INHIBITORY ACTIONS OF

GASTRIN-RELEASING PEPTIDE IN MOUSE

ANTERIOR CINGULATE CORTEX

By

XiaoYan Cao

A thesis submitted in conformity with the requirements for the

degree of Master of Science

Department of Physiology

University of Toronto

© Copyright by XiaoYan Cao, 2011

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“Inhibitory Actions of Gastrin-Releasing Peptide in Mouse Anterior Cingulate Cortex”,

MSc, 2011, Xiaoyan Cao, Department of Physiology, University of Toronto

ABSTRACT

The anterior cingulate cortex (ACC) expresses high density of Gastrin-releasing

peptide (GRP) and GRP receptor mRNA. To address possible function, this investigation

used patch clamp recordings in mouse brain slice preparations to evaluate intrinsic

properties of ACC neurons and neuronal responses to bath-applied GRP peptide. The

ACC neurons were divided according to their morphology, the properties of action

potentials and their firing pattern in response to depolarizing current pulses. Two

physiological groups of interneurons and three groups of pyramidal neurons were

defined. Application of the GRP induced depolarization and increased firing of the

interneurons while hyperpolarization and reduced firing in pyramidal neurons. Moreover,

activation of GRP receptor facilitated GABAergic neurotransmission via a postsynaptic

mechanism. The results suggest that GRP receptor is an important regulator of neuronal

circuits in the ACC and may consequently play an important role for ACC neurons in the

central processing of high brain function.

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TABLE OF CONTENTS

CHAPTER 1;

1.1 Preface-overview

INTRODUCTION

1.2 Anterior cingulate cortex (ACC)

1.2.1 Cellular Anatomy

1.2.2 Neuronal input to the ACC

1.2.3 Neuronal output from the ACC

1.2.4 Synaptic transmission in the ACC

1.2.4.1 Excitatory transmission in the ACC

1.2.4.2 Inhibitory transmission in the ACC

1.2.5 LTP of excitatory transmission

1.2.6 Long-term plastic changes after nerve injury

1.3 Gastrin-releasing peptide (GRP)

1.3.1 GRP peptide

1.3.2 GRP receptor

1.3.3 GRP receptor signaling pathway

1.3.4 Cellular electrophysiology and receptor-mediated actions

1.3.4.1 Dorsal raphe nucleus

1.3.4.2 Suprachiasmatic nucleus (SCN)

1.3.4.3 Neuropeptide Y neurons of arcuate nucleus of the hypothalamus

1.3.4.4 Proopiomelanocortin neurons of arcuate nucleus of the

hypothalamus

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1.3.4.5 Hippocampus

1.3.4.6 Lateral nucleus of amygdale

1.4 Hypothesis and Objectives

CHAPTER 2

2.1 Animals

; METHODOLOGY

2.2 Behavioral allodynic responses

2.3 Whole-cell patch clamp recording

2.4 Immunohistochemistry

2.5 Passive and Active membrane properties

2.6 Histology and immunohistochemistry

2.7 Confocal microscopy

2.8 Pharmacological Drugs

2.9 Data Analysis

CHAPTER 3;

3.1 Introduction

PROPERTIES OF ANTERIOR CINGULATE CORTICAL

NEURONS

3.2 Results

3.2.1 Morphological properties of the ACC layer II/III pyramidal neurons and

interneurons (unpublished data)

3.2.2 Electrophysiological properties of interneurons in layer II/III of ACC

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3.2.2.1 Fast spiking cells (FS)

3.2.2.2 Non—fast spiking cells (non-FS).

3.2.3 General characteristics of pyramidal cells in layer II/III of ACC

3.2.3.1 Regular Spiking (RS) neuron

3.2.3.2 Intrinsic bursting (IB) neuron

3.2.3.3 Intermediate (IM) neuron

3.2.3.4 Differential electrophysiological properties among RS, IM and IB

pyramidal cells

3.2.4 Intrinsic properties of ACC pyramidal neurons after neuropathic pain

CHAPTER 4

4.1 Introduction

; RESPONSE TO EXOGENOUS GASTRIN-RELEASING PEPTIDE

(GRP)

4.2 Results

4.2.1 The Expression of GRP and GRP Receptors in the ACC

4.2.2 Morphological and electrophysiological properties of interneurons and

pyramidal neurons in ACC

4.2.3 Activation of GRPR induces inward currents in interneurons but small or

undetectable currents in pyramidal neurons

4.2.4 Activation of GRP receptors enhances spontaneous GABAergic, but not

glutamatergic transmission

4.2.5 Increase of GABA Release by GRP is action potential dependent

4.2.6 Activation of GRPR also increases the frequency of sIPSCs in pyramidal

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neurons on the basal lateral amygdala and insular cortex

CHAPTER 5;

5.1 Discussion of Properties of Anterior Cingulate Cortical Neurons

DISCUSSION

5.1.1 ACC, synaptic transmission, plasticity and spike

5.1.2 Classification of three types of ACC pyramidal cells

5.1.3 Synaptic and nonsynaptic plasticity

5.2 Discussion of Response to Exogenous Gastrin-Releasing Peptide (GRP)

5.2.1 Mechanisms of Modulation of GABA Release by GRPR in the ACC

5.2.2 Functional Significance of GRPR Modulation in the ACC

REFERENCES

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FIGURES

Figure 1.1 Immunohistochemistry reveals a dense expression of the GRP and GRPR

CHAPTER 1

in the ACC (from the Allen Mouse Brain Atlas)

Figure 1.2 Diagram of the human anterior cingulate cortex (ACC).

Figure 1.3 Diagram showing pain transmission and modulation pathways in the

central nervous system.

Figure 2.1 The setup for electrophysiological recordings

CHAPTER 2

Figure 3.1 Schematic diagram demonstrating the recording of ACC neurons.

CHAPTER 3

Figure 3.2 Morphological subtypes of layer II/III pyramidal cells in the ACC

Figure 3.3 Morphological subtypes of layer II/III interneurons in the ACC

Figure 3.4 Firing properties of the typical FS and non-FS neurons in ACC layer

II/III interneurons.

Figure 3.5 Action potential properties of the typical FS and non-FS neurons in

ACC layer II/III interneurons.

Figure 3.6 Characteristics of the typical RS neurons in the ACC.

Figure 3.7 Electrophysiological characteristics of typical IB ACC neurons.

Figure 3.8 Electrophysiological characteristics of the IM cells.

Figure 3.9 Differential intrinsic membrane properties between RS, IM and IB

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pyramidal cells.

Figure 3.10 Changes of action potential properties of IM pyramidal neurons in the

ACC after neuropathic pain.

Figure 3.11 Summary of alterations seen in firing patterns in ACC pyramidal neurons

after neuropathic pain.

Figure 4.1 GABA immunoreactivity in the GRPR neurons.

CHAPTER 4

Figure 4.2 Morphological and electrophysiological properties of interneurons and

pyramidal neurons in the ACC.

Figure 4.3 Activation of GRPR induced inward current in interneruons but

undetectable current change in pyramidal neurons.

Figure 4.4 Activation of GRP receptor by GRP reversibly increased sIPSCs in

pyramidal neurons on the ACC.

Figure 4.5 The GRP-induced sIPSC facilitation is blocked by RC3095 and picrotoxin.

Figure 4.6 Activation of GRP receptor by GRP had no effect on sEPSCs in

pyramidal neurons of ACC.

Figure 4.7 Increase of GABA release by GRP is action potential dependent.

Figure 4.8 Activation of GRP receptors increased GABA release in BLA neurons.

Figure 4.9 Activation of GRP receptors increased GABA release in IC neurons.

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TABLE

Table 3.1 Summary of basic morphological parameters of ACC layer II/III

pyramidal neurons.

Table 3.2 Summary of basic electrophysiological and morphological parameters of

ACC layer II/III interneurons.

Table 3.3 Summary of basic electrophysiological parameters of ACC layer II/III

pyramidal neurons in control and neuropathic pain mice.

Table 4.1 Basic electrophysiological properties of ACC neurons.

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ABBREVIATIONS

ACC anterior cingulate cortex

aCSF artificial cerebrospinal fluid

ADP afterdepolarization

AHP afterhyperpolarizations

AMPA a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid

AP action potential

AP-5 D-2- amino-5-phosphonopentanoic-acid

ARC arcuate nucleus

BLA basal lateral amygdale

CNQX 6-cyano-7-nitroquinoxaline-2,3-dione

CNS central nervous system

CaM calmodulin

CaMKIV Ca2+-calmodulin-dependent protein kinase IV

CPN common peroneal nerve

DAB diaminobenzidine

DAG diacylglyceride

DRN dorsal raphe nucleus

DRG dorsal root ganglion

egr1 early growth response gene 1

EPSCs excitatory postsynaptic currents

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EPSP excitatory postsynaptic potential

ERK extracellular signal-regulated protein kinase

FMRP fragile X mental retardation protein

FS fast spiking

GABA g-Aminobutyric acid

GAD glutamate decarboxylase

GPCR G protein-coupled receptor

GRP Gastrin-Releasing Peptide

IP3 inositol triphosphate

IPSCs inhibitory postsynaptic currents

IB intrinsic bursting

IC insular cortex

IM intermediate

ISI interspike interval

KA kainate

LTP long-term potentiation

MAPK mitogen-activated protein kinase

αMSH alpha-melanocyte-stimulating hormone

mGluRs metabotropic glutamate receptors

NMDA N-methyl-D-aspartate

NPY neuropeptide Y

NTS nucleus tractus solitarius

PBS phosphate buffered saline

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PFC prefrontal cortex

PK2 prokineticin

PKA protein kinase A

PKC protein kinase C

PLC phospholipase C

POMC pro-opiomelanocortin

RMP resting membrane potential

RS regular spiking

SCN suprachiasmatic nucleus

SFA Spike frequency adaptation

TBS θ burst stimulation

TTX tetrodotoxin

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ACKNOWLEDGEMENTS

I wish to thank Dr.Min Zhuo, who provided equipment, laboratory space and financial support to help me complete the graduate traning. His guidance, helpful criticism and concern have been of great help in completion of this research project. This research experience provided me with the opportunity to learn a lot of things about science and scientists.

