: comparison with effects on

GABA-activated Cl- current. J Neurosci, 13(8), 3211-3221. ffrench-Mullen, J. M., Danks, P., & Spence, K. T. (1994). Neurosteroids modulate

calcium currents in hippocampal CA1 neurons via a pertussis toxin-sensitive

G-protein-coupled mechanism. J Neurosci, 14(4), 1963-1977.

172

File, S. E. (1985). Tolerance to the behavioral actions of benzodiazepines. Neurosci

Biobehav Rev, 9(1), 113-121.

File, S. E., Kenny, P. J., & Cheeta, S. (2000). The role of the dorsal hippocampal

serotonergic and cholinergic systems in the modulation of anxiety. Pharmacol

Biochem Behav, 66(1), 65-72.

Fink, C. C., & Meyer, T. (2002). Molecular mechanisms of CaMKII activation in

neuronal plasticity. Curr Opin Neurobiol, 12(3), 293-299.

Friedman, L. K., Gibbs, T. T., & Farb, D. H. (1996). gamma-aminobutyric acidA

receptor regulation: heterologous uncoupling of modulatory site interactions

induced by chronic steroid, barbiturate, benzodiazepine, or GABA treatment

in culture. Brain Res, 707(1), 100-109.

Gallager, D. W., Lakoski, J. M., Gonsalves, S. F., & Rauch, S. L. (1984). Chronic

benzodiazepine treatment decreases postsynaptic GABA sensitivity. Nature,

308(5954), 74-77.

Gao, L., Blair, L. A., Salinas, G. D., Needleman, L. A., & Marshall, J. (2006).

Insulin-like growth factor-1 modulation of CaV1.3 calcium channels depends

on Ca2+ release from IP3-sensitive stores and calcium/calmodulin kinase II

phosphorylation of the alpha1 subunit EF hand. J Neurosci, 26(23), 6259-

6268.

Gao, T., Yatani, A., Dell'Acqua, M. L., Sako, H., Green, S. A., Dascal, N., et al.

(1997). cAMP-dependent regulation of cardiac L-type Ca2+ channels requires

membrane targeting of PKA and phosphorylation of channel subunits.

Neuron, 19(1), 185-196.

173

Gavish, M., & Snyder, S. H. (1981). gamma-Aminobutyric acid and benzodiazepine

receptors: copurification and characterization. Proc Natl Acad Sci U S A,

78(3), 1939-1942.

Gee, K. W., Bolger, M. B., Brinton, R. E., Coirini, H., & McEwen, B. S. (1988).

Steroid modulation of the chloride ionophore in rat brain: structure-activity

requirements, regional dependence and mechanism of action. J Pharmacol

Exp Ther, 246(2), 803-812.

Grant, A. J., Koski, G., & Treistman, S. N. (1993). Effect of chronic ethanol on

calcium currents and calcium uptake in undifferentiated PC12 cells. Brain

Res, 600(2), 280-284.

Grasshoff, C., Drexler, B., Rudolph, U., & Antkowiak, B. (2006). Anaesthetic drugs:

linking molecular actions to clinical effects. Curr Pharm Des, 12(28), 3665-

3679.

Greenblatt, D. J., Laughren, T. P., Allen, M. D., Harmatz, J. S., & Shader, R. I.

(1981). Plasma diazepam and desmethyldiazepam concentrations during long-

term diazepam therapy. Br J Clin Pharmacol, 11(1), 35-40.

Greenfield, L. J., Jr., Zaman, S. H., Sutherland, M. L., Lummis, S. C., Niemeyer, M.

I., Barnard, E. A., et al. (2002). Mutation of the GABAA receptor M1

transmembrane proline increases GABA affinity and reduces barbiturate

enhancement. Neuropharmacology, 42(4), 502-521.

Griesemer, D., & Mautes, A. M. (2007). Closed head injury causes hyperexcitability

in rat hippocampal CA1 but not in CA3 pyramidal cells. J Neurotrauma,

24(12), 1823-1832.

174

Griffiths, R. R., & Johnson, M. W. (2005). Relative abuse liability of hypnotic drugs:

a conceptual framework and algorithm for differentiating among compounds.

J Clin Psychiatry, 66 Suppl 9, 31-41.

Griffiths, R. R., & Weerts, E. M. (1997). Benzodiazepine self-administration in

humans and laboratory animals--implications for problems of long-term use

and abuse. Psychopharmacology (Berl), 134(1), 1-37.

Groc, L., Heine, M., Cognet, L., Brickley, K., Stephenson, F. A., Lounis, B., et al.

(2004). Differential activity-dependent regulation of the lateral mobilities of

AMPA and NMDA receptors. Nat Neurosci, 7(7), 695-696.

Gross, R. A., & Macdonald, R. L. (1988). Differential actions of pentobarbitone on

calcium current components of mouse sensory neurones in culture. J Physiol,

405, 187-203.

Grover, L. M., & Teyler, T. J. (1990). Two components of long-term potentiation

induced by different patterns of afferent activation. Nature, 347(6292), 477-

479.

Grueter, C. E., Abiria, S. A., Wu, Y., Anderson, M. E., & Colbran, R. J. (2008).

Differential regulated interactions of calcium/calmodulin-dependent protein

kinase II with isoforms of voltage-gated calcium channel beta subunits.

Biochemistry, 47(6), 1760-1767.

Gulinello, M., Gong, Q. H., Li, X., & Smith, S. S. (2001). Short-term exposure to a

neuroactive steroid increases alpha4 GABA(A) receptor subunit levels in

association with increased anxiety in the female rat. Brain Res, 910(1-2), 55-

66.

175

Gupta, M. B., Nath, C., Patnaik, G. K., & Saxena, R. C. (1996). Effect of calcium

channel blockers on withdrawal syndrome of lorazepam in rats. Indian J Med

Res, 103, 310-314.

Hall, D. D., Davare, M. A., Shi, M., Allen, M. L., Weisenhaus, M., McKnight, G. S.,

et al. (2007). Critical role of cAMP-dependent protein kinase anchoring to the

L-type calcium channel Cav1.2 via A-kinase anchor protein 150 in neurons.

Biochemistry, 46(6), 1635-1646.

Hall, D. D., Feekes, J. A., Arachchige Don, A. S., Shi, M., Hamid, J., Chen, L., et al.

(2006). Binding of protein phosphatase 2A to the L-type calcium channel

Cav1.2 next to Ser1928, its main PKA site, is critical for Ser1928

dephosphorylation. Biochemistry, 45(10), 3448-3459.

Hallstrom, C., Lader, M. H., & Curry, S. H. (1980). Diazepam and N-

desmethyldiazepam concentrations in saliva, plasma and CSF. Br J Clin

Pharmacol, 9(4), 333-339.

Hanchar, H. J., Dodson, P. D., Olsen, R. W., Otis, T. S., & Wallner, M. (2005).

Alcohol-induced motor impairment caused by increased extrasynaptic

GABA(A) receptor activity. Nat Neurosci, 8(3), 339-345.

Hanson, P. I., & Schulman, H. (1992). Inhibitory autophosphorylation of

multifunctional Ca2+/calmodulin-dependent protein kinase analyzed by site-

directed mutagenesis. J Biol Chem, 267(24), 17216-17224.

Hara, Y., Kobayashi, H., Ooshiro, S., Futamura, K., Nishino, T., Chugun, A., et al.

