Download STUDY OF THE NEUROMODULATORY EFFECTS OF DOPAMINE

STUDY OF THE NEUROMODULATORY EFFECTS OF DOPAMINE
IN THE OVAL BED NUCLEUS OF THE STRIA TERMINALIS IN
DRUG NAÏVE AND COCAINE DEPENDENT LONG-EVANS RATS
by
Michal Krawczyk
A thesis submitted to the Center of Neuroscience
in conformity with the requirements for
the degree of Master of Science
Queen’s University
Kingston, Ontario, Canada
(May, 2011)
Copyright © Michal Krawczyk, 2011
Abstract
The bed nucleus of the stria terminalis (BST), especially its oval (ov) subregion, receives
a robust dopaminergic input from the periaqueducal, retrorubral, and ventral tegmental midbrain
areas (Hasue & Shammah-Lagnado, 2002). Given the critical role of dopamine in motivated
behaviors, we combined behavioral testing of operant responding towards natural and
pharmacological rewards, and brain slices patch-clamp electrophysiology to identify specific
alterations in dopaminergic regulation of inhibitory synaptic transmission within the ovBST as a
result of chronic cocaine self-administration. In drug naïve rats we observed that DA dosedependently decreased GABAA-inhibitory post-synaptic currents (IPSC) through the actions of
pre-synaptic D2 receptors. However in rats maintaining cocaine self-administration, DA (1!M)
increased the amplitude of GABAA-IPSC and this increase resulted from a loss of functional presynaptic D2 receptors and the de novo addition of D1 receptors. Furthermore, direct activation of
D1 receptors only in rats maintaining cocaine self-administration resulted in a sustained increase
of GABAA-IPSC (LTPGABA). The D1-induced LTPGABA was blocked by intracellular application
of a Src-tyrosine kinase antagonist in the recording pipette. Based on this observation we
concluded that the D1 receptor was located post-synaptically. However, the measured LTPGABA
was associated with modifications in the coefficient of variation and paired pulse ratio of evoked
GABAA IPSCs, suggesting that it is maintained by persistently increased GABA release from
pre-synaptic terminals. Therefore to explain this apparent contradiction we propose the existence
of a currently unknown retrograde messenger that is released from the post-synaptic neuron, upon
D1 receptor activation, and travels backward to increase GABA release from presynaptic
terminals. Moreover, application of a D2 agonist blocked the D1-induced LTPGABA, and coapplication of a G-protein antagonist in the intracellular pipette prevented this D2 mediated
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inhibition, suggesting that due to maintenance of cocaine self-administration there was an
emergence of a new post-synaptic D2 component whose functional role seems to inhibit the D1
receptor increase of GABAA-IPSCs. Importantly, modulation of synaptic transmission by
dopamine was identical to drug-naïve conditions when intravenous cocaine administration was
not contingent upon operant responding (yoked), before the maintenance phase of cocaine selfadministration (acquisition), or in rats maintaining operant responding for sucrose under the same
reinforcement schedule. All together we identified robust alterations in the effects of dopamine on
inhibitory synaptic transmission following voluntary cocaine self-administration, these results
represent to our knowledge, the first evidence of a change in dopaminergic regulation of synaptic
transmission specific to voluntary drug intake.
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Co-Authorship
This thesis is based on research conducted by Michal Krawczyk under the supervision of Dr. Eric
Dumont. Robyn Sharma and Xenos Mason, who were both undergraduates in the lab, aided in
research collection. Their specific contributions are specified below:
Figure 4: Robyn contributed 50% to this figure !
Figure 5: Robyn contributed 50% to this figure !
Figure 6: Robyn contributed 50% to this figure !
Figure 7: Robyn contributed 50% to this figure !
Figure 8: Robyn contributed 50% to this figure !
Figure 9: Robyn contributed 25% to this figure
Figure 10: Robyn contributed 50% to this figure
Figure 11: Robyn contributed 50% to this figure
Figure 12: Robyn contributed 50% to this figure!
Figure 13: Robyn contributed 50% to this figure!
Figure 14: Robyn contributed 50% to this figure
Figure 15: Robyn contributed 25% and Xenos contributed 25% to this figure!
Figure 16. Robyn contributed 25% and Xenos contributed 25% to this figure!
!
Figure 17: Robyn contributed 25% and Xenos contributed 25% to this figure!
!
Figure 18: Robyn contributed 25% and Xenos contributed 25% to this figure
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Acknowledgements
To: Eric: Words cannot fully express my gratitude. Through the course of my Masters, you have
not only been an excellent supervisor to me, through your patients, unwavering support, and
dedications to my project. But you have also been my mentor, cultivating in me a genuine passion
for the scientific pursuit of knowledge. I am sincerely thankful for everything.
To: Robyn: Thank you for all the long hours we worked together, all your hard work and input
with regards to the project. But most of all thank you for our friendship.
To: Xenos: Thank you for your help and your friendship.
To: Cindy, Dasha, Rob, Julian, and Andrea for training the rats and making this lab fun to be
in.
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Table of Contents
Abstract ............................................................................................................................................ ii
Co-Authorship................................................................................................................................. iv
Acknowledgements.......................................................................................................................... v
Table of Contents............................................................................................................................ vi
List of Figures ............................................................................................................................... viii
List of Abberviations ...................................................................................................................... ix
Chapter 1 Introduction and Literature Review ................................................................................ 1!
1.1 Addiction................................................................................................................................ 1!
1.2 Brain Circuits of Reward & Addiction .................................................................................. 1!
1.3 Bed Nucleus of the Stria Terminalis (BST)........................................................................... 3!
1.4 BST & Addiction ................................................................................................................... 4!
1.5 Function of the Dopaminergic Pathway ................................................................................ 6!
1.6 Dopamine & the Neurobiology of Addiction ........................................................................ 7!
1.7 Dopamine Receptor Signaling ............................................................................................... 8!
1.8 GABAA Receptors ............................................................................................................... 10!
1.9 Dopamine Modulation of GABAA Channels....................................................................... 11!
1.10 Dopamine Receptors & Addiction..................................................................................... 13!
Rationale ........................................................................................................................................ 14!
Hypothesis...................................................................................................................................... 14!
Specific Aims................................................................................................................................. 14!
Chapter 2 Methods......................................................................................................................... 16!
Chapter 3 Results ........................................................................................................................... 20!
1.1 Passive Properties of ovBST Neurons in Naïve and Cocaine Rats ..................................... 20!
1.2 In Drug-Naïve, Yoked, and Acquisition Rats DA Decreased Inhibitory Transmission in the
ovBST by Activating Pre-Synaptic D2 Receptors..................................................................... 20!
1.3 Only in Rats that Self-Administered Cocaine Under a PR Schedule of Reinforcement was
the DA-Induced Modulation of Inhibitory Transmission Altered ............................................. 22!
1.4 Cocaine Self-Administration Revealed a Loss of Pre-Synaptic D2-like Receptors and a De
Novo Post-Synaptic D1-like and D2-like components. ............................................................. 23!
1.5 D1-Mediated Signal Transduction Pathway involved Src-Tyrosine Kinases in a G-proteinindependent way while the D2-Mediated Signal was G-protein dependent.............................. 24!
Chapter 4 Discussion ..................................................................................................................... 46!
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1.5 D1-Mediated Signal Transduction Pathway involved Src-Tyrosine Kinases in a G-proteinindependent way while the D2-Mediated Signal was G-protein dependent. ............................. 24!
Chapter 4 Discussion...................................................................................................................... 46!
Chapter 5 Future Directions ........................................................................................................... 52!
References ...................................................................................................................................... 54!
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List of Figures
Figure 1: Anatomical localization of the ovBST .......................................................................... 26!
Figure 2: Dopaminergic inputs in the dorsolateral BST ............................................................... 27!
Figure 3: Immunohistochemical localization of D1R and D2R in the ovBST ............................. 28!
Figure 4: Passive Properties of ovBST neurons in Naïve and Cocaine Rats ................................ 29!
Figure 5: Effects of DA on GABAA-IPSC in drug naïve rats....................................................... 30!
Figure 6: The synaptic location of the effect of DA on GABAA-IPSC in drug naïve rats ........... 31!
Figure 7: Effects of D2R and D1R agonists on GABAA-IPSC in drug naïve rats........................ 32!
Figure 8: The synaptic location of the effect of a D2 agonist on GABAA-IPSC in drug naïve rats
........................................................................................................................................................ 33!
Figure 9: Pharmacological characterization of the effects of DA on the amplitude of evoked
GABAA-IPSC in the ovBST of drug naïve rats ............................................................................. 34!
Figure 10: Effects of DA on GABAA-IPSC in all experimental groups....................................... 35!
Figure 11: The synaptic location of the effect of DA on GABAA-IPSC in all experimental groups
........................................................................................................................................................ 36!
Figure 12: Effects of D2R agonists on GABAA-IPSC in all experimental groups ....................... 37!
Figure 13: The synaptic location of the effect of a D2 agonist on GABAA-IPSC in all
experimental groups ....................................................................................................................... 38!
Figure 14: D2R activation during the overstimulation of the adenylyl cyclase pathway ............. 39!
Figure 15: Contribution of D1R in the effect of DA on GABAA-IPSC in all experimental groups
........................................................................................................................................................ 40!
Figure 16. Characterization of the contribution of D1 and D2 receptors in regulating LTPGABA . 41!
!
Figure 17: Pharmacological characterization of LTPGABA ........................................................... 43!
!
Figure 18: Synaptic location of the D1 induced LTPGABA ............................................................ 45!