I am indebted to Dr. Martin Wojtowicz for his willingness to share his expertise with regards to the writing of the thesis and for many valuable discussions and suggestions.

I would like to thank the present and past members of the Zhuo laboratory, Hendrik Steenland, Yongheng Jia, Xiangyao Li, Tao Chen, Giannina Descalzi, Valentina Mercaldo, Longjun Wu, Bo Gong, Hansen Wang, Kunjumon Vadakkan, Emily England, for themendous help, warm friendship and great fun.

I thank my son, Albert YeLe Tian, for being the ultimate reason for finishing this project and my husband, Zhu Tian, a very special friend, for his support during these studies. I would like to extend my gratitude to my parents, MeiLian Liu and Jiang Cao for their unconditional love and support throughout my life and in pursuit of my graduate degree.

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PUBLICATIONS

1 Cao XY, Mercaldo V, Li PY, Wu LJ and Zhuo M. Postsynaptic Regulation of the

Inhibitory Transmission by Gastrin-releasing peptide Receptors in the Anterior Cingulate

Cortex of Adult Mice. Mol Pain. 2010 Sep 13;6(1):52. *

2 Cao XY, Xu H, Wu LJ, Li XY, Chen T, Zhuo M. Characterization of intrinsic

properties of cingulate pyramidal neurons in adult mice. Mol Pain. 2009 Dec 16;5:73. *

3 Hines RM, Wu L, Hines DJ, Steenland H, Mansour S, Dahlhaus R, Singaraja RR, Cao

X, Sammler E, Hormuzdi SG, Zhuo M, El-Husseini A. Synaptic imbalance, stereotypies,

and impaired social interactions in mice with altered neuroligin 2 expression. J Neurosci.

2008 Jun 11;28(24):6055-67.

4 Wu LJ, Ren M, Wang H, Kim SS, Cao X, Zhuo M. Neurabin contributes to

hippocampal long-term potentiation and contextual fear memory. PLoS ONE. 2008 Jan

9;3(1):e1407.

5 Wu LJ, Xu H, Ren M, Cao X, Zhuo M. Pharmacological isolation of postsynaptic

currents mediated by NR2A- and NR2B-containing NMDA receptors in the anterior

cingulate cortex. Mol Pain. 2007 Apr 30;3:11.

ABSTRACTS 1 Cao XY, Mercaldo V, Li PY, Wu LJ and Zhuo M. Postsynaptic Regulation of the

Inhibitory Transmission by Gastrin-releasing peptide Receptors in the Anterior Cingulate

Cortex of Adult Mice. Society for Neuroscience Annual Meeting, San Diego, 2010

(Abstract, poster and oral presentation)

2 Cao XY, Wu LJ, Zhuo M. Characterization of intrinsic properties of cingulate

pyramidal neurons in adult mice. Society for Neuroscience Annual Meeting, Chicago,

2009 (Abstract)

1

CHAPTER 1

INTRODUCTION

1.1 Preface-overview

Anterior cingulate cortex (ACC) is a highly wired cortical region around the rostrum

of the corpus callosum. Neurons in the ACC receive inputs directly or indirectly from

other regions of brain, including the thalamus, amygdala, hippocampus, and other cortical

regions (Wei, Li et al. 1999; Koechlin, Ody et al. 2003; Wang and Shyu 2004; Likhtik,

Pelletier et al. 2005; Vogt 2005; Zhuo 2005). Different behavioral functions of the ACC

subregions have been proposed, mostly from human imaging studies (Bush, Luu et al.

2000), including sensory, memory, emotional and cognitive functions (Rainville, Duncan

et al. 1997; Courtney, Petit et al. 1998; Zhuo 2002; Zhao, Toyoda et al. 2005). However,

it still remains to be determined at synaptic and molecular levels whether such function-

specific regions indeed exist within the ACC.

Studies have shown that the ACC expresses high density of Gastrin-Releasing Peptide

(GRP) and GRP Receptors (see Fig.1.1), the neuropeptide system, which has been

proposed to be considered as a therapeutic target in a subset of neuropsychiatric disorders

(Roesler, Lessa et al. 2004). The research described in this thesis is an initial description

of the membrane properties of ACC neurons recorded in a mouse brain slice preparation

and how these neurons respond to exogenous applications of GRP, which may involved

in the modulation of the ACC related functional roles.

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Figure 1.1 Expression of the GRP and GRPR in the ACC (from the Allen Mouse Brain Atlas). (A) In situ hybridization on a coronal section of mouse brain, showing the expression of the Grp gene in the anterior cingulate cortex (ACC) of wild-type mice (rectangled area http://mouse.brain-map.org/viewImage.do?imageId=79611383). (B) In situ hybridization on a sagittal section of mouse brain, showing expression of the Grp receptor gene in ACC of wild-type mice (rectangled area http://mouse.brain-map.org/viewImage.do?imageId=73493204). 1.2 Anterior cingulate cortex (ACC)

The cingulate gyrus, the most prominent cortical feature on the medial surface of the

brain, is an arch-shaped convolution, lying over the body of the corpus callosum and is

separated by the callosal fissure. It commences the lamina terminalis rostral to the

anterior commissure, curves around the genu of the corpus callosum, extends along the

body of the callosum, and finally turns downward behind the splenium (see Fig.1.2). The

ACC is around the genu of the corpus callosum, highly conserved through mammalian

evolution and is thought to be a homologue of structure in primates and rodents on the

basis of its hemispheric location, agranular cytoarchitecture (Rose and Woolsey 1948;

Niimi, Niimi et al. 1978; Niimi 1978; Robertson and Kaitz 1981; Musil and Olson 1988).

http://mouse.brain-map.org/viewImage.do?imageId=79611383�http://mouse.brain-map.org/viewImage.do?imageId=73493204�http://mouse.brain-map.org/viewImage.do?imageId=73493204�

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The ACC contains layers of pyramidal cells mixed with local interneurons. In layer I,

there are mainly inerneurons. The layers II-III contain mainly pyramidal cells. Neurons in

layer II-III receive sensory inputs from the medial thalamus, a key relay nucleus for

somatosensory information (Shibata 1993; Yamamura, Iwata et al. 1996; Wang and Shyu

2004). The soma of the pyramidal cells may be strictly located at the layer II-III, its distal

dendritic branches can extend to layer I. Neurons in layer V are larger than neuronts in

layers II-III and VI. Many non-pyramidal cells are inhibitory neurons that contain g-

Aminobutyric acid (GABA) and/or neuropeptides. Within the local circuits, inhibitory

neurons often receive innervations from the pyramidal cells (glutamatergic), then release

GABA onto the perisomatic region of the pyramidal cells.

Figure1.2 Diagram of the human anterior cingulate cortex (ACC). A midline Sagittal Surface of the brain contains the ACC and adjacent brain areas. The cingulate cortex is shown in dark brown. 1.2.1 Neuronal input to the ACC

Anatomical tracer studies reveal that the ACC receives widespread inputs from

cortical and subcortical brain areas. ACC neurons receive sensory inputs from the medial

thalamus, a key relay nucleus for somatosensory information including pain (Shibata

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1993; Yamamura, Iwata et al. 1996; Wang and Shyu 2004). ACC neurons form

interconnection with the other ACC neurons in the opposite side of the hemisphere

through callosal projecting fibers, as well as other cortical areas at the same side and

opposite side of the brains. The ACC receive input from the hippocampus, the locus

coeruleus, raphe nuclei, zona incerta, intralaminar nuclei, the claustrum, the globus

pallidus, the nucleus of the diagonal band of Broca, region of the medial forebrain bundle

and the hypothalamus (Petras 1965; Finch, Derian et al. 1984; Li, Takada et al. 1986;

Horikawa, Kinjo et al. 1988).

1.2.2 Neuronal output from the ACC

ACC neurons project to subcortical structures such as the hypothalamus and

periaqueductal gray, which may contribute to descending modulation of spinal sensory

transmission (for example, see (Calejesan, Kim et al. 2000). ACC neurons also form

interconnections with neurons in the amygdala, a structure critical for emotional fear and

anxiety. ACC neurons also form projections to autonomic brainstem nuclei and play an

important role in the visceromotor control (Neafsey et al., 1993). Furthermore, ACC

neurons project to motor cortex to generate motor responses, vocalization etc. Projections

of ACC also include those to the nucleus of the solitary tract, the dorsal motor nucleus of

the vagus, and the sympathetic thoracic intermediolateral cell column (Vogt, Nimchinsky

et al. 1995).

1.2.4 Synaptic transmission in the ACC

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Brain slices from mouse, rats, guinea pigs, and rabbit have been used to investigate

synaptic transmission in the ACC (Vogt and Gorman 1982; Sah and Nicoll 1991; Tanaka

and North 1994; Wei, Li et al. 1999; Wu, Zhao et al. 2005; Zhao, Toyoda et al. 2005).