(2001). Negative inotropic effect of diazepam in isolated guinea pig heart. J

Vet Med Sci, 63(2), 135-143.

176

Harris, R. A. (1990). Distinct actions of alcohols, barbiturates and benzodiazepines on

GABA-activated chloride channels. Alcohol, 7(3), 273-275.

Harris, R. A., Trudell, J. R., & Mihic, S. J. (2008). Ethanol's molecular targets. Sci

Signal, 1(28), re7.

Hell, J. W., Westenbroek, R. E., Breeze, L. J., Wang, K. K., Chavkin, C., & Catterall,

W. A. (1996). N-methyl-D-aspartate receptor-induced proteolytic conversion

of postsynaptic class C L-type calcium channels in hippocampal neurons.

Proc Natl Acad Sci U S A, 93(8), 3362-3367.

Hell, J. W., Westenbroek, R. E., Warner, C., Ahlijanian, M. K., Prystay, W., Gilbert,

M. M., et al. (1993a). Identification and differential subcellular localization of

the neuronal class C and class D L-type calcium channel alpha 1 subunits. J

Cell Biol, 123(4), 949-962.

Hell, J. W., Yokoyama, C. T., Wong, S. T., Warner, C., Snutch, T. P., & Catterall, W.

A. (1993b). Differential phosphorylation of two size forms of the neuronal

class C L-type calcium channel alpha 1 subunit. J Biol Chem, 268(26), 19451-

19457.

Helton, T. D., Xu, W., & Lipscombe, D. (2005). Neuronal L-type calcium channels

open quickly and are inhibited slowly. J Neurosci, 25(44), 10247-10251.

Heninger, C., Saito, N., Tallman, J. F., Garrett, K. M., Vitek, M. P., Duman, R. S., et

al. (1990). Effects of continuous diazepam administration on GABAA subunit

mRNA in rat brain. J Mol Neurosci, 2(2), 101-107.

Hitchcott, P. K., Zharkovsky, A., & File, S. E. (1992). Concurrent treatment with

verapamil prevents diazepam withdrawal-induced anxiety, in the absence of

177

altered calcium flux in cortical synaptosomes. Neuropharmacology, 31(1), 55-

60.

Hockerman, G. H., Dilmac, N., Scheuer, T., & Catterall, W. A. (2000). Molecular

determinants of diltiazem block in domains IIIS6 and IVS6 of L-type Ca(2+)

channels. Mol Pharmacol, 58(6), 1264-1270.

Hockerman, G. H., Peterson, B. Z., Johnson, B. D., & Catterall, W. A. (1997).

Molecular determinants of drug binding and action on L-type calcium

channels. Annu Rev Pharmacol Toxicol, 37, 361-396.

Hodding, G. C., Jann, M., & Ackerman, I. P. (1980). Drug withdrawal syndromes-- a

literature review. West J Med, 133(5), 383-391.

Hoffman, P. L., & Tabakoff, B. (1993). Ethanol, sedative hypnotics and glutamate

receptor function in brain and cultured cells. Alcohol Alcohol Suppl, 2, 345-

351.

Hosie, A. M., Wilkins, M. E., da Silva, H. M., & Smart, T. G. (2006). Endogenous

neurosteroids regulate GABAA receptors through two discrete transmembrane

sites. Nature, 444(7118), 486-489.

Houser, C. R. (2007). Interneurons of the dentate gyrus: an overview of cell types,

terminal fields and neurochemical identity. Prog Brain Res, 163, 217-232.

Houston, C. M., He, Q., & Smart, T. G. (2009). CaMKII phosphorylation of the

GABA(A) receptor: receptor subtype- and synapse-specific modulation. J

Physiol, 587(Pt 10), 2115-2125.

Hu, A. Q., Wang, Z. M., Lan, D. M., Fu, Y. M., Zhu, Y. H., Dong, Y., et al. (2007).

Inhibition of evoked glutamate release by neurosteroid allopregnanolone via

178

inhibition of L-type calcium channels in rat medial prefrontal cortex.

Neuropsychopharmacology, 32(7), 1477-1489.

Hu, X. J., & Ticku, M. K. (1994). Chronic benzodiazepine agonist treatment produces

functional uncoupling of the gamma-aminobutyric acid-benzodiazepine

receptor ionophore complex in cortical neurons. Mol Pharmacol, 45(4), 618-

625.

Huang, Y. Y., & Kandel, E. R. (2005). Theta frequency stimulation induces a local

form of late phase LTP in the CA1 region of the hippocampus. Learn Mem,

12(6), 587-593.

Hudmon, A., Schulman, H., Kim, J., Maltez, J. M., Tsien, R. W., & Pitt, G. S. (2005).

CaMKII tethers to L-type Ca2+ channels, establishing a local and dedicated

integrator of Ca2+ signals for facilitation. J Cell Biol, 171(3), 537-547.

Hullin, R., Singer-Lahat, D., Freichel, M., Biel, M., Dascal, N., Hofmann, F., et al.

(1992). Calcium channel beta subunit heterogeneity: functional expression of

cloned cDNA from heart, aorta and brain. EMBO J, 11(3), 885-890.

Hulme, J. T., Westenbroek, R. E., Scheuer, T., & Catterall, W. A. (2006).

Phosphorylation of serine 1928 in the distal C-terminal domain of cardiac

CaV1.2 channels during beta1-adrenergic regulation. Proc Natl Acad Sci U S

A, 103(44), 16574-16579.

Hunt, W. A. (1983). The effect of ethanol on GABAergic transmission. Neurosci

Biobehav Rev, 7(1), 87-95.

Impagnatiello, F., Pesold, C., Longone, P., Caruncho, H., Fritschy, J. M., Costa, E., et

al. (1996). Modifications of gamma-aminobutyric acidA receptor subunit

179

expression in rat neocortex during tolerance to diazepam. Mol Pharmacol,

49(5), 822-831.

Innis, R. B., Nestler, E. J., & Aghajanian, G. K. (1988). Evidence for G protein

mediation of serotonin- and GABAB-induced hyperpolarization of rat dorsal

raphe neurons. Brain Res, 459(1), 27-36.

Ishizawa, Y., Furuya, K., Yamagishi, S., & Dohi, S. (1997). Non-GABAergic effects

of midazolam, diazepam and flumazenil on voltage-dependent ion currents in

NG108-15 cells. Neuroreport, 8(11), 2635-2638.

Ito, T., Suzuki, T., Wellman, S. E., & Ho, I. K. (1996). Chronic pentobarbital

administration alters gamma-aminobutyric acidA receptor alpha 6-subunit

mRNA levels and diazepam-insensitive [3H]Ro15-4513 binding. Synapse,

22(2), 106-113.

Iversen, S., Kupfermann, I., & Kandel, E. (2000). Emotional states and feelings. In E.

R. Kandel, J. H. Schwartz & T. M. Jessell (Eds.), Principles of Neural Science

(3rd ed.). New York: McGraw-Hill.

Izzo, E., Auta, J., Impagnatiello, F., Pesold, C., Guidotti, A., & Costa, E. (2001).

Glutamic acid decarboxylase and glutamate receptor changes during tolerance

and dependence to benzodiazepines. Proc Natl Acad Sci U S A, 98(6), 3483-

3488.

Jarvis, S. E., & Zamponi, G. W. (2007). Trafficking and regulation of neuronal

voltage-gated calcium channels. Curr Opin Cell Biol, 19(4), 474-482.