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List of Abbreviations
5-HT
serotonin
AC
adenylate cyclase
BST
bed nucleus of the stria terminalis
cAMP
cyclic AMP
CNS
central nervous system
CRF
corticotropin-releasing factor
CREB
cAMP response element binding
CV
coefficient of variation
D1
dopamine D1-like receptor
D2
dopamine D2-like receptor
D3
dopamine D3 receptor
DA
dopamine
DAG
diacylglycerol
DARPP -32
dopamine and cAMP-regulated phosphoprotein
of 32 kDa
DSM
diagnostic and statistical manual of mental
disorders
ERK
extracellular signal regulated kinase pathway
GABA
gamma-aminobutyric acid
GRKs
G protein receptor kinases
IP3
inositol trisphosphate
IPSCs
inhibitory post-synaptic potentials
LA
lateral amygdala
LTP
long-term potentiation
MDMA
3,4-methylenedioxymethamphetamine
mGluRs
metabotropic glutamate receptors
NA
noradrenaline
NAc
nucleus accumbens
NETs
noradrenaline transporters
NF-M
neurofilament-M
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NO
nitric oxide
NOS
nitric oxide synthase
ovBST
oval bed nucleus of the stria terminalis
PiP2
phosphatidylinositol 4,5-bisphosphate
PKA
protein kinase A
PKC
protein kinase C
PLC
phospholipase C
PP1
protein-phosphatase-1
PPR
paired-pulse ratio
PSD95
postsynaptic density 95
sIPSC
spontaneous inhibitory post-synaptic potentials
TKs
tyrosine kinases
VTA
ventral tegmental area
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Chapter 1 Introduction and Literature Review
1.1 Addiction
According to the Canadian Center on Substance Abuse, in Canada the annual costs
associated with drug addiction due to loss of life and productivity was estimated to be forty
billion dollars (Rehm et al., 2006). The health problems alone attributed to drug abuse include an
increased risk of mental health disorders, lung cancer, and HIV (Single et al., 1998).
According to the diagnostic and statistical manual of mental disorders (DSM), addiction
is defined as a chronic relapsing disorder that is characterized by persistent drug-seeking and
drug-taking behaviors (American Psychiatric Association, 2000).
Addiction is a complex disease involving many types of social and psychological factors.
However, fundamentally it is a biological process: the effect of a chemical (drug of abuse) on a
biological substrate (a brain) (Koob, 2006). Therefore, it is a goal of this laboratory and many
other research laboratories studying addiction to identify physiological and molecular
mechanisms that contribute to addiction, which may lead to new treatments of this disease.
1.2
Brain Circuits of Reward & Addiction
!
Over two decades ago Olds and colleagues demonstrated that rodents would work to
electrically stimulate relatively discrete areas of the brain, which demonstrates the existence of
so-called, brain-reward regions (Olds and Milner, 1954). Subsequently, other groups found that
rodents also work to self-administer drugs of abuse and that this self-administration behavior is
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disrupted by lesions of these brain-reward regions (Wise, 1998).
The critical regions in the reward circuitry have now been elucidated, one important region
involves dopamine rich neurons originating in the midbrain, a region of the brain called the
ventral tegmental area (VTA). These dopamine neurons extend fibers that connect to various
limbic regions that include the nucleus accumbens (NAc), hippocampus, amygdala, and the bed
nucleus of the stria terminalis (BST), thereby forming a circuit known as the mesolimbic pathway
(Dahlstrom and Fuxe, 1965; Fallon and Moore, 1978; Simon et al., 1979; Ungerstedt, 1971).
The NAc, also known as the ventral striatum, is one of the most important substrates for the
acute rewarding effects of natural and pharmacological rewards. In fact, research over the past
several decades have delineated that all drugs of abuse increase dopamine-mediated transmission
in the NAc, and some drugs also act directly on NAc neurons by dopamine-independent
mechanisms (Di Chiara et al., 1999; Di Chiara et al., 2004). For instance, cocaine is selfadministered directly into the shell of the NAc of the rat (Rodd-Henricks et al., 2002), and
injection of amphetamine into the NAc can induce reinstatement of drug-seeking behavior
(Stewart and Vezina, 1988).
Several additional brain areas interacting with the VTA and NAc are also essential for the
reinforcing effects of drugs of abuse. These regions include the hippocampus, amygdala, and
related structures of the so-called ‘extended amydala’ (Heyser et al., 1999; Hyytia and Koob,
1995; Meil and See, 1997; Penton et al., 2011, Roberts et al., 1996, Thompson et al., 2002;
Thompson et al., 2005; Vorel et al., 2001). The hippocampus and amygdala are critical regions
for the establishment of reward-associated memories (Lee et al., 2005; Milton et al., 2008;
Wittmann et al., 2005).
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Due to similar morphology, immunoreactivity, and connectivity, the BST, the medial part
of the NAc, and the central nucleus of the amygdala are often grouped together as the ‘extended
amydala’ (De Olmos and Heimer, 1999). Evidence for the involvement of the extended amygdala
in addiction has come from animal studies, where animals that prefer morphine-paired
environments show increased neuronal activation in the extended amygdala, as measured through
protein Fos expression (Harris and Aston-Jones, 2003; Heyser et al., 1999; Hyytia and Koob,
1995; Meil and See, 1997; Roberts et al., 1996). Furthermore, GABAergic transmission within
the extended amygdala is altered during the course of dependence to ethanol. Microinjection of
previously ineffective doses of GABA agonists into the central nucleus of the amygdala can
attenuate lever pressing for ethanol in animals that are depended on the drug (Roberts et al.,
1996). This suggests that the GABAergic system has been altered to become more responsive to
agonists during the course of dependence. The location of the BST within the ‘extended
amygdala’ suggests an important role in drug addiction.
1.3
Bed Nucleus of the Stria Terminalis (BST)
The Bed Nucleus of the Stria Terminalis consists of approximately twelve nuclei that have
a wide variety of physiological functions, from the coordination of neuroendocrine, autonomic,
and somatomoter responses to the initiation of reward, stress, and anxiety responses (Deyama et
al., 2007; Dong and Swanson, 2003; Dong and Swanson, 2004; Dong and Swanson, 2006; Erb
and Stewart, 1999; Eiler et al., 2003; Epping-Jordan et al., 1998; Gewirtz et al., 1998; Sajdyk et
al., 2008; Sullivan et al., 2004).
The present study focused on the oval nuclei of the BST (BSTov), which lies dorsal to the
anterior commissure and medial to the internal capsule, and the majority of these neurons stain
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positive for glutamic acid decarboxylase (GAD), the enzyme responsible for the conversion of
glutamate to GABA, suggesting that the neurons are primarily GABAergic (Figure 1) (Dong et
al., 2001; Ju and Swanson, 1989; Larriva-Sahd J, 2006). The BSTov projects to other nuclei
within the BST, and outside the BST, it receives dense reciprocal connections with the central
and basolateral nuclei of the amygdala (Dong et al., 2001; Larriva-Sahd J, 2006).
There is also a robust dopaminergic input to the ovBST, primarily originating from the
VTA, but also coming from the periaqueducal gray and the retrorubral field (Figure 2 & Figure 3)
(Freedman and Cassell, 1994; Hasue & Shammah-Lagnado, 2002; Meloni et al., 2006; Phelix et
al., 1992). This dense dopaminergic input makes the ovBST an attractive substrate to investigate
the effects of dopamine on inhibitory synaptic transmission.
1.4
BST & Addiction
Consistent with the anatomical interconnections mentioned above, emerging data is
demonstrating the importance of the BST in mediating the reinforcing effects of drugs of abuse
(Eiler et al., 2003; Epping-Jordan et al., 1998; Hyytia and Koob, 1995; Walker et al., 2000).
Chronic administration of certain drugs has been shown to modulate noradrenaline transporters
(NETs) and metabotropic glutamate receptors (mGluRs), altering glutamatergic transmission
within the BST (Grueter et al., 2006; Macey et al., 2003). Additional work in the BST
demonstrates that excitatory synaptic transmission is enhanced following cocaine selfadministration and to a lesser degree in rats that self-administer natural reward (sucrose).
Interestingly, this enhancement was not seen when cocaine or sucrose was delivered passively,
suggesting that the cocaine-related changes were not simply due to the pharmacological effects of
cocaine but instead could be due to an associative process acquired during self-administration
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(Dumont et al., 2005). This study highlights the need for proper experimental controls since
contingent, voluntary drug intake and noncontingent drug exposure induced differential changes.
Therefore, in our study we utilized a range of experimental controls that included a control group
where intravenous cocaine administration was not contingent upon operant responding (yoked).
There is accumulating evidence to suggest that the BST plays a key role in mediating
stress-induced relapse to cocaine and heroin seeking-behavior (Shaham et al., 2000), as well as in
stress-induced maintenance and reinstatement of morphine conditioned place preference (Wang et
al., 2001). Relapse induced by stress involves actions of corticotropin-releasing factor (CRF) and
noradrenaline in the brain (Erb et al., 2001; Wang et al., 2001). The BST in particular is a region
in the brain that contains both, numerous immunoreactive cells for CRF, and is densely
innervated by noradrenergic fibers (Delfs et al., 2000; Moga et al., 1989). In fact, it has been
showed that blocking CRF or NA activity in the BST specifically blocked stress-induced
reinstatement to drug seeking (Erb et al., 2001; Leri et al., 2002; Wang et al., 2001)
In addition the BST contributes in the aversive impact of drug withdrawal. Delfs and
colleagues evaluated the role of noradrenergic innervation of the BST during opiate withdrawal in
rats. Withdrawal was associated with pronounced activation of BST neurons as judged by c-fos
staining, and this activation was markedly reduced by systemic injections of the noradrenergic
antagonists. Furthermore, lesions of noradrenergic inputs directly into the BST decreased opiate
withdrawal behavior (Delfs et al., 2000).
As commonly seen in other brain regions that receive dopaminergic innervation, drugs of
abuse increase dialysate DA levels in the BST. It is notable that the magnitude of the effect and
the sensitivity to the drug is higher in this area as compared to the NAc (Di Chiara et al., 1999).
The background presented here, particularly that the BST is involved in drug addiction and that
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drugs of abuse increase dialysate DA levels in the BST, makes the BST and attractive substrate
for measuring how drug-related behaviors alter dopaminergic modulation of synaptic
transmission.