1.2.4.1 Excitatory transmission

Glutamate (Glu) stands out as the main mediator of fast excitatory synaptic

transmission in the ACC (Sah and Nicoll 1991; Wei, Li et al. 1999), which is

demonstrated by the observation that bath application of 6-cyano-7-nitroquinoxaline-2,3-

dione (CNQX) and D-2- amino-5-phosphonopentanoic-acid (AP-5) completely abolishes

fast excitatory postsynaptic currents (EPSCs) recorded in the ACC neurons. Different

types of glutamate receptors, including a-amino-3-hydroxy-5-methyl-4-

isoxazolepropionic acid (AMPA), including GluR1 and GluR2/3 subunits, kainate (KA),

N-methyl-D-aspartate (NMDA) and metabotropic glutamate receptors (mGluRs) are

found in the ACC. Pharmacologically, application of CNQX, a non-selective inhibitor of

AMPA and KA receptors, could completely block the fast synaptic responses induced by

focal stimulation (Wei, Li et al. 1999; Wu, Toyoda et al. 2005; Wu, Zhao et al. 2005). In

the presence of the picrotoxin, AP-5, and a selective AMPA receptor antagonist, GYKI

53655, small KA receptor-mediated excitatory postsynaptic currents (KA EPSCs) could

be induced by the single-shock stimulation (Wu, Zhao et al. 2005). Genetic deletion of

the KA GluR6 and GluR5 subunits completely abolished KA EPSCs and KA-activated

currents (Wu, Zhao et al. 2005). NMDA receptor mediated slow synaptic responses were

recorded at physiological temperatures from the ACC in vitro brain slices and in vivo

freely moving mice (Liauw, Wu et al. 2005; Wu, Toyoda et al. 2005), suggesting that

6

NMDA receptors are tonically active in this region. In addition, a recent report found that

NMDA receptors in the ACC neurons contribute to generation of action potentials

induced by a depolarizing step applied to the soma of ACC neurons (Wang and Zhuo

2006).

1.2.4.2 Inhibitory transmission

Besides excitatory transmission in the ACC, GABA is the major inhibitory transmitter

in the ACC, which is nicely demonstrated by the observation that bath application of

picrotoxin completely abolishes spontaneous inhibitory postsynaptic currents (sIPSCs)

and eIPSCs recorded in the ACC neurons. The IPSCs are mainly mediated by the

postsynaptic GABAA receptors (Wu, Xu et al. 2007). GABAB receptors are also

reported in the ACC, although the roles of GABAB have not been investigated. Unlike

excitatory synaptic transmission, few studies have been reported about the inhibitory

transmission. Recent studies using KA knockout mice reported that GluR5 containing

KA receptors modulate GABAergic transmission in the ACC (Wu, Xu et al. 2007).

1.2.5 LTP of excitatory transmission

Different stimulation protocols have been found for inducing long lasting potentiation

of synaptic responses in the ACC cells，including pairing training protocol (synaptic

activity paired with postsynaptic depolarization), the spike-excitatory postsynaptic

potential (EPSP) pairing protocol, and θ burst stimulation (TBS) protocol all induce LTP

in ACC pyramidal neurons (Wei and Zhuo 2001; Wei, Qiu et al. 2002; Zhao, Toyoda et

al. 2005; Zhao, Toyoda et al. 2005). Genetic, pharmacological and electrophysiological

7

approaches have been used to investigate the basic mechanisms for LTP in ACC

synapses. Bath application of NR2A antagonist NVP-AAM077 and NR2B antagonist

ifenprodil/Ro compounds almost completely blocked NMDA receptor-mediated EPSCs

as well as LTP, suggesting the critical role of NMDA receptors (Zhao, Toyoda et al.

2005). Synaptic injection of BAPTA, a selective Ca2+ chelator, completely blocked the

induction of LTP, indicating the importance of elevated postsynaptic Ca2+

concentrations (Zhao, Toyoda et al. 2005). Studies found that ACC LTP is absent in

adenylyl cyclase subtype 1 (AC1) (Wei, Qiu et al. 2002; Ko, Vadakkan et al. 2005; Zhao,

Toyoda et al. 2005) and GluR1 knockout mice (Zhao et al., unpublished). Several other

signaling proteins or protein kinases are found to be involved in ACC LTP, including the

calcium binding sites of calmodulin (CaM) (Wei, Qiu et al. 2002), Ca2+-calmodulin-

dependent protein kinase IV (CaMKIV), early growth response gene 1 (egr1), mitogen-

activated protein kinase (MAP kinase) and fragile X mental retardation protein (FMRP)

(Zhao, Toyoda et al. 2005; Toyoda, Wu et al. 2007). At least four different synaptic

mechanisms might contribute to the expression of LTP: (i) presynaptic enhancement of

the glutamate release; (ii) postsynaptic enhancement of glutamate receptor-mediated

responses; (iii) recruitment of functional glutamate receptors; and (iv) structural changes

in synapses (Zhuo 2007).

1.2.6 Long-term plastic changes after nerve injury

ACC neurons are multi-functional and studies from animals and humans consistently

demonstrate that ACC neurons play important roles in various sensory, memory, emotion

and cognitive functions (Peoples 2002; Sanders, Gallup et al. 2002; Botvinick, Cohen et

8

al. 2004; Dalley, Cardinal et al. 2004). In this thesis, I mainly focus on the roles of ACC

in sensory functions including pain. The anterior cingulate cortex (ACC) is important for

the affective and emotional component of physiological and pathological pain (Rainville,

Duncan et al. 1997; Casey 1999; Hutchison, Davis et al. 1999; Johansen and Fields 2004;

Tang, Ko et al. 2005). Brain imaging and electrophysiological studies have shown that

the ACC responds to painful stimuli in humans (Rainville, Duncan et al. 1997; Lenz,

Rios et al. 1998; Eisenberger, Lieberman et al. 2003) and nociceptive stimuli in animals

as well (Sikes and Vogt 1992; Yamamura, Iwata et al. 1996; Koyama, Tanaka et al.

1998). Activation of neurons in the ACC produced pain-like aversive behaviors or fear

memory, while inhibition of excitatory transmission produced the blockade of pain-

aversive learning or analgesic effects (Johansen, Fields et al. 2001; Johansen and Fields

2004; Tang, Ko et al. 2005). Moreover, there are long-term changes in synaptic plasticity

and transmitter release in the ACC in chronic pain conditions: amputation causes long-

term facilitation of local stimulation-induced ACC synaptic responses and specific loss of

long-term synaptic depression (Wei, Li et al. 1999; Wei and Zhuo 2001); peripheral

inflammation of the hind paw with complete Freund's adjuvant in adult mice causes the

upregulation of the postsynaptic NMDA receptor NR2B subunit (Wu, Toyoda et al.

2005). Furthermore, increased presynaptic glutamate releases were found in ACC

neurons after inflammation (Zhao, Ko et al. 2006; Xu, Wu et al. 2008) or nerve injury

(Xu, Wu et al. 2008).

Neuropathic pain occurs as a consequence of the injury to peripheral or central

nervous system. Symptoms include spontaneous pain, abnormal hypersensitivity to

innocuous touch (allodynia) and to noxious mechanical or thermal stimulation

9

(hyperalgesia) (Treede, Meyer et al. 1992; Woolf and Mannion 1999). Clinical

neuropathic pain can arise from a variety of different disease states (e.g., diabetic

neuropathy, trigeminal neuralgia, postherpetic neuralgia, AIDS) or traumatic injuries,

nerve compression, or chemotherapy (Jain 2008). Long-term plastic changes along the

sensory pathways are suggested to contribute to the neuropathic pain, including the

peripheral nociceptors, spinal dorsal horn, subcortical areas and cortical areas (Ko, Ao et

al. 2005; Toyoda, Zhao et al. 2005; Zhuo 2006). Of particular interest here is that the

ACC is undergoing dramatic changes under chronic pain conditions (Ko, Ao et al. 2005;

Zhuo 2006). However, little is known about the intrinsic electrophysiological properties

of ACC neurons and possible changes in intrinsic properties after nerve injury. One major

hypothesis is that altered neuronal excitability after peripheral nerve injury may

contribute to the plastic changes of ACC neurons under chronic pain conditions. To test

the idea, by using the whole-cell patch-clamp recordings in slices under current clamp

mode, we examined the firing activity of ACC neurons in a mouse model of neuropathic

pain reported previously (Vadakkan, Jia et al. 2005) (see Fig.1.3).

1.3 Gastrin-releasing peptide (GRP)

Bombesin is a 14 amino acid-containing peptide first isolated from the skin of the frog

Bombina bombina (Anastasi, Erspamer et al. 1971). Gastrin-releasing peptide (GRP) is a

member of the mammalian bombesin-related peptides, which is mediated by GRP -

receptor (Battey and Wada 1991). Immunohistochemistry and in situ hybridization

studies have shown that GRP and its preferential binding receptor, GRP receptor, are

widely distributed throughout the central nervous system (CNS) (Moody, O'Donohue et

10

al. 1981; Panula, Yang et al. 1982; Moody, Getz et al. 1988; Moran, Moody et al. 1988;

Wada, Way et al. 1990; Battey and Wada 1991; Wada, Way et al. 1991; Ladenheim,

Jensen et al. 1992; Kamichi, Wada et al. 2005). The potent CNS effects of GRP include

regulation of the circadian rhythm, satiety, thermoregulatory effects, grooming behaviors,

11

Figure 1.3 Diagram showing pain transmission and modulation pathways in the central nervous system. A: Schematic diagram of the left lumbar plexus, sciatic nerve and its branches. Abbreviations: Sa.N, saphenous nerve; C.P.N, common peroneal nerve; D.P.N, deep peroneal nerve; S.N, sural nerve; T.N, tibial nerve; S.P.N, superficial peroneal nerve. B: Sensory inputs from the periphery form the first sensory synapses in the spinal cord dorsal horn between the presynaptic terminals of the dorsal root ganglion (DRG) cells and dorsal horn neurons. Some of the spinal dorsal horn neurons send ascending projection to the neurons in the thalamus. The outputs from the thalamus further synapse on the neurons in the anterior cingulate cortex (ACC), and other cortical neurons (like somatosensory cortex). chronic itch sensation, and effects on gastrointestinal function such as acid secretion

(Martinez and Tache 2000; Yegen 2003; Moody and Merali 2004; Karatsoreos, Romeo et

al. 2006; Roesler, Henriques et al. 2006; Roesler, Luft et al. 2006; Kallingal and Mintz

2007; Presti-Torres, de Lima et al. 2007; Sun and Chen 2007).