180

Jenkins, M. A., Christel, C. J., Jiao, Y., Abiria, S., Kim, K. Y., Usachev, Y. M., et al.

(2010). Ca2+-dependent facilitation of Cav1.3 Ca2+ channels by densin and

Ca2+/calmodulin-dependent protein kinase II. J Neurosci, 30(15), 5125-5135.

Jonas, P., & Spruston, N. (1994). Mechanisms shaping glutamate-mediated excitatory

postsynaptic currents in the CNS. Curr Opin Neurobiol, 4(3), 366-372.

Jones, M. V., & Westbrook, G. L. (1996). The impact of receptor desensitization on

fast synaptic transmission. Trends Neurosci, 19(3), 96-101.

Jones, S. W. (1998). Overview of voltage-dependent calcium channels. J Bioenerg

Biomembr, 30(4), 299-312.

Ju, W., Morishita, W., Tsui, J., Gaietta, G., Deerinck, T. J., Adams, S. R., et al.

(2004). Activity-dependent regulation of dendritic synthesis and trafficking of

AMPA receptors. Nat Neurosci, 7(3), 244-253.

Kaila, K. (1994). Ionic basis of GABAA receptor channel function in the nervous

system. Prog Neurobiol, 42(4), 489-537.

Kaila, K., & Voipio, J. (1987). Postsynaptic fall in intracellular pH induced by

GABA-activated bicarbonate conductance. Nature, 330(6144), 163-165.

Kaila, K., Voipio, J., Paalasmaa, P., Pasternack, M., & Deisz, R. A. (1993). The role

of bicarbonate in GABAA receptor-mediated IPSPs of rat neocortical

neurones. J Physiol, 464, 273-289.

Kan, C. C., Hilberink, S. R., & Breteler, M. H. (2004). Determination of the main risk

factors for benzodiazepine dependence using a multivariate and

multidimensional approach. Compr Psychiatry, 45(2), 88-94.

181

Katsura, M., Shibasaki, M., Hayashida, S., Torigoe, F., Tsujimura, A., & Ohkuma, S.

(2006). Increase in expression of alpha1 and alpha2/delta1 subunits of L-type

high voltage-gated calcium channels after sustained ethanol exposure in

cerebral cortical neurons. J Pharmacol Sci, 102(2), 221-230.

Katsura, M., Shibasaki, M., Kurokawa, K., Tsujimura, A., & Ohkuma, S. (2007). Up-

regulation of L-type high voltage-gated calcium channel subunits by sustained

exposure to 1,4- and 1,5-benzodiazepines in cerebrocortical neurons. J

Neurochem, 103(6), 2518-2528.

Kauer, J. A., & Malenka, R. C. (2007). Synaptic plasticity and addiction. Nat Rev

Neurosci, 8(11), 844-858.

Kennedy, M. B., Bennett, M. K., & Erondu, N. E. (1983a). Biochemical and

immunochemical evidence that the "major postsynaptic density protein" is a

subunit of a calmodulin-dependent protein kinase. Proc Natl Acad Sci U S A,

80(23), 7357-7361.

Kennedy, M. B., McGuinness, T., & Greengard, P. (1983b). A calcium/calmodulin-

dependent protein kinase from mammalian brain that phosphorylates Synapsin

I: partial purification and characterization. J Neurosci, 3(4), 818-831.

Kepplinger, K. J., Forstner, G., Kahr, H., Leitner, K., Pammer, P., Groschner, K., et

al. (2000). Molecular determinant for run-down of L-type Ca2+ channels

localized in the carboxyl terminus of the 1C subunit. J Physiol, 529 Pt 1, 119-

130.

182

Klein, R. L., & Harris, R. A. (1996). Regulation of GABAA receptor structure and

function by chronic drug treatments in vivo and with stably transfected cells.

Jpn J Pharmacol, 70(1), 1-15.

Klockner, U., Mikala, G., Varadi, M., Varadi, G., & Schwartz, A. (1995).

Involvement of the carboxyl-terminal region of the alpha 1 subunit in voltage-

dependent inactivation of cardiac calcium channels. J Biol Chem, 270(29),

17306-17310.

Koschak, A., Reimer, D., Huber, I., Grabner, M., Glossmann, H., Engel, J., et al.

(2001). alpha 1D (Cav1.3) subunits can form l-type Ca2+ channels activating

at negative voltages. J Biol Chem, 276(25), 22100-22106.

Lacinova, L. (2005). Voltage-dependent calcium channels. Gen Physiol Biophys, 24

Suppl 1, 1-78.

Lader, M. (1994). Anxiolytic drugs: dependence, addiction and abuse. Eur

Neuropsychopharmacol, 4(2), 85-91.

Lambert, J. J., Peters, J. A., & Cotreel, G. A. (1987). Neuorsteroids and GABAA

receptor function. Trends Pharmacol Sci, 8, 224-227.

Larsson, M., & Broman, J. (2006). Pathway-specific bidirectional regulation of

Ca2+/calmodulin-dependent protein kinase II at spinal nociceptive synapses

after acute noxious stimulation. J Neurosci, 26(16), 4198-4205.

Lau, C. E., Falk, J. L., Dolan, S., & Tang, M. (1987). Simultaneous determination of

flurazepam and five metabolites in serum by high-performance liquid

chromatography and its application to pharmacokinetic studies in rats. J

Chromatogr, 423, 251-259.

183

Lavoie, A. M., & Twyman, R. E. (1996). Direct evidence for diazepam modulation of

GABAA receptor microscopic affinity. Neuropharmacology, 35(9-10), 1383-

1392.

Lee, K. S., & Tsien, R. W. (1983). Mechanism of calcium channel blockade by

verapamil, D600, diltiazem and nitrendipine in single dialysed heart cells.

Nature, 302(5911), 790-794.

Lee, T. S., Karl, R., Moosmang, S., Lenhardt, P., Klugbauer, N., Hofmann, F., et al.

(2006). Calmodulin kinase II is involved in voltage-dependent facilitation of

the L-type Cav1.2 calcium channel: Identification of the phosphorylation

sites. J Biol Chem, 281(35), 25560-25567.

Leitch, B., Szostek, A., Lin, R., & Shevtsova, O. (2009). Subcellular distribution of

L-type calcium channel subtypes in rat hippocampal neurons. Neuroscience,

164(2), 641-657.

Lerma, J., Herranz, A. S., Herreras, O., Abraira, V., & Martin del Rio, R. (1986). In

vivo determination of extracellular concentration of amino acids in the rat

hippocampus. A method based on brain dialysis and computerized analysis.

Brain Res, 384(1), 145-155.

Lerma, J., Morales, M., Vicente, M. A., & Herreras, O. (1997). Glutamate receptors

of the kainate type and synaptic transmission. Trends Neurosci, 20(1), 9-12.

Liang, Y., & Tavalin, S. J. (2007). Auxiliary beta subunits differentially determine

pka utilization of distinct regulatory sites on Cav1.3 L type Ca2+ channels.

Channels (Austin), 1(2), 102-112.

184

Lin, L. H., & Wang, L. H. (1996). Region-specific changes in GABAA receptor delta

subunit mRNA level by tolerance to and withdrawal from pentobarbital.

Neurosci Lett, 202(3), 149-152.

Lin, Y., McDonough, S. I., & Lipscombe, D. (2004). Alternative splicing in the

voltage-sensing region of N-Type CaV2.2 channels modulates channel

kinetics. J Neurophysiol, 92(5), 2820-2830.