1.5
Function of the Dopaminergic Pathway
The neuromodulator dopamine was initially thought to function solely in the hedonic
(pleasure) perception of rewards, but this has been shown not to be the case, as animals can still
exhibit positive hedonic responses in the absence of dopamine. These dopamine deficient
animals, however, cannot use information about rewards to motivate goal-directed behaviors
(Berridge & Robinson, 1998). Further evidence for the role of dopamine originating in the VTA
came from, Shultz and colleagues, who directly recorded the activity of individual dopamine
neurons in animals performing Pavlovian conditioning behavioral tasks (Schultz, 1998). DA
neurons within these regions will respond to the presentation of unpredicted rewards with
precisely timed phasic bursts of activity. However, after training, dopaminergic neurons will
similarly respond to conditioned stimuli, and no longer to the predicted reward itself (Fiorillo et
al., 2003; Schultz, 1998; Tobler et al., 2005; Waelti et al., 2001). Overall these results suggest a
more complicated role for dopamine then simply encoding an internal representation of reward.
Currently, there are a number of theories that try to account for the role of dopamine in the
VTA, from dopamine mediating some aspect of reward-related learning to mediating the
incentive salience of reward, many of these theories are not necessarily mutually exclusive
(Berridge & Robinson, 1998; Robinson & Berridge, 2000; Berridge et al., 2009). The incentive
salience hypothesis suggests that the process of reward can be dissociated into separate
components of ‘wanting’ and ‘liking’, and that these two psychological processes are mediated by
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different neural systems (Olmstead et al., 2000). It suggests that dopamine mediates the ‘wanting’
but not the ‘liking’ component of rewards. This theory accounts for the fact that animals can still
like something in the absence of dopamine transmission, however, animals cannot use this
information to motivate the behaviors necessary to obtain it. In this view, dopamine is released
unto hedonic centers of the brain, where it integrates the pleasure of a specific object or behavior
with its ‘wanting’, and thus motivates the animal to pursue the rewarded object or behavior
(Berridge & Robinson, 1998; Robinson & Berridge, 2000; Berridge et al., 2009).
1.6
Dopamine & the Neurobiology of Addiction
!
Investigations using diverse methods have converged on the conclusion that addictive
drugs influence behavior as a result of their ability to increase synaptic dopamine in the nucleus
accumbens (NAc), bed nucleus of the stria terminalis (BST), and other various limbic regions
associated with the mesolimbic dopaminergic pathway. (Di Chiara et al., 1999; Chiara et al.,
2004).
It is now well known that, weather acting directly or indirectly, all addictive drugs increase
levels of synaptic dopamine within these regions (Di Chiara et al., 1999; Chiara et al., 2004). For
instance, drugs such as cocaine, amphetamine, methamphetamine and MDMA increase dopamine
directly by inhibiting dopamine reuptake or promoting dopamine release through their effects on
dopamine transporters (Kreek et al., 2002). Other drugs, such as nicotine, alcohol, opiates and
marijuana, work indirectly by stimulating neurons that modulate dopamine neurons in the VTA
(Kreek et al., 2002).
How does repeated dopamine release unto these regions due to drug-use, consolidate drugtaking behavior into compulsive use? Dopamine is a neuromodulater and through intracellular
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signaling mechanisms can produce synaptic and other forms of neural plasticity (Chao & Nestler,
2004). Therefore, suprathreshold levels of dopamine released unto dopaminergic circuits could
lead to long-term alterations in neural function, and as a result cause the behaviors associated
with addiction. As mentioned above, there is good evidence to view increases in dopamine not
directly related to reward per se, but rather involved in incentive salience (Berridge & Robinson,
1998; Robinson & Berridge, 2000; Berridge et al., 2009). This provides a different perspective
about drugs, as it implies that drug-induced increases in dopamine will inherently motivate
further procurement of more drugs, regardless of whether or not the effects of the drug are
consciously perceived to be pleasurable. Indeed, some addicted individuals report that they seek
drugs even though its effects are no longer pleasurable (Gawin, 1991). The principal role of
dopamine in the molecular underpinnings of addiction is the primary reason why research
investigators have intensely focused on how dopaminergic modulation of synaptic transmission is
altered in addicted animals, and why our lab continues this focus of research.
1.7
Dopamine Receptor Signaling !
An integral part of the present study is to determine the intracellular signaling
mechanisms that are utilized by dopamine in the ovBST. Therefore this section will provide a
short summary of the dopamine receptor subtypes and the known intracellular signaling cascades.
Dopamine receptors belong to the super-family of G-protein-coupled receptors, which
contain seven transmembrane regions, an extracellular N-terminal domain and an intracellular Cterminal domain (Civelli et al., 1993; Schwartz et al., 1993).
Based on genetic, pharmacological and biochemical criteria, mammalian dopamine
receptors are classified into two families, the D1-like and D2-like receptors (Civelli et al., 1993;
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Schwartz et al., 1993). The D1-like receptor family is comprised of the D1 and D5 receptor
subtypes, while the D2-like receptor family is comprised of the D2, D3, and D4 receptor
subtypes. At the molecular level, most signaling properties are shared among all of the receptor
subtypes within a family, although some differences among subtypes within a family have been
identified (Civelli et al., 1993; Schwartz et al., 1993; Neve et al., 2004). For the sake of brevity
this section will focus on the intracellular signaling properties of each family as a whole rather
than treating each receptor subtype separately.
The effects of D1-like receptors are mainly mediated by coupling to G"s/olf, which causes
sequential activation of adenylate cyclase (AC) which is able to free the catalytic subunit of
protein kinase A (PKA) and activate it through the conversion of ATP to cyclic AMP (cAMP).
This active form of PKA can go on to directly phosphorylate a number of voltage and ligandgated ion channels, proteins involved in signal transduction, and proteins involved in the
regulation of gene expression (Corvol et al., 2001; Jin et al., 2001; Neve et al., 2004; Sidhi, 1998;
Wang et al., 2001).
One such important substrate of PKA is the bifunctional signalling protein DARPP -32
(dopamine and cAMP-regulated phosphoprotein of 32 kDa). Depending on its phosphorylation
status DARPP-32 can either inhibit PKA or can inhibit protein-phosphatase-1 (PP1). Through
inhibition of PP1, DARPP-32 can alter phosphorylation of voltage-gated ion channels or
ionotropic receptors, and also influence gene expression. The activation of DARPP-32 is thus
crucial for the amplification of D1-like receptor signaling (Greengard et al., 1999; Neve et al.,
2004).
However, some D1-like receptors, especially those expressed in the striatum and
amygdala, are not always coupled to adenylate cyclase but to phospholipase C (PLC) through the
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interaction with another heterotrimeric G protein, G"q. Activation of PLC stimulates inositol
triphosphate formation, resulting in mobilization of intracellular calcium stores, which can further
alter signal transduction pathways (Jin et al., 2001; Jin et al., 2003; Leonard et al., 2003 Mahan et
al., 1990; Neve et al., 2004; Undie and Friedman, 1900; Undie et al., 1994; Wang et al., 1995).
The D2-like receptors are coupled to G"i/o which inhibit adenylate cylase activity, and
consequently function to antagonize the D1/cAMP/DARPP-32 signalling cascade (Dessauer et
al., 2002; Obadiah et al., 1999; Nishi et al., 1999; Stoof and Kebabian, 1981). For example,
stimulation of D2-like receptors has been shown to decrease the PKA-induced phosphorylation of
DARPP-32 and simultaneously to increase the phosphorylation of DARPP-32 at a site that leads
to the inhibition of PKA. The net affect of both actions is to inhibit the D1/cAMP/DARPP-32
signalling cascade (Greengard et al., 1999; Neve et al., 2004; Nishi et al., 1999). The !" subunits
that are released by receptor activation of G"i/o also participate in the modulation of many
signaling pathways including the Mitiogen Activated Protein Kinase pathway, PLC, and in the
regulation of ion channels (Neve et al., 2004).
1.8
GABAA Receptors !
The present study focused on how dopamine modulates GABAA receptors within the
ovBST, as such this section will provide a short summary of what is known about GABAA
receptors within the mammalian brain.
The GABAA receptors are members of the Cys-loop superfamily of ligand-gated ion
channels, whose endogenous ligand is Gamma-aminobutyric acid (GABA), the most abundant
inhibitory neurotransmitter in the central nervous system (CNS) (Chebib and Johnston, 1999;
Mohler, 2006). Following release from presynaptic vesicles, GABA exerts fast inhibitory effects
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by interacting with GABAA receptors, whose primary function is to hyperpolarize neuronal
membranes in mature CNS neurons, thereby diminishing the chance of a successful action
potential occurring and hindering the spread of excitability. GABAA receptors are found both
presynaptically, where they decrease the likelihood of neurotransmitter release, and
postsynaptically, where they decrease the likelihood of neuronal firing. Activation of GABAA
receptors causes membrane hyperpolarization by allowing Cl- influx, reflecting the relatively low
concentration of Cl- found intracellularly in most adult CNS neurons. GABAA receptors mediate
the majority of GABAergic signaling and thus function in maintaining the inhibitory tone of the
mammalian brain (Costa, 1998).
GABAA receptors are heteropentameric, that are assembled from a large family of eight
subunits: #, !, ", $ %, &, ', (. Five of these subunits combine to form unique GABAA channels.
The minimal requirement to produce a fully functional GABAA receptor is the inclusion of both #
and ! subunits, but the most common type in the mammalian brain is a pentamer comprising of
two #’s, two !’s, and one " subunit. The large structural diversity of subunits that compose
GABAA channels, and the multiple splice variants of each subunit provides an enormous potential
for receptor heterogeneity, that is believed to be responsible for determining the receptors agonist
affinity, chance of opening, conductance, and other properties (Mohler, 2006).
1.9
Dopamine Modulation of GABAA Channels !