1.3.1 GRP peptide in the brain

Human, canine, porcine, and avian GRPs are amidated, 27-amino acid peptides and

are remarkably well conserved in the primary structure especially in the carboxy termini

(Spindel, Zilberberg et al. 1986). The human gene for GRP is located on chromosome 18

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and contains three exons and two introns (Naylor, Sakaguchi et al. 1987). The

distribution of GRP mRNA was determined using in situ hybridization techniques

(Moody, Getz et al. 1988; Wada, Way et al. 1990). High levels of GRP mRNA were

present in the isocortex (layers II and III), the anterior olfactory nucleus, the

suprachiasmatic and medial preoptic nucleus of the hypothalamus, hippocampal

formation, amygdale and nucleus of the solitary tract (NST). Low levels of GRP mRNA

were present in the isocortex (layers V and VI), paraventricular nucleus of the

hypothalamus, magnocellular preoptic nucleus and central gray. These results suggest

that GRP ligand may be supplied mainly by the local network.

1.3.2 GRP receptor

The GRP-R gene has been mapped to chromosome Xp22 and its structure has been

delineated (Xiao et al., 2001; Mouse Genome Database, August 1997. Mouse Genome

Informatics, The Jackson Laboratory, Bar Harbor, MA. http://www.informatics.jax.org/).

In adult animals, GRP-Rs are widely expressed in the central nervous system (Battey and

Wada 1991)(Wada et al., 1991) and gastrointestinal tract (Moran, Moody et al. 1988).

Using in situ hybridization techniques in the rat brain, high densities of GRP receptor

mRNA were observed in layer II of the isocortex, the dentate gyrus, nucleus of the lateral

olfactory tract, magnocellular preoptic nucleus of the basal ganglia, nucleus ambiguous

and throughout the hypothalamus including the suprachiasmatic nucleus, supraoptic

nucleus, paraventricular nucleus, medial preoptic nucleus and lateral mammillary nucleus

(Battey and Wada 1991). Low density of GRP mRNA were present in the nucleus

accumbens, the central gray and nucleus tractus solitarius (NST). The

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13

immunohistochemistry study in the mouse brain shows that, in most instances, the

distribution of GRP-R immnunoreactivity in the mouse brain agrees closely with that

described for GRP-R gene expression in the rat brain (Kamichi, Wada et al. 2005).

However, some disparities were found. For example, GRP-R mRNA was only observed

in the layers 2 and 3, but GRP-R immunoreactivities were detected in all layers of the

isocortex. In addition, GRP-R mRNA expression was very weak in the rat piriform cortex

while immunoreactivities were strong in the mouse piriform cortex. These disparities

may be caused by the difference in the sensitivity of the techniques between in situ

hybridization and immunohistochemistry.

1.3.3 GRP receptor signaling pathway

Previous studies have shown that GRPR is a Gαq protein-coupled receptor (Hellmich,

Ives et al. 1999; Lee, Dixon et al. 1999). Agonist binding to GRP-R stimulates

phospholipase C-β (PLC-β) resulting in the production of inositol 1,4,5-trisphosphate and

diacylglycerol (DAG), an increase in the concentration of free cytosolic Ca2+([Ca2+]i),

and the activation of both protein kinase C (PKC) (Aprikian, Han et al. 1996; Kim, Evers

et al. 1996; Li, Wollheim et al. 1996) and mitogen-activated protein kinase

(MAPK)/extracellular signal-regulated protein kinase (ERK) pathways (Nemoz-Gaillard,

Cordier-Bussat et al. 1998; Hellmich, Ives et al. 1999; Qu, Xiao et al. 2002).

Among the various GRP antagonists synthesized (Radulovic, Cai et al. 1991; Cai,

Radulovic et al. 1992), the pseudononapeptide [D-Tpi6, Leu13, psi(CH2NH)-Leu14]

(RC-3095) is a potent competitive GRP receptor antagonist, which has been shown to be

effective in inhibiting tumor growth in several experimental models (Qin, Ertl et al. 1994;

14

Szepeshazi, Schally et al. 1997; Chatzistamou, Schally et al. 2001; Bajo, Schally et al.

2002).

1.3.4 Cellular electrophysiology and receptor-mediated actions

The objective of the research described here is to use electrophysiology to examine the

actions of GRP on ACC neurons. This section of the thesis provides an overview of the

literature already available on the electrophysiology of GRP.

1.3.4.1 Dorsal raphe nucleus

Intracellular recordings made from neurons in the dorsal raphe from rat brain slices

indicate that GRP induced a depolarization of membrane potential and increase in firing

rate of action potentials (Pinnock and Woodruff 1991; Pinnock, Reynolds et al. 1994).

The response to the peptides persisted in the presence of tetrodotoxin (TTX) and low-

calcium, high-magnesium-containing artificial cerebrospinal fluid (ACSF), suggestive of

a postsynaptic site of action. Under voltage clamp conditions the peptides caused a

decrease in membrane conductance accompanied by an inward current; an associated

decrease in a potassium conductance was proposed as a mechanism (Pinnock, Reynolds

et al. 1994).

1.3.4.2 Suprachiasmatic nucleus (SCN)

Several papers have reported the GRP-induced excitation in SCN neurons. In

hamster/rats slice preparations, extracellular single-unit recording studies reveal that GRP

administration increased the spontaneous firing of neurons in the SCN (Albers, Liou et al.

15

1991; Piggins and Rusak 1993; Tang and Pan 1993). The excitation of SCN neurons

produced by peptide applicaition was dose dependent.

1.3.4.3 Neuropeptide Y neurons (NPY) of arcuate nucleus of the hypothalamus

Using extracellular single-unit recording in rat brain slices, bombesin related peptides

have been shown to activate unidentified neurons of the arcuate nucleus (Lin and Pan

1993). Data obtained in whole cell patch clamp recording in mouse slice preparations by

Van den Pol et al (2009) indicate that GRP increased the spike frequency and depolarized

NPY neurons in a dose-dependent manner. The data also suggest that GRP excites NPY

neurons through both activation of the Na+/Ca2+ exchanger and opening of sodium-

dependent nonselective cation channels. Note of an increase in the frequency of

spontaneous inhibitory postsynaptic currents (IPSCs), blockable with TTX, may reflect

an action on cells that projected to NPY neurons, and not by acting on presynaptic

terminals (van den Pol, Yao et al. 2009).

1.3.4.4 Proopiomelanocortin neurons (POMC) of arcuate nucleus of the

hypothalamus

Similar to NPY cells, bombesin depolarized and increased the spike frequency of

POMC neurons. The depolarization was TTX-resistant, suggesting a direct postsynaptic

effect of the peptide (van den Pol, Yao et al. 2009).

1.3.4.5 Hippocampus

16

Previous electrophysiological study shows that GRP excited non-pyramidal neurones

in rat hippocampus (Dreifuss and Raggenbass 1986). Consistent with these findings, in a

subsequent analysis, reverse transcription followed by the polymerase chain reaction

using cytoplasm from single hippocampal interneurones demonstrated the expression of

GRP receptor (Lee, Dixon et al. 1999). In addition, data from whole cell recordings

reveal that GRP elicited a marked increase in the frequency of GABAA receptor-mediated

IPSCs recorded in principal neurons of hippocampus. The effect was mediated by

depolarization and induced repetitive firing of GABAergic interneurons due to the

activation of a calcium-activated non-selective cation channel rather than any Na+-Ca2+

exchange mechanism (Lee, Dixon et al. 1999).

1.3.4.6 Lateral nucleus of amygdale

In situ hybridization study shows that GRPR gene was expressed by interneurons of

the lateral nucleus of amygdale in mice (Shumyatsky, Tsvetkov et al. 2002). Whole-cell

recordings show that GRP could significantly increase the frequency of sIPSCs recorded

in pyramidal neurons. The facilitation of sIPSC was abolished after application of GRPR

antagonist or in GRPR knock out mice, confirming that the observed effects are indeed

mediated by GRP receptor. Consistent with the notion that the sIPSCs are mediated by

the GABAA receptors, these currents were completely blocked by γ-aminobutyric acid-A

(GABAA) receptor antagonist, picrotoxin. When TTX was applied to the recording bath,

the facilitatory effect of GRP on sIPSCs was completely reversed by blockade of sodium

channels, suggesting the possibility of postsynaptic sites of action (Shumyatsky,

Tsvetkov et al. 2002).

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1.4 Hypothesis and Objectives

The anterior cingulate cortex (ACC), an integral part of the limbic system, wraps

around the rostrum of the corpus callosum (Broca, 1878) and is involved in most theories

of emotion (MacLean, 1993). Little is known about the intrinsic electrophysiology of

ACC neurons, and about possible changes which may underlie the regulation of many

ACC-related cognitive functions, such as neuropathic pain. Gastrin-releasing peptide

(GRP) and its preferential binding receptor (GRPR) have recently emerged as a system

importantly involved in psychiatric and neurological disorders (Roesler, Henriques et al.

2004). Of particular note for this research is that in situ hybridization analysis has shown

the high density of GRP and GRP receptor mRNA in the ACC (see Fig.1.1). Considering

that the roles of GRP receptors in neuronal excitability have been studied in many regions

of the CNS (see below), this research was undertaken to test the hypotheses that GRP

plays an important role in the regulation of the neuronal excitability and synaptic

neurotransmission in ACC, which may result from the modulation of ACC plasticity, by

facilitating the GABAergic transmission. The objectives of this research are, first, to

characterize the intrinsic electrophysiological properties of ACC neurons and, second, to

assess the effects and mechanisms of activation of GRP receptors on ACC cell

excitability.