Liu, J., Bangalore, R., Rutledge, A., & Triggle, D. J. (1994). Modulation of L-type

Ca2+ channels in clonal rat pituitary cells by membrane depolarization. Mol

Pharmacol, 45(6), 1198-1206.

Lorenz, J. M., Riddervold, M. H., Beckett, E. A., Baker, S. A., & Perrino, B. A.

(2002). Differential autophosphorylation of CaM kinase II from phasic and

tonic smooth muscle tissues. Am J Physiol Cell Physiol, 283(5), C1399-1413.

Low, K., Crestani, F., Keist, R., Benke, D., Brunig, I., Benson, J. A., et al. (2000).

Molecular and neuronal substrate for the selective attenuation of anxiety.

Science, 290(5489), 131-134.

Ludwig, A., Flockerzi, V., & Hofmann, F. (1997). Regional expression and cellular

localization of the alpha1 and beta subunit of high voltage-activated calcium

channels in rat brain. J Neurosci, 17(4), 1339-1349.

Lundgren, S. (1987). Serum concentration and drug effect after intravenous and rectal

administration of diazepam. Anesth Prog, 34(4), 128-133.

MacDonald, R. L., Rogers, C. J., & Twyman, R. E. (1989). Barbiturate regulation of

kinetic properties of the GABAA receptor channel of mouse spinal neurones

in culture. J Physiol, 417, 483-500.

185

Maitra, R., & Reynolds, J. N. (1998). Modulation of GABA(A) receptor function by

neuroactive steroids: evidence for heterogeneity of steroid sensitivity of

recombinant GABA(A) receptor isoforms. Can J Physiol Pharmacol, 76(9),

909-920.

Malayev, A., Gibbs, T. T., & Farb, D. H. (2002). Inhibition of the NMDA response

by pregnenolone sulphate reveals subtype selective modulation of NMDA

receptors by sulphated steroids. Br J Pharmacol, 135(4), 901-909.

Malenka, R. C., & Bear, M. F. (2004). LTP and LTD: an embarrassment of riches.

Neuron, 44(1), 5-21.

Malenka, R. C., & Nicoll, R. A. (1999). Long-term potentiation--a decade of

progress? Science, 285(5435), 1870-1874.

Mameli, M., Bellone, C., Brown, M. T., & Luscher, C. (2011). Cocaine inverts rules

for synaptic plasticity of glutamate transmission in the ventral tegmental area.

Nat Neurosci, 14(4), 414-416.

Mann, E. O., & Paulsen, O. (2007). Role of GABAergic inhibition in hippocampal

network oscillations. Trends Neurosci, 30(7), 343-349.

Matthews, D. B., Devaud, L. L., Fritschy, J. M., Sieghart, W., & Morrow, A. L.

(1998). Differential regulation of GABA(A) receptor gene expression by

ethanol in the rat hippocampus versus cerebral cortex. J Neurochem, 70(3),

1160-1166.

McKeon, A., Frye, M. A., & Delanty, N. (2008). The alcohol withdrawal syndrome. J

Neurol Neurosurg Psychiatry, 79(8), 854-862.

186

McKernan, R. M., Rosahl, T. W., Reynolds, D. S., Sur, C., Wafford, K. A., Atack, J.

R., et al. (2000). Sedative but not anxiolytic properties of benzodiazepines are

mediated by the GABA(A) receptor alpha1 subtype. Nat Neurosci, 3(6), 587-

592.

McKernan, R. M., & Whiting, P. J. (1996). Which GABAA-receptor subtypes really

occur in the brain? Trends Neurosci, 19(4), 139-143.

Megias, M., Emri, Z., Freund, T. F., & Gulyas, A. I. (2001). Total number and

distribution of inhibitory and excitatory synapses on hippocampal CA1

pyramidal cells. Neuroscience, 102(3), 527-540.

Messing, R. O., Carpenter, C. L., Diamond, I., & Greenberg, D. A. (1986). Ethanol

regulates calcium channels in clonal neural cells. Proc Natl Acad Sci U S A,

83(16), 6213-6215.

Mihic, S. J., Ye, Q., Wick, M. J., Koltchine, V. V., Krasowski, M. D., Finn, S. E., et

al. (1997). Sites of alcohol and volatile anaesthetic action on GABA(A) and

glycine receptors. Nature, 389(6649), 385-389.

Miller, P. S., & Smart, T. G. (2010). Binding, activation and modulation of Cys-loop

receptors. Trends Pharmacol Sci, 31(4), 161-174.

Miller, S., Yasuda, M., Coats, J. K., Jones, Y., Martone, M. E., & Mayford, M.

(2002). Disruption of dendritic translation of CaMKIIalpha impairs

stabilization of synaptic plasticity and memory consolidation. Neuron, 36(3),

507-519.

187

Mitchell, E. A., Herd, M. B., Gunn, B. G., Lambert, J. J., & Belelli, D. (2008).

Neurosteroid modulation of GABAA receptors: molecular determinants and

significance in health and disease. Neurochem Int, 52(4-5), 588-595.

Mitterdorfer, J., Wang, Z., Sinnegger, M. J., Hering, S., Striessnig, J., Grabner, M., et

al. (1996). Two amino acid residues in the IIIS5 segment of L-type calcium

channels differentially contribute to 1,4-dihydropyridine sensitivity. J Biol

Chem, 271(48), 30330-30335.

Mizoguchi, H., Yoshiike, M., Suzuki, T., & Misawa, M. (1993). Effects of Ca2+

channel blockers on physical dependence on diazepam in mice. Life Sci,

53(22), PL365-370.

Mohler, H., Fritschy, J. M., Crestani, F., Hensch, T., & Rudolph, U. (2004). Specific

GABA(A) circuits in brain development and therapy. Biochem Pharmacol,

68(8), 1685-1690.

Mohler, H., & Okada, T. (1977). Benzodiazepine receptor: demonstration in the

central nervous system. Science, 198(4319), 849-851.

Monyer, H., Burnashev, N., Laurie, D. J., Sakmann, B., & Seeburg, P. H. (1994).

Developmental and regional expression in the rat brain and functional

properties of four NMDA receptors. Neuron, 12(3), 529-540.

Moosmang, S., Haider, N., Klugbauer, N., Adelsberger, H., Langwieser, N., Muller,

J., et al. (2005). Role of hippocampal Cav1.2 Ca2+ channels in NMDA

receptor-independent synaptic plasticity and spatial memory. J Neurosci,

25(43), 9883-9892.

188

Morad, M., & Soldatov, N. (2005). Calcium channel inactivation: possible role in

signal transduction and Ca2+ signaling. Cell Calcium, 38(3-4), 223-231.

Morgan, S. L., & Teyler, T. J. (2001). Electrical stimuli patterned after the theta-

rhythm induce multiple forms of LTP. J Neurophysiol, 86(3), 1289-1296.

Morrow, A. L., Suzdak, P. D., Karanian, J. W., & Paul, S. M. (1988). Chronic ethanol

administration alters gamma-aminobutyric acid, pentobarbital and ethanol-

mediated 36Cl- uptake in cerebral cortical synaptoneurosomes. J Pharmacol

Exp Ther, 246(1), 158-164.

Nagy, J. (2004). The NR2B subtype of NMDA receptor: a potential target for the

treatment of alcohol dependence. Curr Drug Targets CNS Neurol Disord,

3(3), 169-179.