Dopamine exerts varied effects on GABAA currents depending on the cell type and brain
area being examined. Since our study is interested in the ovBST, this section will only focus on
the reported modulation of GABAA receptor-mediated synaptic activity by dopamine in brain
structures that are associated with the mesolimbic pathway.
11
The major intracellular loops of GABAA receptors contain many consensus sites for protein
phosphorylation, and GABAA receptor-mediated currents are modified by a variety of
extracellular and intracellular factors after the activation of PKA, PKC, and tyrosine kinase
dependent pathways (Poisbeau et al., 1999; Smart, 1997).
In the medium spiny neurons of the nucleus accumbens (NAc), DA has been found to
depress GABAergic inhibitory transmission through a pre-synaptic D1-like receptor (Nicola and
Malenka, 1997). Once activated, the D1-like receptor decreases GABAergic transmission by
decreasing N- and P/Q-type calcium currents, which causes a decrease in the amount of presynaptic GABA release (Surmeier et al., 1995). D3 receptors have also been implicated to
contribute to the reduction of GABAA receptor current in NAc neurons. However, the D3
regulation of GABAA receptors occurs through the inhibition of PKA, which results in the dephosphorylation of GABAA receptors and to an increased endocytosis of the receptor (Chen et al.,
2006).
In the inhibitory interneurons of the lateral amygdala (LA), D1-like receptor activation has
been shown to increase the cellular excitability of the inhibitory network, as measured by
increases in the number of spontaneous inhibitory post-synaptic potentials (sIPSC). Surprisingly,
this effect was found to be independent of the cAMP/PKA signal transduction cascade, but
involved activation of the protein tyrosine kinase Src. The effect of activating D2-like receptors
in the inhibitory network of the amygdala was found to be dependent on the activation of D1-like
receptors. Co-activation of D1-like and D2-like receptors leads to a synergistic increase in sIPSC
frequency. Conversely, specific activation of D2-like receptors was found to induce a suppression
of spontaneous inhibition (Loretan et al., 2004).
12
1.10
Dopamine Receptors & Addiction !
Abusive drugs mediate reinforcement through direct or indirect activation of dopamine
receptors. The D1, D2, and D3 subtypes of dopamine receptors have all been implicated in the
reinforcing actions of drugs, as measured by intravenous self-administration studies (Britton et
al., 1991; Maldonado et al., 1993; Vorel et al., 2002). D1 dopamine antagonists are particularly
effective in blocking cocaine self-administration when the antagonist is administered directly into
the shell of the nucleus accumbens or in the central nucleus of the amygdala (Caine et al., 1995;
Maldonado et al., 1993). Moreover, the actions of D1 receptors within the BST seem to be crucial
for mediating the reinforcing properties of cocaine or alcohol (Chiang, 2010; Eiler et al., 2003;
Epping-Jordan et al., 1998).
Furthermore, downstream mechanisms of dopamine receptor signaling have also been
implicated in the reinforcing effects of cocaine and amphetamines. For instance, chronic cocaine
use resulted in decreased levels of the G-protein linked to inhibition of adenylate cyclase activity,
G"i, which coincided with an increase of adenylate cyclase and PKA activity (Striplin and
Kalivas, 1993; Unterwald et al., 1996). Consistent with the effects of increased PKA activity
following chronic cocaine use, it was found that injection of the nonhydrolyzable cAMP analogue
Sp-cAMPS into the NAc increased cocaine self-administration (Self et al., 1998). Another target
downstream of D1 receptors is the extracellular signal regulated kinase pathway (ERK). Recent
discoveries show D1 receptor dependent enhancement of ERK activation in a number of brain
regions including the BST following exposure to addictive drugs (Valjent et al., 2004). Cocaine
has been shown to alter gene expression downstream of DA receptor and cAMP signaling in the
13
NAc (Chao & Nestler, 2004). For instance overexpression of CREB in this region decreases the
rewarding effects of cocaine, while reducing CREB signaling by overexpression of a dominantnegative mutant CREB increases the rewarding effects of cocaine (Carlezon et al., 1998).
Rationale
As mentioned above, the BST receives a robust input of dopaminergic fibers, and drugs of
abuse increase the levels of dopamine within the BST. Furthermore, there is adequate behavioral
evidence to suggest that dopamine receptors within the BST contribute to the reinforcing effects
of drugs of abuse. However, to date, no study has been conducted to measure how dopaminergic
receptors modulate inhibitory synaptic transmission within the BST, and as a consequence
whether or not that modulation is altered through the course of addiction. Such information will
help lead to a better understanding of the etiology as to how dopamine receptors in the BST
contribute to addiction.
Hypothesis
We hypothesize that cocaine self-administration will alter dopaminergic modulation of inhibitory
transmission in the rat ovBST.
Specific Aims
1.) To measure the effect of dopamine on the amplitude of GABAA-IPSCs in the ovBST, and
14
to identify the dopaminergic receptor subtype and the synaptic locus that mediates the
effect.
2.) To characterize how dopaminergic modulation of GABAA-IPSCs is altered in rats with a
history of cocaine self-administration.
3.) Finally, to investigate the intracellular signaling pathway that mediates the effect of
dopamine on GABAA-IPSCs in rats that self-administer cocaine.
15
Chapter 2 Methods
Subjects. Long Evans rats (Charles River Laboratories, www.criver.com) weighing 250-300 g,
were housed individually in a climate-controlled colony room. The animals were maintained on a
12 hr reversed light/dark cycle (09.00h lights off – 21.00h lights on) and all behavioural testing
occurred during the dark phase. Rat chow and water were provided ad libitum in the home cages
and in the test chambers for the duration of the experimental sessions. All the experiments were
conducted in accordance with the Canadian Council on Animal Care guidelines for use of animals
in experiments and approved by the Queen’s University Animal Care Committee.
Surgeries. Before surgical procedures, animals were allowed to acclimatize to surroundings for a
minimum of 3 days. Rats were weighted and anesthetized with isoflurane (2-3%, 5L/min). We
used manufactured indwelling catheters for intra-jugular cannulations (Model IVSA28, Camcath,
Inc., www.camcath.com). The end of the tubing was inserted 32mm into the right jugular vein,
towards the right atria, and tied with 4.0 suturing silks. The rest of the tubing was fed
subcutaneously to a back-mounted 28Ga cannula. All incisions were closed with 4-0 absorbable
sutures silk. Upon surgery completion and recovery from anesthesia, rats were returned to the
colony room. Subjects received 5mg/kg Anafen injections, subcutaneously, for three days postoperatively and also received fruits to supplement normal chow diet during recovery. IV cannulae
were flushed daily with a sterile heparin-saline solution (20 IU heparin/ml) to prevent clots and
conserve patency.
16
Behavioural training. Behavioural testing for sucrose or cocaine self-administration was
conducted in operant chambers, each equipped with a house light, a response lever with cue light,
and a food dispenser for sucrose pellet reinforcement (MedAssociates Inc., www.medassociates.com). Rats were placed in the operant chambers for daily 4-hour sessions. Rats learned
sucrose- or cocaine-reinforced operant responding on a fixed ratio-1 (FR-1) schedule of
reinforcement where each lever press illuminated the cue light and delivered the reward, either
one sucrose pellet (75mg) or a cocaine-HCl infusion (0.75mg/kg in 0.12ml sterile saline in 4 sec).
Upon each reward delivery, the lever was retracted for 20 sec, during which the cue light
remained illuminated; no additional responses could occur during this holding period. Training
was considered acquired when rats responded 25 times, in a titrated way (equally spaced
infusions), for 3 consecutive days. Upon acquisition of operant responding, the rats graduated to a
progressive ratio schedule of reinforcement (PR) in which lever pressing to obtain each
subsequent cocaine injection increased according to; Response Ratio=[5e(injection numberx0.2)]-5 18.
Yoked rats received cocaine in exactly the same amount and frequency as their self-administering
counterparts. Levers were not available but reward delivery was signaled similarly, by a 20-sec
cue light illumination. Rats assigned to the acquisition group only performed the acquisition (FR1) part of the training.
Slices preparation and electrophysiology. Approximately 20 hours after the end of their last
training session, rats were anesthetized with isoflurane and their brains rapidly removed. Coronal
slices (250 !m) containing the BST were prepared in a physiological solution containing (in mM)
126 NaCl, 2.5 KCl, 1.2 MgCl2, 6 CaCl2, 1.2 NaH2PO4, 25 NaHCO3 and, 11 D-glucose at 15°C.
Slices were incubated at 34°C for 60 minutes and transferred to a chamber that was constantly
perfused (1.5ml/min) with physiological solution maintained at 34°C and equilibrated with
17
95%O2/5%CO2. Whole-cell voltage-clamp recordings were made using microelectrodes filled
with a solution containing (in mM) 70 K+-gluconate, 80 KCl, 1 EGTA, 5 HEPES, 2 MgATP, and
0.3 GTP. Post-synaptic currents were evoked by local fiber stimulation with tungsten bipolar
electrodes. Electrodes were placed in the ovBST, 100-500 !m lateral from the recorded neuron,
and paired electrical stimuli (0.1msec duration, 50msec interval) were evoked at 0.1Hz. GABAAIPSC were pharmacologically isolated with the AMPA antagonist DNQX (50 !M). All drugs
were bath-applied through the perfusion system or, in many cases, included in the internal
solution. Recordings were made using a Multiclamp 700B amplifier and a Digidata 1440A
(Molecular Devices Scientific, www.mds.com). Data were acquired and analyzed with Axograph
X (www.axographx.com) running on an Apple computer (www.apple.com).