1

CHAPTER 2

METHODOLOGY

Adult male C57BL/6 mice purchased from Charles River (6-10 weeks old) were

used in experiments. All protocols were approved by The Animal Care and use

Committee at the University of Toronto and conform to NIH guidelines.

2.1 Animal preparation

Adult (6-8 weeks old) male C57BL/6 mice were purchased from Charles River. Mice

were maintained on a 12 h light/dark cycle. Food and water were provided ad libitum.

Experiments were performed under protocols approved by the University of Toronto

Animal Care Committee. A model of neuropathic pain was induced by the ligation of the

common peroneal nerve (CPN) as previously described (Vadakkan, Jia et al. 2005).

Briefly, mice were anaesthetized by intraperitoneal injection of a mixture saline of

ketamine (0.16 mg/kg, Bimeda MTC, Cambridge, Ontario) and xylazine (0.01 mg/kg,

Bayer, Toronto, Canada). 1 cm skin incision was made in the left hind leg to expose the

CPN. The CPN was ligated with chromic gut suture (5-0, Ethicon, Somerville, New

Jersey) slowly until contraction of the dorsiflexors of the foot was visible as twitching of

the digits. Sham surgery was conducted in the same manner but the nerve was not ligated.

The mechanical allodynia was tested on post-surgical day 7 and the mice were used for

electrophysiological studies on post-surgical days 7-14.

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2.2 Behavioral allodynic responses

Mice were placed in a plexi-glass restrainer and allowed to acclimate for 30 minutes

prior to behavioral testing. Mechanical allodynia was assessed based on the

responsiveness of the hind paw to the application of von Frey filaments (Stoelting, Wood

Dale, Illinois) to the point of bending. Positive responses include licking, biting and

sudden withdrawal of the hind paw. Experiments were carried out to characterize the

threshold stimulus. Mechanical pressure from a 1.65 filament (force 0.008 g) was found

to be innocuous in control mice. This filament was then used to test the mechanical

allodynia after neuropathic pain. Ten trials were carried out each time at an interval of 5

minutes and the results are expressed as percentage of positive responses.

2.3 Whole-cell patch-clamp recording

The animals were decapitated and the brain was quickly removed and immersed in

oxygenated (95% O2-5% CO2) cooled (4~6°C) artificial cerebrospinal fluid (ACSF) for

2-3 minutes. ACSF contained (in mM): 124 NaCl, 2.5 KCl, 2 CaCl2, 2 MgSO4, 25

NaHCO3, 1 NaH2PO4, 10 glucose, pH 7.4, 300-310 mOsm. A block of brain tissue

containing the ACC, amygdala or insular cortex was dissected, glued to a small stage

(LOCTITE 404 cyanoacrylate glue) and covered with ACSF. Coronal slices 300 microns

thick containing the ACC, amygdala or insular cortex were made with a vibratome

(Series 1000) and preincubated in oxygenated ACSF at room temperature (22~26°C) for

at least 1 hour, then transferred to a submerged chamber and superfused (2~3 ml/min)

with oxygenated ACSF at room temperature. Experiments were performed in a recording

chamber on the stage of a BX51W1 microscope equipped with infrared DIC optics for

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visualization. Using the whole-cell patch-clamp technique (Fig. 2.1), recordings were

obtained with an Axon 200B amplifier (Axon Instruments, CA). Patch pipettes with

resistances of 3~5 MΩ. were filled with the following solution (in mM): 120 K-gluconate,

5 NaCl, 1 MgCl2, 0.2 EGTA, 10 HEPES, 2 Mg-ATP, 0.1 Na3-GTP and 10

phosphocreatine disodium (adjusted to pH 7.2 with KOH). Biocytin (0.2%) was included

in the pipette solution to label recorded neurons. The membrane potentials were held at -

70 mV throughout all experiments. When recording GABAA receptor-mediated currents,

K-gluconate was replaced by Cs-MeSO3 and a holding potential of 10 mV. Spontaneous

EPSCs were recorded in the presence of GABAA receptor antagonist, picrotoxin (100 μM)

and spontaneous IPSCs were recorded in the presence of a NMDA receptor antagonist,

AP5 (100 μM) and a non-NMDA receptor antagonist, CNQX (20 μM). GRP and its

receptor antagonist RC3095 were purchased from Sigma. To examine the mIPSCs, TTX

(1 μM) was bath-applied in the perfusion solution. The sIPSCs/mIPSCs were analyzed

with the Mini Analysis Program v5.2.4 (Synaptosoft Inc., Decatur, GA). Access

resistance

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2.4 Passive membrane properties

Off-line analysis was performed using Clampfit version 9 (Axon Instruments). Resting

membrane potential (RMP) was the low-pass readout of the electrode amplifier and was

not corrected for liquid junction potential (~12 mV) after terminating the recording. The

membrane potential was measured immediately after establishing the whole-cell

configuration. Only neurons that had a resting membrane potential more negative than -

60 mV were further investigated. Conductance was determined from the linear slope

(between -60 mV to -80 mV) of the current-voltage (I-V; Vhold = -70 mV) relationships.

2.5 Active membrane properties and firing patterns

Action potentials (APs) were detected in response to suprathreshold current injections

from a holding potential around -70 mV. Depolarizing currents of 5~200 pA (400-ms

duration) were delivered in increments of 5 pA until an AP was evoked. The rheobase

was defined as the minimum current required to evoke an action potential. The AP

voltage threshold (Vthreshold) was defined as the first point on the rising phase of the spike

at which the change in voltage exceeded 50 mV/ms. The spike amplitude was quantified

as the difference between the Vthreshold and the peak voltage. The duration of the AP was

measured at the threshold voltage. The spike width was measured at 1/2 of the total spike

amplitude (measured from the Vthreshold level). The time to the peak of fast component of

the afterhyperpolarization (fAHP) was estimated as the time from the peak of the action

potential to the most negative voltage reached during the fAHP (defined as the peak of

fAHP). The amplitude of fAHP was estimated as the difference between the Vthreshold and

the peak of fAHP. If an afterdepolarization (ADP) was present, its amplitude was

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determined as a distance between the peak of fAHP to the peak of ADP. Amplitude of

slow component of the afterhyperpolarization (sAHP) was measured from the Vthreshold to

the peak of sAHP. The time to the peak of sAHP was estimated as the time from the peak

of the action potential to the peak of sAHP. The waveform characteristics of the action

potentials recorded from neurons of control and neuropathic pain mice, i.e., maximum

rise slope, maximum decay slope, rise time, rise slope, decay time and decay slope, were

determined using Clampfit 9.2 software (Axon Instruments). The properties of firing

patterns and hyperpolarizing responses were analyzed from voltage responses to injected

current pulses. Instantaneous firing frequency was calculated as the reciprocal of the

interspike interval (ISI). The spike firing frequencies were plotted against the interval

number since train onset.

2.6 Histology and immunohistochemistry

After electrophysiological recordings, slices containing biocytin-filled ACC neurons

were fixed overnight in a cold solution containing 4% paraformaldehyde. The slices were

then collected in 1% stock Tris buffered saline for 1 hour and washed twice (10 minutes

each time) in Tris buffered saline. Slices were then incubated in phosphate buffered

saline (PBS) with 0.1% Triton X-100 (JT Baker; Phillipsburg, NJ) for a period of 2 hours

to enhance the penetration of the subsequent streptavidin. The biocytin filled cells were

rendered fluorescent by incubating overnight in a Cy3-conjugated streptavidin (Jackson

ImmunoResearch Labs; West Grove, PA) solution (1 mg/ml of PBS) at 4°C. The

following day, slices were equilibrated in 1% Tris buffered saline and mounted on glass

slides.

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For dual fluorescent immunohistochemistry, mice were deeply anesthetized with

halothane and perfused transcardially with 50-100 ml saline followed by 150-500 ml of

cold 0.1 M phosphate buffer (PB) containing 4% paraformaldehyde. Brains were

removed and post-fixed in 4% paraformaldehyde/PBS and then will be placed in 30%

sucrose in 0.2 M PBS, embedded in the OCT compound and frozen. Coronal sections (30

μM thickness) were cut using a cryostat. Sections were then incubated overnight with

anti-GRPR (1:50; rabbit polycolonal, Santa Cruz Biotecnology) and anti- glutamate

decarboxylase (GAD) 67 (1:300; mouse monoclonal, Chemicon) antibodies at 4°C.

Sections were then be washed 3 times with PBS 0.1 M and incubated for 2 hours with

anti Mouse-FITC and rabbit-rhodamine conjugated secondary antibodies (1:200;

Chemicon). Images of the ACC areas of at 0.7-μm intervals with 20× lens were obtained

with Bio-Rad Laboratories MRC 1000 laser-scanning confocal fluorescent imaging

system.

2.7 Confocal microscopy

Labeled neurons were imaged by a confocal microscope (Fluoview FV 1000,

Olumpus, Tokyo, Japan). Optical sections, usually at consecutive intervals of 1-2 μm,

were imaged through the depth of the labeled neurons and saved as image stacks.

Collapsing this stack using z projection on the confocal software onto a single plane

generated a two-dimensional reconstruction of the labeled neuron. The image stack was

also reconstructed in 3-D with appropriate software, to define areas of interest in the

neuron. Although the effects of laser illumination on fixed tissue are not known, to

prevent possible ultrastructural damage we tried to minimize both the scanning time and

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the laser intensity. The horizontal extent of axons was measured as the average distance

between the three most distal axonal endings on each side from the soma of individual

pyramidal neurons. The photomicrograph was assembled by using Adobe Photoshope.