O'Brien C, P. (2005). Benzodiazepine use, abuse, and dependence. J Clin Psychiatry,

66 Suppl 2, 28-33.

Obermair, G. J., Szabo, Z., Bourinet, E., & Flucher, B. E. (2004). Differential

targeting of the L-type Ca2+ channel alpha 1C (CaV1.2) to synaptic and

extrasynaptic compartments in hippocampal neurons. Eur J Neurosci, 19(8),

2109-2122.

Oliveria, S. F., Dell'Acqua, M. L., & Sather, W. A. (2007). AKAP79/150 anchoring

of calcineurin controls neuronal L-type Ca2+ channel activity and nuclear

signaling. Neuron, 55(2), 261-275.

Olsen, R. W., & Sieghart, W. (2008). International Union of Pharmacology. LXX.

Subtypes of gamma-aminobutyric acid(A) receptors: classification on the

189

basis of subunit composition, pharmacology, and function. Update.

Pharmacol Rev, 60(3), 243-260.

Olson, P. A., Tkatch, T., Hernandez-Lopez, S., Ulrich, S., Ilijic, E., Mugnaini, E., et

al. (2005). G-protein-coupled receptor modulation of striatal CaV1.3 L-type

Ca2+ channels is dependent on a Shank-binding domain. J Neurosci, 25(5),

1050-1062.

Ouyang, W., Wang, G., & Hemmings, H. C., Jr. (2003). Isoflurane and propofol

inhibit voltage-gated sodium channels in isolated rat neurohypophysial nerve

terminals. Mol Pharmacol, 64(2), 373-381.

Papadopoulos, V., Lecanu, L., Brown, R. C., Han, Z., & Yao, Z. X. (2006).

Peripheral-type benzodiazepine receptor in neurosteroid biosynthesis,

neuropathology and neurological disorders. Neuroscience, 138(3), 749-756.

Peters, J. A., Kirkness, E. F., Callachan, H., Lambert, J. J., & Turner, A. J. (1988).

Modulation of the GABAA receptor by depressant barbiturates and pregnane

steroids. Br J Pharmacol, 94(4), 1257-1269.

Platzer, J., Engel, J., Schrott-Fischer, A., Stephan, K., Bova, S., Chen, H., et al.

(2000). Congenital deafness and sinoatrial node dysfunction in mice lacking

class D L-type Ca2+ channels. Cell, 102(1), 89-97.

Plotkin, M. D., Snyder, E. Y., Hebert, S. C., & Delpire, E. (1997). Expression of the

Na-K-2Cl cotransporter is developmentally regulated in postnatal rat brains: a

possible mechanism underlying GABA's excitatory role in immature brain. J

Neurobiol, 33(6), 781-795.

190

Poisbeau, P., Williams, S. R., & Mody, I. (1997). Silent GABAA synapses during

flurazepam withdrawal are region-specific in the hippocampal formation. J

Neurosci, 17(10), 3467-3475.

Porter, N. M., Thibault, O., Thibault, V., Chen, K. C., & Landfield, P. W. (1997).

Calcium channel density and hippocampal cell death with age in long-term

culture. J Neurosci, 17(14), 5629-5639.

Putzier, I., Kullmann, P. H., Horn, J. P., & Levitan, E. S. (2009). Cav1.3 channel

voltage dependence, not Ca2+ selectivity, drives pacemaker activity and

amplifies bursts in nigral dopamine neurons. J Neurosci, 29(49), 15414-

15419.

Rabbani, M., Brown, J., Butterworth, A. R., & Little, H. J. (1994). Dihydropyridine-

sensitive calcium channels and barbiturate tolerance and withdrawal.

Pharmacol Biochem Behav, 47(3), 675-680.

Rabbani, M., & Little, H. J. (1999). Increases in neuronal Ca2+ flux after withdrawal

from chronic barbiturate treatment. Eur J Pharmacol, 364(2-3), 221-227.

Ramos, A. (2008). Animal models of anxiety: do I need multiple tests? Trends

Pharmacol Sci, 29(10), 493-498.

Reddy, D. S., & Rogawski, M. A. (2009). Neurosteroid replacement therapy for

catamenial epilepsy. Neurotherapeutics, 6(2), 392-401.

Remondes, M., & Schuman, E. M. (2003). Molecular mechanisms contributing to

long-lasting synaptic plasticity at the temporoammonic-CA1 synapse. Learn

Mem, 10(4), 247-252.

191

Reuveny, E., Twombly, D. A., & Narahashi, T. (1993). Chlordiazepoxide block of

two types of calcium channels in neuroblastoma cells. J Pharmacol Exp Ther,

264(1), 22-28.

Rey, E., Treluyer, J. M., & Pons, G. (1999). Pharmacokinetic optimization of

benzodiazepine therapy for acute seizures. Focus on delivery routes. Clin

Pharmacokinet, 36(6), 409-424.

Richards, C. D. (1972). On the mechanism of barbiturate anaesthesia. J Physiol,

227(3), 749-767.

Rivera, C., Voipio, J., Payne, J. A., Ruusuvuori, E., Lahtinen, H., Lamsa, K., et al.

(1999). The K+/Cl- co-transporter KCC2 renders GABA hyperpolarizing

during neuronal maturation. Nature, 397(6716), 251-255.

Robison, A. J., Bass, M. A., Jiao, Y., MacMillan, L. B., Carmody, L. C., Bartlett, R.

K., et al. (2005). Multivalent interactions of calcium/calmodulin-dependent

protein kinase II with the postsynaptic density proteins NR2B, densin-180,

and alpha-actinin-2. J Biol Chem, 280(42), 35329-35336.

Rosenberg, H. C., & Chiu, T. H. (1985). Time course for development of

benzodiazepine tolerance and physical dependence. Neurosci Biobehav Rev,

9(1), 123-131.

Rosenberg, H. C., Tietz, E. I., & Chiu, T. H. (1991). Differential tolerance to the

antipentylenetetrazol activity of benzodiazepines in flurazepam-treated rats.

Pharmacol Biochem Behav, 39(3), 711-716.

192

Rudolph, U., Crestani, F., Benke, D., Brunig, I., Benson, J. A., Fritschy, J. M., et al.

(1999). Benzodiazepine actions mediated by specific gamma-aminobutyric

acid(A) receptor subtypes. Nature, 401(6755), 796-800.

Rupprecht, R., Papadopoulos, V., Rammes, G., Baghai, T. C., Fan, J., Akula, N., et al.

(2010). Translocator protein (18 kDa) (TSPO) as a therapeutic target for

neurological and psychiatric disorders. Nat Rev Drug Discov, 9(12), 971-988.

Rutherford, D. M., Okoko, A., & Tyrer, P. J. (1978). Plasma concentrations of

diazepam and desmethyldiazepam during chronic diazepam therapy. Br J Clin

Pharmacol, 6(1), 69-73.

Safa, P., Boulter, J., & Hales, T. G. (2001). Functional properties of Cav1.3

(alpha1D) L-type Ca2+ channel splice variants expressed by rat brain and

neuroendocrine GH3 cells. J Biol Chem, 276(42), 38727-38737.

Saliba, R. S., Gu, Z., Yan, Z., & Moss, S. J. (2009). Blocking L-type voltage-gated

Ca2+ channels with dihydropyridines reduces gamma-aminobutyric acid type

A receptor expression and synaptic inhibition. J Biol Chem, 284(47), 32544-

32550.