Drugs. Stock solution of DA (10 mM), quinpirole (1mM), SCH-23390 (10mM), prazosin
(1mM), noradrenaline (10mM), 5-HT (10mM), propranolol (1mM), and GDP-!-s (10mM) were
prepared in double distilled water. Stock solution of DNQX (100mM), forskolin (100mM), SKF81297 (1mM), sulpiride (1mM), yohimbine (1mM), methysergide (10mM), H89 (10mM), U73122 (10mM), genistein (10mM), daidzein (10mM), PP2 (10mM), and PP3 (10mM) were
prepared in DMSO (100%). Every drug further dissolved in the physiological solutions at the
desired concentration and the final DMSO concentration never exceeded 0.1%. Cocaine-HCl was
dissolved at 2.5mg/ml in sterile saline and pH was adjusted to 7.3 with NaOH. Drugs were
obtained from Sigma-Aldrich Co. (www.sigmaaldrich.com) or Tocris Biosciences
(www.Tocris.com) except cocaine-HCl (Medisca, www.medisca.com).
Statistical Analysis. We measured drug-induced change in post-synaptic currents (PSC) peak
amplitude from baseline in percentage (((Peak amplitudedrug-Peak amplitudebaseline)/Peak
amplitudebaseline)*100) 5-10 min after bath application of the drugs. We assessed drug effects
18
using two-tailed t-tests with hypothesized values of 0 (H0: ) GABAA-IPSC (%)=0). We
minimized Type I error with a Bonferroni-adjusted # level (#=0.05/number of t-tests). We
calculated paired-pulse ratios (PPR) by dividing the second (S2) by the first (S1) peak amplitude
that we normalized to baseline. We calculated peak amplitudes for S1 and S2 from a baseline
value measured 10 ms after the end of a 1mV test pulse. We assessed drug effects on PPR using
one-tailed t-tests with hypothesized values of 1 (H0: ) PPR (Normalized)=1). We minimized
Type I error with a Bonferroni-adjusted # level (#=0.05/number of t-tests). We used multiple ttests because we only measured the full dose-response effect in Control, Sucrose, and Cocaine
rats. We analyzed coefficient of variation (CV) by plotting r ((1/CV2 drug)/(1/CV2baseline)) against #
(Peak amplitudedrug/Peak amplitudebaseline) and computed bivariate linear Fits of r by # (Faber and
Korn, 1991). We used one-way ANOVAs to compare multiple means and conducted appropriate
statistical tests for multiple comparisons (indicated in Results) when ANOVAs deemed
significance. All statistical analyses were done with JMP 9.0.
19
Chapter 3 Results
1.1
Passive Properties of ovBST Neurons in Naïve and Cocaine Rats
All our recordings were done in neurons of the oval nuclei of the BST (ovBST), which
lies dorsal to the anterior commissure and medial to the internal capsule (Figure 1a). No agonist
tested in the present study significantly changed passive properties (membrane input resistance
(Rin) or membrane holding currents (Hc)) of ovBST neurons voltage-clamped at -70mV (I will
change this once I complete the figure)
1.2
In Drug-Naïve, Yoked, and Acquisition Rats DA Decreased Inhibitory
Transmission in the ovBST by Activating Pre-Synaptic D2 Receptors
Exogenously applied DA (0.1 to 100!M) dose-dependently decreased the amplitude of
evoked and pharmacologically isolated GABAA-IPSC in drug-naïve rats (Figure 5). GABAA IPSC rapidly returned to baseline upon when DA was washed out of the perfusion chamber. The
DA-induced reduction of GABAA-IPSC reached a plateau at 30!M (-42.8±6.5%) and increasing
DA concentration to 100!M (-42.4±6.5%) did not produce any further effect (Figure 5). At
concentrations where DA decreased GABAA-IPSC, we observed statistically significant increases
in paired-pulse ratio (PPR50msec) suggesting it acts pre-synaptically (Figure 6a). Coefficient of
variation (CV) analyses revealed a significant positive correlation, also suggesting a pre-synaptic
effect of DA (Figure 6b).
20
In rats trained to self-administer sucrose (operant) for 20±4 days, that were yoked to
cocaine self-administering rats (yoke), or that only acquired operant responding for cocaine
(acquisition; 5±0.6 days on a fixed ratio-1 schedule of reinforcement, acquisition), DA dosedependently reduced GABAA-IPSC in a similar manner to that seen in drug-naïve rats (Figure 10
& Figure 11).
The D2-like agonist quinpirole (0.1: -15±7.3%; t(4)=-2.2; P<0.025 and 1!M: -48±6.2%;
t5=8.1; P<0.0001) dose-dependently and pre-synaptically mimicked the effects of DA on
GABAA-IPSC in drug naïve rats (Figure 7 & Figure 8). Likewise, quinpirole (1!M) presynaptically reduced GABAA-IPSC amplitude in the ovBST of operant (-34.6±4.8%; t(13)=-7.4;
P<0.0001), yoke (-30.1±6.4%; t(12)=-4.9; P<0.0001), and acquisition (-53.2±6.2%; t(5)=-9;
P<0.0001) rats (Figure 12).
To further elucidate the receptor subtype that mediates the effect of DA, DA 30!M was
co-applied with either the D1R antagonist SCH-23390 (10!M) or the D2R antagonist sulpiride
(10!M). As well, DA 30 !M was co-applied with a cocktail of noradrenergic antagonists
(prazosin 0.1!M, yohimbine 1!M, and propranolol 1!M) or the 5-HT1,2,7 receptor antagonist
(methysergide 10!M), since DA is known to bind non-specifically to these receptors (Cornil &
Ball, 2008; Cornil et al., 2002; Guiard et al., 2008). Only the D2R antagonist, sulpiride blocked
the DA-induced reduction in GABAA-IPSC (F(4,19)=7.6, P=0.0008) (Figure 9). The D1R
antagonist SCH-23390, noradrenergic antagonists, and the 5-HT1,2,7 receptor antagonist
methysergide all failed to significantly block the DA-induced reduction in GABAA-IPSC (Figure
9b). Furthermore, there was a modest reduction in GABAA-IPSC amplitude caused by application
of NA (10!M) (Figure 9b). On average, 5-HT (10!M) had no significant effect on GABAA-IPSC
amplitude in the ovBST (Figure 9b). However, 5-HT decreased (-26.0±6.8, n=8) or slightly
21
increased (11.0±6.1, n=3) GABAA-IPSC in the ovBST. Sulpiride and SCH-23390 did not
produce any effects on their own (Figure 9b). Thus, in drug-naïve, operant, and acquisition rats,
D2-like receptors are responsible for the effects of DA on GABAA-IPSC in the ovBST.
1.3
Only in Rats that Self-Administered Cocaine Under a PR Schedule of
Reinforcement was the DA-Induced Modulation of Inhibitory Transmission Altered
In direct contrast to drug-naïve animals and all other experimental groups, DA (1!M)
increased (17.2±4.1%; t(20)=4.3, P<0.001) the amplitude of GABAA-IPSC in the ovBST of rats
that maintained cocaine self-administration (cocaine) for 15±2 days under a progressive-ratio
(PR) schedule of reinforcement where the effort required to obtain subsequent cocaine infusions
increased exponentially (Figure 10). At concentrations where DA increased GABAA-IPSC, we
observed statistically significant decreases in paired-pulse ratio (PPR50msec) suggesting the locus
of action was pre-synaptic (Figure 11a). Furthermore, the coefficient of variation (CV) analyses
revealed a significant positive correlation, also suggesting a pre-synaptic effect of DA in cocaine
maintenance rats (Figure 11b)
At higher concentrations, DA produced no effect (10!M; 0.4±2.5%, t6=0.16) or partly
decreased (30!M; -23±2.4%; t10=-9.6, P<0.001) GABAA-IPSC amplitudes (Figure 10).
Altogether, statistical analyses revealed that the effect of DA on GABAA-IPSC amplitude in the
cocaine maintenance group was significantly different to all other groups at 1!M (F4,61=24.9,
P<0.0001). Likewise, the effect of DA in the cocaine maintenance group was different than the
drug-naïve and the operant group at 10!M (F2,16=24.9, P<0.0001) and 30!M (F2,19=5.9, P=0.012).
In contrast, DA produced similar effects in the drug-naïve, operant, and cocaine maintenance
groups at 0.1 (F2,7=0.9, P=0.08) (Figure 10).
22
1.4
Cocaine Self-Administration Revealed a Loss of Pre-Synaptic D2-like
Receptors and a De Novo Post-Synaptic D1-like and D2-like components.
Next we sought to investigate the dopaminergic receptor subtypes that mediated the
effects of DA on GABAA-IPSC in cocaine rats. In contrast to drug-naïve and other experimental
groups, quinpirole was ineffective in reducing GABAA-IPSC amplitude in cocaine maintenance
rats (0.1: 2.1±3.7%; t(8)=0.3; ns and 1!M: -6.2±5.3%; t(11)=-1.4; ns) (Figure 12). Overstimulation
of the adenylyl cyclase pathway through bath application of the adenylyl cyclase activator
forskolin (100nM) rescued the D2-like reduction in GABAA-IPSC in cocaine maintenance rats
(Figure 14). In contrast, forskolin was ineffective at rescuing the D2-like response when applied
intracellularly (Figure 14). Since forskolin was ineffective when applied post-synaptically (in the
recording pipette), these observations also support a pre-synaptic localization of D2-like receptors
in the ovBST.
Surprisingly, when the D2-like antagonist sulpiride (10!M) was added in the perfusion,
DA (1!M) produced a large and sustained increase in GABAA-IPSC that could be completely
blocked by the co-application of the D1 antagonist- SCH-23390 (10!M)(Figure 15 & Figure 16).
Similarly, when we bath applied the selective D1/D5 agonist SKF-81297 (1!M), we also
observed this long-term potentiation (104±8%) of GABAA-IPSC (LTPGABA)(Figure 16). This
suggest that in addition to a loss of pre-synaptic D2-like receptors, maintenance of cocaine selfadministration revealed a de novo D1-like component that was not present or at least not
functional in drug-naïve rats. The D1 induced LTPGABA was mediated by statistically significant
decreases in paired-pulse ratio (PPR50msec) suggesting the locus of action was pre-synaptic (Figure
23
18a & 18b). Furthermore, the coefficient of variation (CV) analyses revealed a significant
positive correlation, also suggesting a pre-synaptic effect (Figure 18c).