Only brightness and contrast were adjusted.

2.8 Data analysis

Results were expressed as mean ± standard error of the mean (S. E. M.). Statistical

comparisons were performed with the use of ANOVA, the Student t-test and χ2 test with

the post-hoc Scheffe F-test in immunocytochemical experiments. Analysis of

mIPSCs/sEPSCs was performed with cumulative probability plots and was compared

using the Kolmogorov-Smirnov (K-S) test for significant differences. In all cases, P <

0.05 was considered statistically significant.

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CHAPTER 3

PROPERTIES OF THE ANTERIOR

CINGULATE CORTEX (ACC)

3.1 Introduction

The anterior cingulate cortex (ACC), a highly wired cortical region around the

rostrum of the corpus callous, receives widespread inputs from cortical and subcortical

brain areas and telencephalic areas known for their role in emotional aspects of behavior

(Frankland, Bontempi et al. 2004; Maviel, Durkin et al. 2004; Zhuo 2006). Although

much information exists on synaptic transmission of ACC neurons, there is relatively

little known about the intrinc properties of cingulate neurons. Knowledge of basic

properties of neurons is important for understanding how they regulate their excitability,

integrate afferent information and formulate appropriate output firing patterns. This

chapter provides an initial description of the intrinsic passive and active properties of a

sample (interneuronsm, n=30; pyramidal cells, n=120) of neurons from layer II/III of the

ACC (Fig. 3.1A, B). We decided to focus on Layer II-III cells, because that (i) neurons in

this regions receive sensory inputs from thalamus (Yang, Shih et al. 2006; Lee, Chang et

al. 2007); (ii) our previous studies showed that synapses in Layer II-III undergo plastic

changes after LTP induction (Ko, Ao et al. 2005; Wu, Toyoda et al. 2005; Zhao, Ko et al.

2006; Zhuo 2006; Zhuo 2007) or peripheral nerve injury (Xu, Wu et al. 2008). After

stable recordings were obtained, these neurons were electrophysiologically characterized

and simultaneously injected with biocytin for histochemical processing and

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morphological analysis (Fig. 3.1C). All neurons described in this study were recorded for

at least 20 minutes and had a minimum resting membrane potential of -55mV for

interneurons and -60 mV for pyramidal neurons.

Figure 3.1 Schematic diagram demonstrating the recording of ACC neurons. A, Representative coronal section showing the placement of a whole-cell patch recording in a cingulate slice; B, Diagram representation of the location of the recorded neurons in layer II/III; C, Photomicrograph of a representative biocytin--labeled layer II/III ACC pyramidal neuron as visualized with confocal laser scanning microscopy. This figure was originally published in “Mol Pain. 2009 Dec 16; 5:73”. Biomed Central is the original publisher. 3.2 Results

Two main classes of ACC cells were distinguished, pyramidal cells and interneurons,

according to: (i) their morphology; and (ii) the properties of action potentials and their

firing pattern in response to depolarizing current pulses. Furthermore, two physiological

groups of interneurons and three groups of pyramidal cells were defined according to

their action potential firing behavior.

Furthermore, two types of layer II/III interneurons were observed according to their

firing patterns and electrophysiological properties: (i) fast spiking cells (FS) that fired a

train of spikes with high frequencies and little or no frequency adaptation once threshold

was reached; (ii) non—fast spiking cells (non-FS) that fired a train of APs with low

frequencies and moderate frequency adaptation after reaching the threshold (Fig. 3.4).

The pyramidal cells were further divided into three groups (Fig. 3.6, 3.7 and 3.8)

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according to their firing pattern: (i) RS (regular spiking) cells that fired single spikes,

with each spike followed by a prominent sAHP and an obvious deflection of fAHP and

ADP during the hyperpolarizing phase; (ii) IM (intermediate) cells that fired single spikes

and the action potential was followed by a fast AHP, ADP, and sAHP; (iii) IB (intrinsic

bursting) cells that fired an initial spike doublet, followed by single spikes.

3.2.1 Morphological properties of the ACC layer II/III pyramidal neurons and

interneurons

Pyramidal and nonpyramidal cells in layer II/III were first identified visually using

infrared illumination and video-enhanced differential interference contrast optics (Stuart,

Dodt et al. 1993). In this study, it was intended to record smaller cells for interneurons,

which was due to the difficulty of distinguishing large nonpyramidal cells from

pyramidal cells by direct visualization. The later biocytin staining clearly distinguished

pyramidal cells and interneurons with respect to soma shape and dendritic arborizations.

Pyramidal neurons were the most abundant cell type in our sample and these neurons had

a typical pyramidal soma. All pyramidal cells had a prominent apical dendrite, which

ascended toward the superficial layers, gave off some branches usually within layer I and

formed apical tufts. Their basal dendrites were mainly located within the same layer as

the soma. Axonal branches which were very far from the soma were hard to follow. In

most cases, the main axon was directed toward the deeper layers, such as V and VI.

According to this study, the dendritic structure did not clearly distinguish RS, IM and IB

cells, which was more dependent on relatively how far the cell was located from the layer

I. Despite the cell types, the cells which were closer to layer I (Fig. 3.2) had shorter apical

28

dendrites and wider extension of apical tufts than those of cells which were far from the

layer I (Fig. 3.2; table 3.1).

Somata of interneurons were usually ovoid in shape and the soma size was

smaller than that of pyramidal cells. In contrast to the pyramidal cells, interneuron was

lack of the apical dendrite. Cells displayed 2-5 primary dendrites which were mostly

smooth and extended in all directions with the layer as the soma (Fig. 3.3). Their main

axons did not go toward the white matter and showed variable branching patterns. Many

more axonal branches appeared close to the soma (Fig. 3.3). Morphologically, it was

difficult to distinguish FS neurons from non-FS neurons (see table 3.2).

29

Figure 3.2 Morphological subtypes of layer II/III pyramidal cells in the ACC A-C: Computer aided reconstruction of RS, IM and IB cells respectively. Dashed lines represent the borders of the indicated layers. Pyramidal cell located in layer II/III with its apical dendrite reaching and forming a terminal tuft in layer I.

30

Table 3.1 Summary of basic morphological parameters of ACC layer II/III pyramidal neurons. RS cells

IM cells

IB cells

Significant Difference

Numbe of neurons tested 6 11 12 Soma area (μm2) 250.1±14.6 315.7±43.3 290.0±27.1 P>0.05 Major axis (μm) 21.4±1.5 19.7±1.4 18.7±0.6 P>0.05 Minor axis (μm) 11.2±10.7 14.5±1.2 13.1±0.9 P>0.05 Primary Dendrites (n) 5.4±0.2 6.0±0.3 6.2±0.2 P>0.05 Total dendritic length (μm) 2509.3±233.9 2939.5±249.3 2851.0±148.0 P>0.05 Values are Mean ± SEM. Significant differences were obtained by One Way Analysis of Variance. RS, regular spiking; IM, intermediate; IB, intrinsic bursting.

31

Figure 3.3 Morphological subtypes of anterior cingulate cortex layer II/III interneuron cells. A-E: Computer aided reconstruction of FS (A, B), non-FS (C-E) cells respectively. Dashed lines represent the borders of the indicated layers. Interneuron located in Layer II/III characterized by a multipolar dendritic organization and lack of a prominent apical dendrite.

32

Table 3.2 Summary of basic electrophysiological and morphological parameters of ACC layer II/III interneurons. Fast spike cells Non-fast spike

cells Significant Difference

(n=5) (n=14) RMP (mV) -66±4 -59±2 P>0.05 Threshold AP (mV) -40±2 -44±1 P0.05 Spike Amplitude (mV) 77.3±4.5 92.2±2.5 P

33

(ii) non—fast spiking cells (non-FS) that fired a train of APs with low frequencies and

moderate frequency adaptation after reaching the threshold (Fig. 3.4).

3.2.2.1 Fast spiking cells (FS)

Comparing the firing patterns enabled two groups of nonpyramidal cells to be

provisionally identified: fast spiking cells (FS, n=5) and non—fast spiking cells (non-FS,

n=14). FS cells, compared with non-FS neurons, required fewer depolarizing current

steps to reach spike threshold (Figs. 3.4C and 3.4D). Once threshold was reached, FS

cells usually fired trains of spikes with high frequencies and little or no frequency

adaptation (Figs. 3.4A and 3.4C). At higher stimulation intensities, all FS cells exhibited

a continuous repetitive discharge (Fig. 3.4A), with maximal spike frequency of at least

500-- 600 Hz for hundreds of milliseconds (Fig. 3.4C). FS cells are thus able to perform a

relatively faithful conversion of input to output over a wide dynamic range; in strong

contrast to non-FS and pyramidal cells, the output of these neurons is likely to retain the

precise temporal features of their input (Figs. 3.4Aand B, 3.6F, 3.7F, and 3.8F firing

patterns).

3.2.2.2 Non—fast spiking cells (non-FS)

Non-FS cells required strong depolarizing current steps to reach spike threshold

(Figs. 3.4, Table 3.3). After reaching the threshold, non-FS cells fired a train of APs with

low frequencies and moderate frequency adaptation, which further increased with a

higher intensity of the stimulating current (Fig. 3.4, B and D). A marked adaptation of the

firing frequency is also observed in pyramidal cells (Figs.3.6F, 3.7F, 3.8F and table 3.2).

non-FS and pyramidal neurons, however, could be differentiated by the amplitude and

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duration of their action potentials. Action potentials emitted by pyramidal cells were

slower and had a smaller AHP than those of non-FS cells (Table 3.2 and 3.3).