Sanna, A., Congeddu, E., Saba, L., Porcella, A., Marchese, G., Ruiu, S., et al.

(2004a). The cerebellar GABAA alpha6 subunit is differentially modulated by

chronic ethanol exposure in normal (R100R) and mutated (Q100Q) sNP rats.

Brain Res, 998(2), 148-154.

Sanna, E., Talani, G., Busonero, F., Pisu, M. G., Purdy, R. H., Serra, M., et al.

(2004b). Brain steroidogenesis mediates ethanol modulation of GABAA

receptor activity in rat hippocampus. J Neurosci, 24(29), 6521-6530.

193

Saunders, P. A., & Ho, I. K. (1990). Barbiturates and the GABAA receptor complex.

Prog Drug Res, 34, 261-286.

Saxena, N. C., & Macdonald, R. L. (1994). Assembly of GABAA receptor subunits:

role of the delta subunit. J Neurosci, 14(11 Pt 2), 7077-7086.

Saxena, N. C., & Macdonald, R. L. (1996). Properties of putative cerebellar gamma-

aminobutyric acid A receptor isoforms. Mol Pharmacol, 49(3), 567-579.

Schmid, S. M., & Hollmann, M. (2008). To gate or not to gate: are the delta subunits

in the glutamate receptor family functional ion channels? Mol Neurobiol,

37(2-3), 126-141.

Schulz, D. W., & Macdonald, R. L. (1981). Barbiturate enhancement of GABA-

mediated inhibition and activation of chloride ion conductance: correlation

with anticonvulsant and anesthetic actions. Brain Res, 209(1), 177-188.

Seeburg, P. H., & Hartner, J. (2003). Regulation of ion channel/neurotransmitter

receptor function by RNA editing. Curr Opin Neurobiol, 13(3), 279-283.

Serafini, R., Bracamontes, J., & Steinbach, J. H. (2000). Structural domains of the

human GABAA receptor 3 subunit involved in the actions of pentobarbital. J

Physiol, 524 Pt 3, 649-676.

Shen, G., Mohamed, M. S., Das, P., & Tietz, E. I. (2009). Positive allosteric

activation of GABAA receptors bi-directionally modulates hippocampal

glutamate plasticity and behaviour. Biochem Soc Trans, 37(Pt 6), 1394-1398.

Shen, G., & Tietz, E. I. (2011). Down-regulation of synaptic GluN2B subunit-

containing N-methyl-D-aspartate receptors: a physiological brake on CA1

neuron alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid

194

hyperexcitability during benzodiazepine withdrawal. J Pharmacol Exp Ther,

336(1), 265-273.

Shen, G., Van Sickle, B. J., & Tietz, E. I. (2010). Calcium/calmodulin-dependent

protein kinase II mediates hippocampal glutamatergic plasticity during

benzodiazepine withdrawal. Neuropsychopharmacology, 35(9), 1897-1909.

Shen, K., Teruel, M. N., Subramanian, K., & Meyer, T. (1998). CaMKIIbeta

functions as an F-actin targeting module that localizes CaMKIIalpha/beta

heterooligomers to dendritic spines. Neuron, 21(3), 593-606.

Sheng, M., Cummings, J., Roldan, L. A., Jan, Y. N., & Jan, L. Y. (1994). Changing

subunit composition of heteromeric NMDA receptors during development of

rat cortex. Nature, 368(6467), 144-147.

Sigel, E. (2002). Mapping of the benzodiazepine recognition site on GABA(A)

receptors. Curr Top Med Chem, 2(8), 833-839.

Silva, A. J., Stevens, C. F., Tonegawa, S., & Wang, Y. (1992). Deficient hippocampal

long-term potentiation in alpha-calcium-calmodulin kinase II mutant mice.

Science, 257(5067), 201-206.

Singh, A., Gebhart, M., Fritsch, R., Sinnegger-Brauns, M. J., Poggiani, C., Hoda, J.

C., et al. (2008). Modulation of voltage- and Ca2+-dependent gating of

CaV1.3 L-type calcium channels by alternative splicing of a C-terminal

regulatory domain. J Biol Chem, 283(30), 20733-20744.

Smith, M. A., & Stoops, W. W. (2001). Sensitivity to the effects of sedative-

hypnotics on motor performance: influence of task difficulty and chronic

phenobarbital administration. Behav Pharmacol, 12(2), 125-134.

195

Smith, S. S. (2002). Withdrawal properties of a neuroactive steroid: implications for

GABA(A) receptor gene regulation in the brain and anxiety behavior.

Steroids, 67(6), 519-528.

Smith, S. S., Gong, Q. H., Hsu, F. C., Markowitz, R. S., ffrench-Mullen, J. M., & Li,

X. (1998). GABA(A) receptor alpha4 subunit suppression prevents

withdrawal properties of an endogenous steroid. Nature, 392(6679), 926-930.

Snutch, T. P., Tomlinson, W. J., Leonard, J. P., & Gilbert, M. M. (1991). Distinct

calcium channels are generated by alternative splicing and are differentially

expressed in the mammalian CNS. Neuron, 7(1), 45-57.

Soderpalm, B., Lof, E., & Ericson, M. (2009). Mechanistic studies of ethanol's

interaction with the mesolimbic dopamine reward system.

Pharmacopsychiatry, 42 Suppl 1, S87-94.

Somogyi, P., & Klausberger, T. (2005). Defined types of cortical interneurone

structure space and spike timing in the hippocampus. J Physiol, 562(Pt 1), 9-

26.

Song, J., Shen, G., Greenfield, L. J., Jr., & Tietz, E. I. (2007). Benzodiazepine

withdrawal-induced glutamatergic plasticity involves up-regulation of GluR1-

containing alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid

receptors in Hippocampal CA1 neurons. J Pharmacol Exp Ther, 322(2), 569-

581.

Staley, K. J., & Proctor, W. R. (1999). Modulation of mammalian dendritic

GABA(A) receptor function by the kinetics of Cl- and HCO3- transport. J

Physiol, 519 Pt 3, 693-712.

196

Staley, K. J., Soldo, B. L., & Proctor, W. R. (1995). Ionic mechanisms of neuronal

excitation by inhibitory GABAA receptors. Science, 269(5226), 977-981.

Steinbach, J. H., & Akk, G. (2001). Modulation of GABA(A) receptor channel gating

by pentobarbital. J Physiol, 537(Pt 3), 715-733.

Stelzer, A., Kay, A. R., & Wong, R. K. (1988). GABAA-receptor function in

hippocampal cells is maintained by phosphorylation factors. Science,

241(4863), 339-341.

Sternbach, L. H. (1972). The discovery of librium. Agents Actions, 2(4), 193-196.

Steward, O., & Schuman, E. M. (2001). Protein synthesis at synaptic sites on

dendrites. Annu Rev Neurosci, 24, 299-325.

Strack, S., McNeill, R. B., & Colbran, R. J. (2000). Mechanism and regulation of

calcium/calmodulin-dependent protein kinase II targeting to the NR2B subunit

of the N-methyl-D-aspartate receptor. J Biol Chem, 275(31), 23798-23806.

Striessnig, J., & Koschak, A. (2008). Exploring the function and pharmacotherapeutic

potential of voltage-gated Ca2+ channels with gene knockout models.

Channels (Austin), 2(4), 233-251.