The observation that DA (1!M) when co-applied with the D2 antagonist sulpiride resulted in
LTPGABA was unexpected since bath application of quinpirole alone was ineffective in
modulating GABAA-IPSC in cocaine maintenance rats (Figure 12). However when we co-applied
quinpirole (1!M) into the perfusion with SKF, we blunted the D1-induced LTPGABA (Figure 16a2
& Figure 17a). These results suggest that due to cocaine self-administration there is a loss of
functional pre-synaptic D2-like receptors, but an emergence of a new D2-like component, who’s
functional role seems to antagonize or regulate the D1 receptor increase of GABAA-IPSCs.
Interestingly, this novel D2-like receptor component was post-synaptic as shown by the next
series of experiments.
1.5
D1-Mediated Signal Transduction Pathway involved Src-Tyrosine Kinases
in a G-protein-independent way while the D2-Mediated Signal was G-protein
dependent.
Next, we wanted to elucidate the intracellular signaling mechanisms that mediate our D1
and D2 effects in cocaine rats. When we blocked G-protein activity by adding a non-hydrolizable
GTP analog, GDP-!-s (250!M), in the recording micropipette, we were still able to induce the
D1-dependent LTPGABA, by either application of SKF-81297 (1!M) alone or co-application of
DA 1!M with sulpiride (10!M) (Figure 17a). However, with the inclusion of GDP-!-s in the
recording pipette, quinpirole was no longer effective at blocking the D1 dependent LTPGABA
(Figure 17a). Moreover, when G-proteins were pharmacologically blocked, we also observed that
24
application of DA without sulpiride was now able to induce LTPGABA (Figure 17a). This
experiment demonstrated first, that D1- and D2-like signals were respectively independent and
dependent upon G-protein activation, and second, that the D2-like component in cocaine
maintenance rats was post-synaptic (GDP-!-s was in the recording pipette and could not act presynaptically). Thus, cocaine self-administration revealed an intriguing D1-induced LTPGABA, that
involved intracellular signaling independent of G-proteins.
We further investigated this D1 intracellular signaling by adding the potent protein kinase
A inhibitor H89 (10!M) in the recording micropipette. H89 did not affect DA-induced increase in
GABAA-IPSC in the ovBST of cocaine maintenance rats (Figure 17b). The effects of DA on
GABAA-IPSC in the ovBST of cocaine rats also remained intact upon inhibition of the hydrolysis
of PiP2 to IP3 and DAG with the phospholipase C inhibitor U-73122 (10!M) (Figure 17b).
Conversely, intracellular application of the non-specific tyrosine kinases (TKs) inhibitor genistein
(50!M) or the specific Src-TKs inhibitor PP2 completely blocked DA-induced LTPGABA (Figure
17b & 17c). The inactive analogs of genistein and PP2, respectively daidzein and PP3, did not
affect DA-induced LTPGABA (Figure 17b & 17c). These experiments demonstrated that the D1induced LTPGABA effect was mediated intracellularly by the tyrosine kinase, Src, and as a
consequence these results also suggest that the D1 receptor was located post-synaptically.
25
ic
ovBST
ac
Figure 1: Anatomical localization of the ovBST. Schematic illustrating the anatomical
localization of the oval bed nucleus of the stria terminalis. ac: anterior commissure; ic: internal
capsule.
26
L.J. Freedman, M.D. Cassell / Bram Research 633 (1994) 243-252
ally [20,30,53] and in the extended amygdala specifically [14,44]. Although previous descriptions of TH
immunoreactivity as being heavier in CeM appear to
disagree with our results, published photomicrographs
247
(Fig. 5 of Fuxe et al. [18], Fig. 8a,b of Fallon of Ciofi
[14]) show a distribution of fibers fairly similar to our
own. Most likely, the difference in specific localization
was attributable to differences in the definition of the
Figure 2: Dopaminergic inputs in the dorsolateral BST. Photomicrograph of tyrosine
hydroxylase (A) and dopamine/3-hydroxylase (B) immunoreactive fibers in the bed nucleus of
the stria terminalis at rostral (A, B) levels in coronal section. Bar = 500 µm (modified from
Freedman and Cassell, 1994).
27
Fig. 3. Photomicrograph of tyrosine hydroxylase (A, C) and dopamine/3-hydroxylase (B, D) immunoreactive fibers in the bed nucleus of the stria
terminalis at rostral (A, B) and caudal (C, D) levels in coronal section. Bar = 500 ~tm.
REGULATION OF SYNAPTIC TRANSMISSION BY DOPAMINE IN ovBST
FIG. 6.
tion of D1
micrograph
to reveal i
A=) and D
line deline
red dotted
anterior co
oval nucle
A=, 0.1 mm
excitatory synaptic transmission in the ovBST (Fig. 7). This
double dissociation is appreciable at lower concentration of
both DA and NA (e.g., at 1 !M). It is thus likely that in
physiological conditions, the effect of DA is restricted to
inhibitory transmission whereas NA should preferentially modulate excitatory synaptic transmission.
In contrast to DA and NA, 5-HT does not modulate
excitatory transmission and is largely undetected immunohistochemically in the ovBST (Phelix et al. 1992). This
observation contrasts with 5-HT-induced reduction in
evoked EPSC reported by Guo et al. in the anterolateral BST
(Guo and Rainnie 2010), and suggests that 5-HT plays little
or no neuromodulatory role of excitatory transmission in the
ovBST. Nonetheless we observed bidirectional modulation
of inhibitory transmission by 5
It is, however, unlikely t
through 5-HT receptors because m
fere with the DA-mediated reduct
lation of inhibitory transmission in
altogether has not been done and
investigation such as those done f
(Guo and Rainnie 2010).
Anatomical evidence suggests th
in neurocircuits that influence ing
its motivational component (Don
ingly, the BST, and in particular
key within the neural circuits sen
Figure 3: Immunohistochemical localization of D1R and D2R in the ovBST.
Light receives ascending in
The ovBST
micrographs of the rat brain immunostained to reveal immunoreactivity fornal
D1Rtrunk,
(A) andhorizontal
D2R
inputs from
(B). ac, anterior commissure; ic, internal capsula; ov, oval nucleus. Scale bars:
A
and
B,
0.3
mm;
regions, and top-down cortical in
A =, 0.1 mm; B =, 50 mm (modified from Krawczyk et al., 2011
likely conveying excitatory input
viscerosensory portion of the dysg
the olfactory amygdalopiriform tra
et al. 1999), which could, respecti
external cues triggering operant
28
How excitatory inputs into the ovB
network function is currently unk
Sahd extensively described the lo
ovBST, which is compartmentaliz
(Larriva-Sahd 2006). The shell se
mit incoming information to the
projections neurons. ovBST projec
RESULTS ).
20
Control
Holding current (pA) or Rin M )
15
Cocaine
10
5
0
-5
-10
-15
-20
Hc
Rin
(-70mV)
Figure 4: Passive Properties of ovBST neurons in Naïve and Cocaine Rats. Dot plot showing
the effects of dopamine DA (1!M) on membrane holding current (Hc) and membrane input
resistance (Rin) in drug-naïve and cocaine rats. (Robyn Sharma contributed data to this figure).
Figure 4: Passive Properties of ovBST neurons in Naïve and Cocaine Rats. Dot plot showing
the effects of dopamine DA (1!M) on membrane holding current29(Hc) and membrane input
resistance (Rin) in drug-naïve and cocaine rats. (Robyn Sharma contributed data to this figure).
38
a.
S2
200 pA
S1
50 ms
Baseline
DA 30µM
Wash
b.
GABAA-IPSC (%)
20
0
3 13 9 10 5
-20
*
- 40
- 60
*
*
*
0.1 1 10 30 100
[DA]µM
Figure 5: Effects of DA on GABAA-IPSC in drug naïve rats. (a.) representative traces showing
the effects of bath application of DA on electrically evoked GABAA-IPSC in the ovBST. Each
trace is the average of 5 consecutive events. (b.) bar graph summarizing the effects of DA on the
peak amplitude of evoked GABA-IPSC in the ovBST. S1, stimulus 1; S2, stimulus 2. *,
significantly different from 0; P < 0.01.
30
Paired-pulse ratio (Normalized)
a.
* *
1.6
1.4
*
*
1.2
6
1
5
0.1 1 10 30 100
[DA]µM
b.
1.6
r2=0.22
p=0.018
r
1.2
0.8
0.4
0
0 0.4
0.8
1.2
Figure 6: The synaptic location of the effect of DA on GABAA-IPSC in drug naïve rats. (a.)
bar graph summarizing the effects of DA on the paired-pulse ratios of evoked GABAA-IPSC in
the ovBST. (b.) coefficient of variation analysis of the effects of DA (0.1–30 !M) on evoked
GABAA-IPSC in the ovBST. r, [(1/CV2 drug)/(1/CV2baseline)]; ', peak amplitudedrug/Peak
amplitudebaseline. *, significantly different from 0; P < 0.01.
31
150 pA
a.
50 ms
baseline Quin1µM Wash
b.
GABAA-IPSC (%)
20
0
-20
6
5
6
7
*
- 40
- 60
*
0.1 1
0.1 1
[Quin]µM [SKF]µM
Figure 7: Effects of D2R and D1R agonists on GABAA-IPSC in drug naïve rats. (a.)
representative traces showing the effects of bath application of the D2R agonist quinpirole on
electrically evoked GABAA-IPSC in the ovBST. Each trace is the average of 5 consecutive
events. (b.) bar graph summarizing the effects of the D2 agonist quinpirole and the D1 agonist
SKF 81296 on the peak amplitude of evoked GABA-IPSC in the ovBST. *, significantly different
from 0; P < 0.01.