Figure 3.4 Firing properties of the typical FS and non-FS neurons in ACC layer II/III interneurons. A,B: subsequent sweeps with spikes beginning with the response to the first suprathreshold current step recorded from the typical FS, and non-FS neurons in ACC layer II/III interneurons. C, D: plots of spike frequencies in the sweeps illustrated in A,B. After the stimulating current reached spike threshold in each of the 2 cells 4 current steps, each of 10 pA were applied. In response to the same intensities of current FS generated a large number of spikes at high frequencies with no frequency accommodation across the applied current range. FS also required the smaller current to reach threshold compared with non-FS. Non-FS generated spikes with prominent frequency accommodation.

35

We next found that the FS and non-FS cells exhibited significantly different in the

intrinsic electrical properties of the two types of cells (see table 3.2). Typically, the action

potentials of FS neurons had a large AHP and a short duration (Fig. 3.5A-F), which are

general characteristics of FS cells. Action potentials in FS neurons also had fast fAHP,

small amplitudes and low AP thresholds (Fig. 3.5G). The AHP in FS cells had a

prominent fast component and usually lacked the slow component or ADP (Fig. 3.4A).

Action potentials in non-FS cells had a smaller AHP and a longer duration at half

height than those recorded in FS cells (Fig. 3.5A-F)). Non-FS cells also generated action

potentials with slow fAHP, large amplitudes and relative high AP thresholds

(3.5G).Spike AHP of these cells had the shallow fast component and was separated from

the prominent slow component by marked ADP (Fig. 3.4B).

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Figure 3.5 Action potential properties of the typical FS and non-FS neurons in ACC layer II/III interneurons. A, B: illustration of the fAHP in FS and non-FS. C: Histogram summary data of the amplitude of the fAHP in FS and non-FS. In addition the non-FS has afterdepolarization (ADP) and slow components of afterhypolarization (AHPs) that are lacking in the FS. D, E: action potential as illustrated from threshold (arrow) in the FS (D) and non-FS (E). F: Histogram summary data of the half width of the action potential in FS and non-FS. G: Histograms for data reflect time of fAHP, action potential threshold and action potential amplitude in FS and non-FS. (*P

37

potential was followed by a fast AHP, ADP, and sAHP; (iii) IB (intrinsic bursting) cells

that fired an initial spike doublet, followed by single spikes. These cells typically had a

big ADP followed single spike evoked by low intensity of current injection.

In many excitable cells, action potentials are followed by after-potentials that

regulate the excitability of the cell for periods ranging from a few milliseconds to several

seconds (Bourque, Randle et al. 1985; Storm 1990). In the ACC neurons, we found

different patterns of afterhyperpolarization (AHPs), with or without afterdepolarization

(ADP), followed individual action potentials. Three distinct groups of pyramidal neurons

in layers II/III of the ACC were classified based on their evoked firing patterns in

response to depolarizing pulses and characteristics of the AHPs followed the action

potentials (see Figs. 3.6, 3.7 and 3.8 for examples). They have been called regular spiking

(RS), AHPs without ADP; intermediate (IM), AHPs with ADP; and intrinsic bursting

(IB) cells, AHPs with both ADP and the burst activity triggered by the ADP.

3.2.3.1 Regular Spiking (RS) neuron

Twenty ACC neurons (24.7%) were classified as RS cells. The averaged resting

potential of RS cells was -74.4 ± 2.3 mV and their averaged action potential threshold

was -33.4 ± 1.0 mV (see Table 3.3). The voltage-current relationships of these typical RS

neurons were linear at membrane potentials between -65 and -90 mV (Fig. 3.6D),

yielding a mean slope resistance of 120.1 ± 13.6 MΩ. In response to an injection of a

suprathreshold depolarizing current pulse, RS neurons typically fired single spikes

followed by a slow component and an obvious deflection of fAHP and ADP (

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7.4 ms (from 43.3 to 125.7 ms). In RS cells, a just suprathreshold depolarizing current

could only evoke single action potentials and no spike bursting complex could be evoked

even with carefully grading of injected current intensity near the threshold. Gradually

holding the cell to more positive potentials, RS cells fired more spikes in response to the

same depolarizing pulses with longer interspike interval between subsequent spikes (Fig.

3.6E). At the resting membrane potential (RMP), stronger depolarization also triggered a

train of single spikes with marked spike frequency adaptation (Fig. 3.6F).

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Figure 3.6 Characteristics of the typical RS neurons in the ACC. A, Schematic representation of the location of the recorded neurons (a and b); B, Biocytin profiles of two representative ACC RS pyramidal neurons as visualized with confocal laser scanning microscopy. Scale bar = 50 μm. C, Superimposed current-clamp traces in response to a series of intracellular current pulses (1000 ms, 80 pA per step). The neurons exhibited an AHP with a slow component. Note that an obvious deflection was observed during the hyperpolarizing phase; D, I-V plots constructed from the values of traces shown in (C) displayed linearity in the membrane voltage range between -65 and -90 mV; E, Traces evoked by the same current injections (400 ms, 100 pA) as the neurons was depolarized from -85 to -50 mV. The more the cells were depolarized, the more the action potentials were elicited by the same depolarizing pulse. Each spike was followed by a sAHP that lasted for tens of milliseconds. Note that the black traces were for cell (a) and gray traces for cell (b); F, Action potential trains were evoked by 400 ms current injection of 160, 180 and 200 pA from the holding potential of -70 mV. Note the increase in the frequency of action potential discharge with increasing current injections. Note that the black traces were for cell (a) and gray traces for cell (b). This figure was originally published in “Mol Pain. 2009 Dec 16; 5:73”. Biomed Central is the original publisher.

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3.2.3.2 Intrinsic bursting (IB) neuron

Twenty five ACC neurons (30.9%) were classified as IB cells. The passive

membrane properties of bursting cells were similar to those of the RS and IM cells (Table

3.3). IB neurons had a mean RMP of -74.7 ± 1.1 mV, a mean action potential threshold of

-36.0 ± 0.7 mV and a linear voltage-current relationship between -60 and -85 mV with a

mean slope resistance of 129.5 ± 8.0 MΩ (Table 3.3). When activated from the resting

membrane potential by an intracellular current pulse, the action potential was followed by

an AHP with a big ADP (n = 14, 3.4 ± 0.4 mV), which can facilitate an action potential

and therefore, form a burst response (Fig. 3.7C, F). The ability of IB cells to generate

bursting is the primary physiological properties that distinguish bursting cells from the

two other types. The character of the burst was not always all or none. Some cells (n =

14), in response to a just suprathreshold current injection, displayed single spikes with

prominent ADP. However, a burst can always be evoked by slightly increasing the

current intensity. When the membrane potential was gradually depolarized, pulses of

current could only elicit regular spaced spike (Fig. 3.7E), suggesting that, in IB cells, the

generation of burst during a stimulus was dependent upon the previous voltage of the

membrane. At the constant membrane potential of -70 mV, lower current intensity

elicited initial doublet of action potentials, while higher current evoked an initial burst

followed by a train of current potential (Fig. 3.7F).

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Figure 3.7 Electrophysiological characteristics of typical IB ACC neurons. A, Schematic representation of the location of the recorded neurons (a and b); B, Biocytin profiles of two representative IB ACC pyramidal neurons as visualized with confocal laser scanning microscopy. Scale bar = 50 μm; C, Superimposed current-clamp traces in response to a series of intracellular current pulses (1000 ms, 80 pA per step). The neurons exhibited an AHP with a big afterdepolarization (ADP), which could facilitate an action potential and therefore, formed a burst response; D, I-V plots constructed from the values of traces shown in (C) displayed a linear response in the voltage range between -60 and -85 mV; E, Traces evoked by the same current injections (400 ms, 100 pA) as the neurons were depolarized from -85 to -50 mV. When held at a hyperpolarized membrane potential, the burst response was evoked in response to the depolarizing current injections. By contrast, with gradual depolarization (Vm ≥ -65 mV) (left to right), a train of single spikes was evoked in response to the same current pulse and the burst was inactivated. The dotted line denotes the resting membrane potential. Note that the black traces were for cell (a) and gray traces for cell (b); F, Action potential trains were evoked by 400 ms current injection of 60, 80 and 100 pA from the holding potential of -70 mV. Note that a lower current intensity evoked an initial doublet of action potentials while higher current evoked an initial doublet of action potentials followed by a regular discharge. Note that the black traces were for cell (a) and gray traces for cell (b). This figure was originally published in “Mol Pain. 2009 Dec 16; 5:73”. Biomed Central is the original publisher.

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3.2.3.3 Intermediate (IM) neuron

By far the most frequently recorded type of neurons in layers II/III of the ACC (n

= 36, 44.4%) was classified as IM. The reason why these cells are called intermediate is

that their electrophysiological characteristics were intermediate between RS and IB cells

(Fig. 3.8). For the intrinsic membrane properties of IM cells, there was no significant

difference from that of typical RS cells (Table 3.3). The averaged RMP of IM cells was -

70.8 ± 1.8 mV. The mean input resistance was 122.1 ± 12.1 MΩ and the mean action

potential threshold was -35.2 ± 0.6 mV. In response to a just suprathreshold current, the

action potential of these neurons was followed by a fAHP which was subsequently

followed by a small ADP (Fig. (Fig.3.8E; n = 36, 1.5 ± 0.2 mV) and a subsequent sAHP

(Fig. (Fig. 3.8C), a key feature that distinguish the IM cells from RS cells. The presence

of ADP is considered to indicate a tendency for a cell to fire in bursts. However, IM cells

did not generate spike bursts when RMP was manipulated over a wide range (from -85

mV to -55 mV) with tonic current injection. At more depolarized potentials (> -60 mV),

the fAHP and the ADP diminished and the spike trains showed a progressive adaptation

in frequency, which was indistinguishable from RS cells (Fig. (Fig. 3.8E). At the RMP, in

response to different intensities of current pulses, cells fired trains of spikes with the first

spike followed by the postspike fAHP, ADP, and sAHP (Fig. (Fig. 3.8F).