Striessnig, J., Koschak, A., Sinnegger-Brauns, M. J., Hetzenauer, A., Nguyen, N. K.,

Busquet, P., et al. (2006). Role of voltage-gated L-type Ca2+ channel

isoforms for brain function. Biochem Soc Trans, 34(Pt 5), 903-909.

Study, R. E., & Barker, J. L. (1981). Diazepam and (--)-pentobarbital: fluctuation

analysis reveals different mechanisms for potentiation of gamma-

aminobutyric acid responses in cultured central neurons. Proc Natl Acad Sci U

S A, 78(11), 7180-7184.

197

Swanson, L. W., & Cowan, W. M. (1977). An autoradiographic study of the

organization of the efferent connections of the hippocampal formation in the

rat. J Comp Neurol, 172(1), 49-84.

Szewczyk, A., & Wojtczak, L. (2002). Mitochondria as a pharmacological target.

Pharmacol Rev, 54(1), 101-127.

Taha, S., Hanover, J. L., Silva, A. J., & Stryker, M. P. (2002). Autophosphorylation

of alphaCaMKII is required for ocular dominance plasticity. Neuron, 36(3),

483-491.

Tang, Z. Z., Liang, M. C., Lu, S., Yu, D., Yu, C. Y., Yue, D. T., et al. (2004).

Transcript scanning reveals novel and extensive splice variations in human l-

type voltage-gated calcium channel, Cav1.2 alpha1 subunit. J Biol Chem,

279(43), 44335-44343.

Tarabova, B., Lacinova, L., & Engel, J. (2007). Effects of phenylalkylamines and

benzothiazepines on Ca(v)1.3-mediated Ca2+ currents in neonatal mouse

inner hair cells. Eur J Pharmacol, 573(1-3), 39-48.

Tatebayashi, H., Motomura, H., & Narahashi, T. (1998). Alcohol modulation of

single GABA(A) receptor-channel kinetics. Neuroreport, 9(8), 1769-1775.

Thibault, O., & Landfield, P. W. (1996). Increase in single L-type calcium channels

in hippocampal neurons during aging. Science, 272(5264), 1017-1020.

Thomas, C. G., Miller, A. J., & Westbrook, G. L. (2006). Synaptic and extrasynaptic

NMDA receptor NR2 subunits in cultured hippocampal neurons. J

Neurophysiol, 95(3), 1727-1734.

198

Thomas, P., Sundaram, H., Krishek, B. J., Chazot, P., Xie, X., Bevan, P., et al.

(1997). Regulation of neuronal and recombinant GABA(A) receptor ion

channels by xenovulene A, a natural product isolated from Acremonium

strictum. J Pharmacol Exp Ther, 282(2), 513-520.

Tietz, E. I., Huang, X., Chen, S., & Ferencak, W. F., 3rd. (1999a). Temporal and

regional regulation of alpha1, beta2 and beta3, but not alpha2, alpha4, alpha5,

alpha6, beta1 or gamma2 GABA(A) receptor subunit messenger RNAs

following one-week oral flurazepam administration. Neuroscience, 91(1),

327-341.

Tietz, E. I., Rosenberg, H. C., & Chiu, T. H. (1986). Autoradiographic localization of

benzodiazepine receptor downregulation. J Pharmacol Exp Ther, 236(1), 284-

292.

Tietz, E. I., Zeng, X. J., Chen, S., Lilly, S. M., Rosenberg, H. C., & Kometiani, P.

(1999b). Antagonist-induced reversal of functional and structural measures of

hippocampal benzodiazepine tolerance. J Pharmacol Exp Ther, 291(3), 932-

942.

Tippens, A. L., Pare, J. F., Langwieser, N., Moosmang, S., Milner, T. A., Smith, Y.,

et al. (2008). Ultrastructural evidence for pre- and postsynaptic localization of

Cav1.2 L-type Ca2+ channels in the rat hippocampus. J Comp Neurol, 506(4),

569-583.

Tovar, K. R., & Westbrook, G. L. (2002). Mobile NMDA receptors at hippocampal

synapses. Neuron, 34(2), 255-264.

199

Tseng, Y. T., Wellman, S. E., & Ho, I. K. (1993). Differential effects on GABAA

receptor gamma 2-subunit messenger RNA by tolerance to and withdrawal

from pentobarbital--an in situ hybridization study. Life Sci, 53(19), PL321-

326.

Tseng, Y. T., Wellman, S. E., & Ho, I. K. (1994). In situ hybridization evidence of

differential modulation by pentobarbital of GABAA receptor alpha 1- and

beta 3-subunit mRNAs. J Neurochem, 63(1), 301-309.

Twyman, R. E., & Macdonald, R. L. (1992). Neurosteroid regulation of GABAA

receptor single-channel kinetic properties of mouse spinal cord neurons in

culture. J Physiol, 456, 215-245.

Uehara, A., & Hume, J. R. (1985). Interactions of organic calcium channel

antagonists with calcium channels in single frog atrial cells. J Gen Physiol,

85(5), 621-647.

Ueno, S., Wick, M. J., Ye, Q., Harrison, N. L., & Harris, R. A. (1999). Subunit

mutations affect ethanol actions on GABA(A) receptors expressed in Xenopus

oocytes. Br J Pharmacol, 127(2), 377-382.

Ulrichsen, J., Bech, B., Ebert, B., Diemer, N. H., Allerup, P., & Hemmingsen, R.

(1996). Glutamate and benzodiazepine receptor autoradiography in rat brain

after repetition of alcohol dependence. Psychopharmacology (Berl), 126(1),

31-41.

Uusi-Oukari, M., & Korpi, E. R. (2010). Regulation of GABA(A) receptor subunit

expression by pharmacological agents. Pharmacol Rev, 62(1), 97-135.

200

Uzunova, V., Sheline, Y., Davis, J. M., Rasmusson, A., Uzunov, D. P., Costa, E., et

al. (1998). Increase in the cerebrospinal fluid content of neurosteroids in

patients with unipolar major depression who are receiving fluoxetine or

fluvoxamine. Proc Natl Acad Sci U S A, 95(6), 3239-3244.

Van Sickle, B. J., & Tietz, E. I. (2002). Selective enhancement of AMPA receptor-

mediated function in hippocampal CA1 neurons from chronic benzodiazepine-

treated rats. Neuropharmacology, 43(1), 11-27.

Van Sickle, B. J., Xiang, K., & Tietz, E. I. (2004). Transient plasticity of

hippocampal CA1 neuron glutamate receptors contributes to benzodiazepine

withdrawal-anxiety. Neuropsychopharmacology, 29(11), 1994-2006.

Vinet, J., & Sik, A. (2006). Expression pattern of voltage-dependent calcium channel

subunits in hippocampal inhibitory neurons in mice. Neuroscience, 143(1),

189-212.

Wafford, K. A., Thompson, S. A., Thomas, D., Sikela, J., Wilcox, A. S., & Whiting,

P. J. (1996). Functional characterization of human gamma-aminobutyric

acidA receptors containing the alpha 4 subunit. Mol Pharmacol, 50(3), 670-

678.

Wallner, M., Hanchar, H. J., & Olsen, R. W. (2003). Ethanol enhances alpha 4 beta 3

delta and alpha 6 beta 3 delta gamma-aminobutyric acid type A receptors at

low concentrations known to affect humans. Proc Natl Acad Sci U S A,

100(25), 15218-15223.