32
Paired-pulse ratio (Normalized)
*
1.6
1.4
*
1.2
1
0.1 1
[Quin]µM
Figure 8: The synaptic location of the effect of a D2 agonist on GABAA-IPSC in drug naïve
rats. Bar graph summarizing the effects of the D2 agonist quinpirole on the paired-pulse ratios of
evoked GABAA-IPSC in the ovBST. *, significantly different from 0; P < 0.01.
33
a.
GABAA-IPSC (Normalized)
1.4
1.2
1
0.8
0.6
0.4
DA 30µM
DA 30µM + Sulpiride 10µM
DA 30µM + SCH-23390 10µM
0.2
0
0
10
Time (min)
15
20
10
GABAA IPSC (%)
b.
5
0
7
5
5
5
7
7
6 11
-10
-20
-30
*
- 40
-50
*
*
*
+
+
-
+ +
- - + - +
- - -
†
DA(30)
Sulpiride(10)
SCH-23390(10)
NA antagos.(1)
5-HT antago.(10)
NA(10)
5-HT(10)
+
-
+
+
-
+
-
- - - - + - - - - - - + - - +
Figure 9: Pharmacological characterization of the effects of DA on the amplitude of evoked
GABAA-IPSC in the ovBST of drug naïve rats. (a.) Representative dot plot showing the timecourse of the effects of DA on evoked GABAA-IPSC in the ovBST in the absence and the
presence of the D1R antagonist SCH-23390 or the D2R antagonist sulpiride. (b.) Bar chart
summarizing the effect of monoaminergic agonists and antagonists on evoked GABAA-IPSC in
the ovBST. Asterix, Significantly different from 0; P<0.01. Dagger, Significantly different from
DA 30!M; P<0.05.
34
a3.
a2.
a1.
Control
Cocaine
200 pA
Sucrose
40 ms
Baseline
DA1 µM
Wash
Baseline
b.
DA1 µM
Baseline
Wash
†*
30
GABAa-IPSC (%)
Wash
Control
Sucrose
Acquisition
Cocaine
Yoked
20
10
0
DA1 µM
†
3
3
4
13 16 8 21 8
9
7
7
7
5 12
-10
-20
-30
*
-40
**
-50
*
†
*
*
*
*
0.1
1
10
*
30
[Dopamine]µM
Figure 10: Effects of DA on GABAA-IPSC in all experimental groups. Above: Representative
traces showing the effect of bath applied DA on the amplitude of evoked whole-cell GABAAIPSC in brain slices prepared from Control (a1.), Sucrose (a2.), and Cocaine (a3.) rats. (b.) Bar
chart summarizing the effects of DA on the change in amplitude of evoked GABAA-IPSC in all
experimental groups. Asterix, Student’s t-test, P<0.001. Dagger, One-way ANOVA, P<0.01.
35
a.
PPR (normalized)
1.5
1.4
*
1.3
* *
*
**
*
**
Control
Sucrose
Acquisition
Cocaine
Yoked
1.2
1.1
†
1.0
0.9
0.8
†
0.1
1
*
30
10
[Dopamine]µM
b1.
Control
2.0
r2=0.18
p=0.02
b3.
Sucrose
1.6
1.2
r2=0.33
p=0.0008
Cocaine (maintenance)
4.0
r2=0.49
p<0.0001
3.0
r
r
3.0
b2.
0.4
0
0
r
0.8
1.0
2.0
1.0
0
0.2 0.4 0.6 0.8 1.0 1.2
0
0.2
0.4
0.6
0.8 1.0
0
0
0.4
0.8
1.2
1.6
Figure 11: The synaptic location of the effect of DA on GABAA-IPSC in all experimental
groups. (a.) Bar chart summarizing the effects of DA on the change in paired-pulse ratio of
evoked GABAA-IPSC in all experimental groups. Bellow: Dot plot illustrating coefficient of
variation (CV) analyses of the effects of DA (0.1-30!M) on evoked GABAA-IPSC in brain slices
from Control (b1.), Sucrose (b2.), and Cocaine (b3.) rats. Dot plot shows r ((1/CV2
2
drug)/(1/CV baseline)) as a function of $ (Peak amplitudedrug/Peak amplitudebaseline). Asterix,
Student’s t-test, P<0.001. Dagger, One-way ANOVA, P<0.01.
36
a1.
a2.
Control
150 pA
Cocaine
50 ms
baseline Quin 1µM Wash
baseline Quin 1µM Wash
GABAa-IPSC (%)
b.
0
7 14 6 12 13
†
-20
-40
-60
Control
Sucrose
Acquisition
Cocaine
Yoked
* *
*
*
Quinpirole 1 µM
Figure 12: Effects of D2R agonists on GABAA-IPSC in all experimental groups. Above:
representative traces showing the effects of the D2R agonist quinpirole (1!M) on evoked
GABAA-IPSC in brain slices prepared from Control (a1.) and Cocaine (a2.) rats. (b.) Bar charts
summarizing the change in amplitude of GABAA-IPSC produced by bath application of
quinpirole in all experimental groups. Asterix, Student’s t-test, P<0.001. Dagger, One-way
ANOVA, P<0.05.
37
PPR (normalized)
1.6
*
*
* *
Control
Sucrose
Acquisition
Cocaine
Yoked
1.4
1.2
1
†
Quinpirole 1 µM
Figure 13: The synaptic location of the effect of a D2 agonist on GABAA-IPSC in all
experimental groups. Bar charts summarizing the change in paired-pulse ratio of GABAA-IPSC
produced by bath application of quinpirole. Asterix, Student’s t-test, P<0.001. Dagger, One-way
ANOVA, P<0.05.
38
50
*
40
30
Control
*
Cocaine
∆ GABAa- IPSC (%)
20
10
0
-10
†
-20
-30
- 40
*
-50
*
*
-60
extracellular forskolin (100nM)
+ +
+ +
-
-
intracellular forskolin (100nM)
-
-
-
-
+ +
quinpirole (1µM)
-
-
+ +
+ +
Figure 14: D2R activation during the overstimulation of the adenylyl cyclase pathway. Bar
chart summarizing the effect of quinpirole (1!M) on the amplitude of GABAA-IPSC in the
presence of extracellular or intracellular forskolin (100nM). Asterix, Student’s t-test, P<0.001.
Dagger, One-way ANOVA, P<0.05.
39
15
5
30
150 pA
Normalized GABAA-IPSC
0
2
Control
Operant
Acquisition
Cocaine
Yoked
50 ms
D1R activation
1
0
-5
0
5
10
15
Time (min)
20
25
30
Figure 15: Contribution of D1R in the effect of DA on GABAA-IPSC in all experimental
groups. Dot plot illustrating the averaged time-course of the effect of a 5-min co-application of
DA (1!M) and the D2-like antagonist sulpiride (10!M) on the amplitude of GABAA-IPSC tested
in drug-naïve (pale gray), operant (medium gray), acquisition (dark gray), yoke (open), and
cocaine (blue) rats. Asterix, Student’s t-test, P<0.001.
40
Normalized GABAA-IPSC
a1.
3
2
1
0
-5
5
10 15
Time (min)
0
20
DA+sulpiride
DA+sulpiride+SCH-23390
SKF-81297
a2.
*
*
GABAa-IPSC (%)
120
80
40
0
DA(1)
Sulpiride(10)
SCH-23390(10)
SKF-81297(1)
Quinpirole (1)
4
6
4
7
+ + + + - + - - +
-
+
- - - +
41
25
Figure 16. Characterization of the contribution of D1 and D2 receptors in regulating
LTPGABA. Dot plot illustrating the effect of a 5-min application of the D1-like agonist SKF-81297
(blue squares) or a co-application of DA (1!M) and the D2-like antagonist sulpiride (10mM) in
the absence (blue circles) or presence (black circles) of the D1-like antagonist SCH23390 (10!M)
on evoked GABAA-IPSC in cocaine maintenance rats. c. Bar chart summarizing DA receptors
pharmacology underlying D1-mediated LTPGABA in cocaine maintenance rats. Asterix, Student’s
t-test, P<0.001.
42
a.
b.
*
†
120
†
†
†
100
80
60
40
SKF-81297(1)
GDP-ßs (250)
Quinpirole(1)
DA(1)
Sulpiride(10)
†
120
†
80
60
40
4
+
-
6
4
4
8
4
+ + + - + - + - +
- + + - - - - + +
- - - + -
0
DA(1)+Sulpi(10)
H-89(10)
U-73122(10)
Genistein(50)
Daidzein(50)
PP2(10)
PP3(10)
4
6 4
6 4
6
6
+
-
+
+
-
+
+
-
+
+
-
+
+
+
+
-
Normalized GABAA-IPSC
PP3
PP2
2
1
D1R activation
0
-5
†
100
c.
3
*
20
20
0
†
†
140
GABAA-IPSC (%)
GABAA-IPSC (%)
140
*
0
5
10
Time (min)
43
15
20
+
+
-
Figure 17: Pharmacological characterization of LTPGABA a. Bar chart summarizing the effect
of co-application of quinpirole on SKF-81297 (1!M)-induced LTPGABA and intra-cellular Gprotein blockade. b. Bar chart summarizing the pharmacological characterization of DA-induced
LTPGABA. c. Dot plot illustrating the intra-cellular effect of the c-Src inhibitor PP2 (10!M, black
circles) or its inactive analog PP3 (blue circles) on DA-induced LTPGABA. Asterix, Student’s ttest, P<0.001. Dagger, One-way ANOVA, P<0.05.
44
A.
B.
'#
1.6
"#
PPR (S2/S1)
r= -0.68
p=0.0001
$#
PPR (%)
t13=-5.29
P=0.0001
1.8
%#
1.4
1.2
1.0
0.8
*
0.6
&#
0.4
!&#
5
30!
r=0.70
p=0.005
4
r
!%#
!$#
!"#
!&##
0!
C.
3
2
1
!$#
#
$#
&##
&$#
(##
0
0
Initial PPF (%)
0.5
1.0
1.5
2.0
2.5
3.0
!