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Figure 3.8 Electrophysiological characteristics of the IM cells. A, Schematic representation of the location of the recorded neurons (a and b); B, Biocytin profiles of those two representative IM ACC pyramidal neurons as visualized with confocal laser scanning microscopy. Scale bar = 50 μm. C, Superimposed current-clamp traces in response to a series of intracellular current pulses (1000 ms, 80 pA per step). The neurons exhibited an AHP with an early fast and a delayed slow component. Fast and slow components were intercalated by an afterdepolarization (ADP). D, I-V plots constructed from the values of traces shown in (C) showed a linear response in the voltage range between -65 and -85 mV. E, Traces evoked by the same current injections (400 ms, 100 pA) as the neurons were depolarized from -85 to -50 mV. Note that on no occasion did the IM neurons fire in bursts. The black traces were for cell (a) and gray traces for cell (b); F, Action potential trains were evoked by 400 ms current injection of 120, 140, 160 and 180 pA from the holding potential of -70 mV. Note that only the first spike was followed by an ADP. Traces reflect spike frequency adaptation in evoked action potential trains. The black traces were for cell (a) and gray traces for cell (b). This figure was originally published in “Mol Pain. 2009 Dec 16; 5:73”. Biomed Central is the original publisher.

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3.2.3.4 Differential electrophysiological properties among RS, IM and IB pyramidal

cells

Although RS, IM and IB pyramidal cells showed similarities in many prospects

of electrophysiological properties, such as resting membrane potential, membrane

resistance, current threshold and action potential amplitude, these neurons exhibited

significant differences in some of the intrinsic properties. In general, those cells had the

lowest membrane excitability for RS cells and the highest membrane excitability for IB

cells and the properties for IM cells were in between. To evoke action potential, RS cells

needed significantly higher mean intensity of current injection (112.3 ± 8.5 pA) as

compared with IB cells (75.8 ± 3.7 pA; P < 0.01, one way ANOVA) and a trend toward a

lower mean intensity of current injection than IM cells (97.4 ± 7.6 pA; P = 0.08, one way

ANOVA; Fig. 3.9F). In response to the same intensities of current pulses, RS cells

generated the smallest number of spikes with the lowest frequency in both initial and

steady state spike firing (Fig. 3.9A, D) as compared with IM (Fig. 3.9B, D) and IB (Fig.

3.9C, D) cells. IB cells exhibit the larger ADP (Fig. 3.9E; P < 0.001, one way ANOVA)

as compared with IM cells and RS cells, which facilitated IB cells in generation of an

initial doublet of action potentials with high initial frequency (Fig. 3.9C, D). At the same

current injection, IB cells also showed the highest frequency in steady state spike firing

and generated highest number of spikes among those three groups of cells (Fig. 3.9A-D).

For properties of single action potential, the half width of the action potential of RS

cells (Fig. 3.9G, 1.66 ± 0.07 ms) was significantly wider than that in IB neurons (Fig.

3.9G, 1.43 ± 0.04 ms, P < 0.05, one way ANOVA) and in IM cells (Fig. 3.9G, 1.46 ±

0.05 ms, P < 0.01, one way ANOVA). IB cells (see Table 3.3) also showed faster kinetics

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Figure 3.9 Differential intrinsic membrane properties between RS, IM and IB pyramidal cells. A-C, Plots of spike discharge frequencies for each interspike interval during the current injection illustrated in Figures 2F, 3F, 4F for RS, IM and IB cells. After the stimulating current reached spike threshold in each of the 3 representative cells, 3-4 current steps, each of 40 pA were applied. Note that at just above the rheobase, IB cell fired an initial burst with two APs at up to 50 Hz, whereas RS cell and IM cells fired APs regularly at ~5 Hz; D, Plots of the mean spike discharge frequencies, measured from the 1st interspike interval (left) and the last interspike interval (right) for RS (n = 20), IM (n = 36) and IB (n = 25) cells, at different stimulation intensities of depolarizing current (100--350 pA, 400 ms). In response to the same intensities of current RS generated spikes at the lowest frequency and degree of initial frequency moderately increased with increase in current intensity. IB generated an initial doublet of action potentials with highest frequency, which increased with increase in current intensity. The initial frequency of the spikes for IM was in between. Values are given as the mean ± S.E.M. *P < 0.05, significantly different from RS cells; **P < 0.01, significantly different from RS cells; ***P < 0.001, significantly different from RS cells; #P < 0.05, significantly different from IM cells; ###P < 0.001, significantly different from IM cells; two-way ANOVA. E, Histogram showing the distribution of the amplitude of the ADP. Black bars indicate the ADP of IB neurons (n = 14). Gray and white bars indicate the ADP of IM (n = 36) and RS (n = 20) neurons respectively. F, Histogram reflects the rheobase for RS (n = 20), IM (n = 36) and IB (n = 25) cells (**P < 0.01 based on one way ANOVA). Values are given as the mean ± SEM. (G) Histogram summary data of the half widths of the RS (n = 20), IM (n = 36) and IB (n = 25) cells (*P < 0.05 and **P < 0.01 based on one way ANOVA). This figure was originally published in “Mol Pain. 2009 Dec 16; 5:73”. Biomed Central is the original publisher.

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with decay time (1.53 ± 0.08 ms) shorter than either RS (2.09 ± 1.12 ms, P < 0.001, one way ANOVA) cells or IM cells (1.63 ± 0.06 ms, P < 0.01, one way ANOVA). 3.2.4 Intrinsic properties of ACC pyramidal neurons after neuropathic pain

To investigate whether or not the intrinsic properties of ACC pyramidal neurons

are altered after nerve injury, we studied the firing patterns and action potentials in mice

after undergoing neuropathic pain. Animals with the sham surgery were used as the

control. Recordings were performed between 7-14 days after the nerve injury, a time

when the maximal behavioral sensitization can be observed (Vadakkan, Jia et al. 2005).

Before electrophysiological experiments, behavioral allodynic responses were always

evaluated. First, we found that there was no significant change the in proportion of cell

types in the ACC of neuropathic pain mice as compared with control mice. Similar

percentages were found among IM cells (neuropathic: 47.5% V.S. control: 44.4%), IB

cells (neuropathic: 31.2% V.S. control: 30.9%) and RS cells (neuropathic: 21.3% V.S.

control: 24.7%).

Next, we compared the passive membrane and the single action potential

properties. Figure 3.10A-C show representative recordings of APs elicited in RS, IM, and

IB neurons by a 400 ms depolarizing current. There were no significant differences in

parameters tested, such as RMP, membrane resistance, current threshold, action potential

amplitude, half width, rise time and decay time (Fig. 3.10D-E, see Table 3.3). Finally, we

compared the firing pattern of these three types of pyramidal cells in neuropathic and

control animals (Fig. 3.11). Membrane excitability of RS and IB neurons from

neuropathic or control animals was indistinguishable (Fig. 3.11B, D, F). For IM cells,

however, with the same current injection, neuropathic pain cells (n = 29 cells) showed

higher initial frequency compared with control cells (n = 36, p < 0.05, two-way ANOVA;

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Fig. 3.11A). No difference was found for the steady state frequency of IM cells between

neuropathic and control mice (Fig. 3.11C). Moreover, neuropathic pain cells (n = 29)

produced more spikes for the same current injection compared with control cells (n = 36,

p < 0.05, two-way ANOVA; Fig. 3.11E).

Figure 3.10 Changes of action potential properties of IM pyramidal neurons in the ACC after neuropathic pain. A-C, Examples of APs generated from RS, IM, and IB neurons. All responses elicited in response to a 400 ms depolarizing current pulse of appropriate suprathreshold magnitude. Current records omitted for clarity. A, APs of neurons from non-operated mice; B, APs of neurons from sham-operated mice; C, APs of neurons from neuropathic pain mice; D-F, Histograms for data from RS (control, n = 20; neuropathic, n = 13), IM (control, n = 36; neuropathic, n = 29), and IB (control, n = 25; neuropathic, n = 19) neurons reflect rheobase, AP threshold and half width from control mice (non-operated and sham-operated) and neuropathic pain mice (P > 0.05, Student t-test). All values are given as the mean ± SEM. This figure was originally published in “Mol Pain. 2009 Dec 16; 5:73”. Biomed Central is the original publisher.

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Figure 3.11 Summary of alterations seen in firing patterns in ACC pyramidal neurons after neuropathic pain. A-B, Plot of the initial discharge frequency measured from the 1st interspike interval at different stimulation intensities of depolarizing current (100--350 pA, 400 ms). C-D, Plot of the steady-state discharge frequency measured from the last interspike interval at different stimulation intensities of depolarizing current (100--350 pA, 400 ms). E-F, Plot of the number of APs at different stimulation intensities of depolarizing current (100--350 pA, 400 ms); (A), (C) and (E) show relationships for IM neurons; (B), (D) and (F) for RS and IB neurons. Note that in IM cells, the initial discharge frequencies (*P < 0.05, F1,389 = 23.34, two-way ANOVA) and the number of APs (*P < 0.05, F1,389 = 13.47, two-way ANOVA) attained in neuropathic pain cells are greater than those seen in controls. Error bars represent S.E.M. Control data was collected from 20 RS, 36 IM and 25 IB neurons. Neuropathic pain data was collected from 13 RS, 29 IM and19 IB cells, respectively. This figure was originally published in “Mol Pain. 2009 Dec 16; 5:73”. Biomed Central is the original publisher.

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Table 3.3 Summary of basic electrophysiological parameters of ACC layer II/III pyramidal neurons in control and neuropathic pain mice. This table was originally published in “Mol Pain. 2009 Dec 16; 5:73”. Biomed Central is the original publis