201

Wallner, M., Hanchar, H. J., & Olsen, R. W. (2006a). Low-dose alcohol actions on

alpha4beta3delta GABAA receptors are reversed by the behavioral alcohol

antagonist Ro15-4513. Proc Natl Acad Sci U S A, 103(22), 8540-8545.

Wallner, M., Hanchar, H. J., & Olsen, R. W. (2006b). Low dose acute alcohol effects

on GABA A receptor subtypes. Pharmacol Ther, 112(2), 513-528.

Wang, H. G., George, M. S., Kim, J., Wang, C., & Pitt, G. S. (2007).

Ca2+/calmodulin regulates trafficking of Ca(V)1.2 Ca2+ channels in cultured

hippocampal neurons. J Neurosci, 27(34), 9086-9093.

Wenthold, R. J., Petralia, R. S., Blahos, J., II, & Niedzielski, A. S. (1996). Evidence

for multiple AMPA receptor complexes in hippocampal CA1/CA2 neurons. J

Neurosci, 16(6), 1982-1989.

Whittington, M. A., Dolin, S. J., Patch, T. L., Siarey, R. J., Butterworth, A. R., &

Little, H. J. (1991). Chronic dihydropyridine treatment can reverse the

behavioural consequences of and prevent adaptations to, chronic ethanol

treatment. Br J Pharmacol, 103(3), 1669-1676.

Whittington, M. A., & Little, H. J. (1991). Nitrendipine, given during drinking,

decreases the electrophysiological changes in the isolated hippocampal slice,

seen during ethanol withdrawal. Br J Pharmacol, 103(3), 1677-1684.

Wilson, M. A., & Frye, C. A. (1999). Effects of chronic benzodiazepine exposure on

stress-induced neuroactive steroid levels. Brain Res, 824(1), 136-139.

Wolf, M. E. (2003). Effects of psychomotor stimulants on glutamate receptor

expression. Methods Mol Med, 79, 13-31.

202

Woods, J. H., Katz, J. L., & Winger, G. (1992). Benzodiazepines: use, abuse, and

consequences. Pharmacol Rev, 44(2), 151-347.

Woods, J. H., & Winger, G. (1995). Current benzodiazepine issues.

Psychopharmacology (Berl), 118(2), 107-115; discussion 118, 120-101.

Woodward, J. J. (1999). Ionotropic glutamate receptors as sites of action for ethanol

in the brain. Neurochem Int, 35(2), 107-113.

Wu, Y., Rosenberg, H. C., Chiu, T. H., & Zhao, T. J. (1994). Subunit- and brain

region-specific reduction of GABAA receptor subunit mRNAs during chronic

treatment of rats with diazepam. J Mol Neurosci, 5(2), 105-120.

Xiang, K., Earl, D. E., Davis, K. M., Giovannucci, D. R., Greenfield, L. J., Jr., &

Tietz, E. I. (2008). Chronic benzodiazepine administration potentiates high

voltage-activated calcium currents in hippocampal CA1 neurons. J Pharmacol

Exp Ther, 327(3), 872-883.

Xiang, K., & Tietz, E. I. (2007). Benzodiazepine-induced hippocampal CA1 neuron

alpha-amino-3-hydroxy-5-methylisoxasole-4-propionic acid (AMPA) receptor

plasticity linked to severity of withdrawal anxiety: differential role of voltage-

gated calcium channels and N-methyl-D-aspartic acid receptors. Behav

Pharmacol, 18(5-6), 447-460.

Xiang, K., & Tietz, E. I. (2008). Chronic benzodiazepine-induced reduction in

GABA(A) receptor-mediated synaptic currents in hippocampal CA1

pyramidal neurons prevented by prior nimodipine injection. Neuroscience,

157(1), 153-163.

203

Xie, X. H., & Tietz, E. I. (1992). Reduction in potency of selective gamma-

aminobutyric acidA agonists and diazepam in CA1 region of in vitro

hippocampal slices from chronic flurazepam-treated rats. J Pharmacol Exp

Ther, 262(1), 204-211.

Xu, W., & Lipscombe, D. (2001). Neuronal Ca(V)1.3alpha(1) L-type channels

activate at relatively hyperpolarized membrane potentials and are

incompletely inhibited by dihydropyridines. J Neurosci, 21(16), 5944-5951.

Yamakage, M., Matsuzaki, T., Tsujiguchi, N., Honma, Y., & Namiki, A. (1999).

Inhibitory effects of diazepam and midazolam on Ca2+ and K+ channels in

canine tracheal smooth muscle cells. Anesthesiology, 90(1), 197-207.

Yang, L., Liu, G., Zakharov, S. I., Bellinger, A. M., Mongillo, M., & Marx, S. O.

(2007). Protein kinase G phosphorylates Cav1.2 alpha1c and beta2 subunits.

Circ Res, 101(5), 465-474.

Yang, L., Liu, G., Zakharov, S. I., Morrow, J. P., Rybin, V. O., Steinberg, S. F., et al.

(2005). Ser1928 is a common site for Cav1.2 phosphorylation by protein

kinase C isoforms. J Biol Chem, 280(1), 207-214.

Yu, S., & Ho, I. K. (1990). Effects of acute barbiturate administration, tolerance and

dependence on brain GABA system: comparison to alcohol and

benzodiazepines. Alcohol, 7(3), 261-272.

Zeng, X., & Tietz, E. I. (1997). Depression of early and late monosynaptic inhibitory

postsynaptic potentials in hippocampal CA1 neurons following prolonged

benzodiazepine administration: role of a reduction in Cl- driving force.

Synapse, 25(2), 125-136.

204

Zeng, X., Xie, X. H., & Tietz, E. I. (1995). Reduction of GABA-mediated inhibitory

postsynaptic potentials in hippocampal CA1 pyramidal neurons following oral

flurazepam administration. Neuroscience, 66(1), 87-99.

Zeng, X. J., & Tietz, E. I. (1999). Benzodiazepine tolerance at GABAergic synapses

on hippocampal CA1 pyramidal cells. Synapse, 31(4), 263-277.

Zeng, X. J., & Tietz, E. I. (2000). Role of bicarbonate ion in mediating decreased

synaptic conductance in benzodiazepine tolerant hippocampal CA1 pyramidal

neurons. Brain Res, 868(2), 202-214.

Zhang, H., Fu, Y., Altier, C., Platzer, J., Surmeier, D. J., & Bezprozvanny, I. (2006).

Ca1.2 and CaV1.3 neuronal L-type calcium channels: differential targeting

and signaling to pCREB. Eur J Neurosci, 23(9), 2297-2310.

Zhang, H., Maximov, A., Fu, Y., Xu, F., Tang, T. S., Tkatch, T., et al. (2005).

Association of CaV1.3 L-type calcium channels with Shank. J Neurosci,

25(5), 1037-1049.

Zhang, M., Moller, M., Broman, J., Sukiasyan, N., Wienecke, J., & Hultborn, H.

(2008). Expression of calcium channel CaV1.3 in cat spinal cord: light and

electron microscopic immunohistochemical study. J Comp Neurol, 507(1),

1109-1127.

Zhong, J., Russell, S. L., Pritchett, D. B., Molinoff, P. B., & Williams, K. (1994).

Expression of mRNAs encoding subunits of the N-methyl-D-aspartate

receptor in cultured cortical neurons. Mol Pharmacol, 45(5), 846-853.

205