Figure 18: Synaptic location of the D1 induced LTPGABA (A.) Graph plotting the change in
paired-pulse ratio in all drug groups with the initial paired-pulse facilitation (r = -0.68, p =
0.0001). (B.) Paired-pulse ratio at 0 and 30 minutes post DA 1!M with sulpiride 10!M or SKF81297 1!M application (t13 = -5.29, p = 0.0001). (C.) Dot plot illustrating coefficient of variation
(CV) analyses of the effects of DA 1!M with sulpiride 10!M or SKF-81297 1!M on evoked
GABAA-IPSC. Dot plot shows r ((1/CV2 drug)/(1/CV2baseline)) as a function of $ (Peak
amplitudedrug/Peak amplitudebaseline).
45
Chapter 4 Discussion
While several behavioral studies have elegantly demonstrated that D1 receptors are
necessary for mediating the reinforcing effects of drugs of abuse in the BST, to date, there have
been no studies examining the interaction of dopamine on inhibitory synaptic transmission within
the BST (Eiler et al., 2003; Epping-Jordan et al., 1998). Such information would be necessary if
we are to begin to piece together an understanding as to how D1 receptors contribute to
reinforcement within the BST. Therefore in the present study we sought to examine dopaminergic
regulation of inhibitory synaptic transmission within the BST in a rat model of cocaine selfadministration. In summary, the present data demonstrated that dopaminergic modulation of
inhibitory transmission switched from a pre-synaptic D2-like mediated depression, to a postsynaptic D1-like dependent potentiation of GABAA-inhibitory post-synaptic currents (IPSC),
only in rats maintaining operant responding for intravenous cocaine self-administration. These
results represent, to our knowledge, the first evidence of a change in dopaminergic regulation of
synaptic transmission specific to voluntary drug intake.
Importantly, modulation of synaptic transmission by dopamine was identical to drugnaïve conditions when intravenous cocaine administration was not contingent upon operant
responding (yoked), before the maintenance phase of cocaine self-administration (acquisition), or
in rats maintaining operant responding for sucrose under the same reinforcement schedule. This
data strongly suggest that the cocaine-related changes seen in the ovBST were not simply due to
the pharmacological effects of cocaine but instead could be due to an associative process acquired
during self-administration. When rats self-administer cocaine, this results in a distorted and
46
excessive dopamine signal in the BST due to the drugs ability to elevate dopamine by direct
pharmacological actions. As mentioned in the introduction there is good evidence to view
increases in dopamine not directly related to reward per se, but rather involved in incentive
salience. Therefore this distorted dopamine signal, in conjunction with the various inputs the BST
receives during lever pressing for cocaine, would result in the overlearning of drug-related cues,
thus leading to the valuation of drugs above other goals. The continuance of which would
ultimately produce the pathological adaptations that we measured in the BST, and this
exaggerated D1 signal could possibly further narrow the focus of behavior leading to loss of
control over drug use, a core characteristic of addiction.
There were a number of alterations in dopaminergic modulation of inhibitory
transmission within the ovBST in rats maintaining operant responding for intravenous cocaine
self-administration. Firstly, in cocaine maintenance rats there was a lack of a pre-synaptic D2-like
mediated depression of inhibitory transmission as was seen in naïve rats and other experimental
groups. This result is consistent with the dysfunction or decreased availability of D2-like
receptors observed after cocaine exposure in both primates and humans (Nader et al., 2002;
Nader et al., 2006; Volkow et al., 1993). This D2-like response was rescued when the adenylyl
cyclase activator forskolin was added to the bath, suggesting that cocaine self-administration
disrupted the coupling between the Gi-coupled D2-like receptor that was located pre-synaptically.
These results are consistent with the literature that suggest that due to repeated exposure to drugs,
certain G-protein coupled receptors undergo a complex processes of desensitization, that is
believed to play a role in tolerance (Bohn et al., 2000). Currently, there are a number of
mechanisms that account for such desensitization. One such mechanism involves G protein
receptor kinases (GRKs), which phophorylate the ligand bound form of the receptor, leading to
47
the binding of adaptor-like proteins, termed arrestins, ultimately resulting in the uncoupling of the
receptor from its cognate G-protein and decreased functional signaling (Heuss and Gerber, 2000).
Recent information has in fact suggested that the D2 receptor is a substrate for GRKs in cellular
expression systems and that GRK-mediated phosphorylation promotes receptor internalization
(Namkung and Sibley, 2004).
Secondly, in addition to a loss of pre-synaptic D2-like receptors, maintenance of cocaine
self-administration revealed a de novo post-synaptic D1-like component that was not present or at
the least not functional in drug-naïve rats. The lack of a D1-like induced effect in naïve rats is
consistent with previous immunohistochemical studies showing low expression of D1 receptor
protein in the dorsolateral BST (Figure 3) (Krawczyk et al., 2011). This finding is in line with
previous research showing increased sensitivity or availability of D1 dopamine receptors
observed following chronic exposure to drugs of abuse. Henry and White, reported in vitro
electrophysiological evidence for D1 receptor supersenistivity to D1 agonists within the NAc
after repeated cocaine administration, while Higashi and colleagues observed a similar
phenomenon following methamphetamine administration (Henry and White, 1991; Higashi et al.
1989). More recently, cocaine self-administration was found to increase D1 receptor density in
the NAc of Rhesus monkeys (Nader et al. 2002).
This D1-like component when activated on its own resulted in a persistent increase in
GABAA-IPSC (LTPGABA). The D1 subtype of dopamine receptors was found to increase
GABAA-IPSC through a G-protein-independent way that was mediated by activation of Srctyrosine kinases. Most commonly, the effects of D1 receptors are mediated by coupling to G"s/olf ,
which causes sequential activation of the cAMP/PKA pathway. However, cAMP/PKA
independent pathways are known to exist, specifically in the striatum and amygdala, where the
48
receptors are coupled to phospholipase C through the interaction of G"q (Jin et al., 2001; Neve et
al., 2004). Yet, surprisingly, both PLC and G-protein inhibitors failed to block the D1-induced
LTPGABA. Currently, there have been no comprehensive studies or findings on D1 signaling
independent of G-proteins. However, in a study conducted on the inhibitory interneurons of the
lateral amygdala, Loretan and colleagues found that D1-induced increase in inhibitory
transmission in these cells was G-protein independent, as they were unable to block the D1 effect
by application of the G-protein antagonist suramine. Remarkably, as in our study, they found that
the D1-induced increase in inhibitory transmission was mediated by the tyrosine kinase Src
(Loretan et al., 2004). These results are particularly exciting as it highlights a novel signal
transduction pathway through which D1 receptors modulate inhibitory transmission, and it is
possible that this D1 linked activation of Src tyrosine kinases is a more common feature found in
the extended amygdala. However one distinguishing feature between our study and Loretan and
colleagues is that the observed D1 c-Src mediated increase in inhibitory transmission in our study
was not seen in naïve conditions but only after rat’s self-administered cocaine.
Furthermore, an increasing number of observations suggest that metabotropic receptors
do not necessarily associate with G-proteins, but in many cases these receptors transduce
signaling pathways through G-protein independent mechanisms. Metabotropic receptors can
interact with a wide variety of intracellular molecules in addition to G-proteins, which include
associated cytoskeletal, adaptor, and chaperone proteins. For example, the adaptor molecule %arrestin binds to many metabotropic receptors and is primarily involved in targeting these
receptors for endocytosis. However, %-arrestin has also been shown to couple metabotropic
receptors to the activation of Src-tyrosine kinases and to facilitate the formation of
multimolecular complexes. For instance, metabotropic glutamate receptors, cholinergic receptors,
49
adrenergic receptors and peptidergic receptors, all commonly coupled to G-proteins, have all been
shown to be associated with non-G-protein, Src-dependent signalling pathways. (Heuss et al.,
1999, Heuss and Gerber, 2000). Further elucidation of the D1-signalling pathway and whether or
not %-arrestin is involved remains an interesting and open avenue for further study.
Based on the observation that the D1-induced LTPGABA was blocked by intracellular
application of a Src-tyrosine kinase antagonist in the recording pipette, we concluded that the D1
receptor was located post-synaptically. However, the measured LTPGABA was associated with
modifications in the coefficient of variation and paired pulse ratio of evoked GABAA IPSCs,
suggesting that it is maintained by persistently increased GABA release from pre-synaptic
terminals. Therefore to explain this apparent contradiction we propose the existence of a currently
unknown retrograde messenger that is released from the post-synaptic neuron, upon D1 receptor
activation, and travels backward to increase GABA release from presynaptic terminals. There are
a number of possible retrograde messengers that are known to increase the release of GABA, one
particularly interesting candidate is the gas, nitric oxide (NO). Recently, NO was discovered to
play a role in activity dependent long-term potentiation of GABAergic synapses in the VTA
(Nugent et al., 2007).
Thirdly, due to cocaine self-administration there was an emergence of a new postsynaptic D2-like component, whose functional role seems to inhibit the D1 receptor increase of
GABAA-IPSCs. Interestingly, the D3 receptor subtype of dopamine receptors, are expressed postsynaptically in the terminal sites of the mesolimbic pathway, largely overlapping with dopamine
D1 receptors within these regions (Le Moine and Bloch, 1996). A multitude of behavioral
evidence suggests that one possible function of the D3 receptor is to down-regulate excessive
transmission at postsynaptic D1 receptors. For instance, mutant mice that lack functional D3
50
receptors exhibit escalated locomotor activity when the mice received repetitive D1 stimulation
(Xu et al., 1997). Furthermore, D3 deficient mice displayed other neuroadaptive changes such as
increased behavioral responses to cocaine, opiates, and methamphetamine (Chen et al. 2006; Le
Foll et al. 2002; Narita et al. 2003). The findings that D3 mutants exhibit accelerated behavioral
sensitization in the presence of D1 agonists and drugs indicates that D3 receptors may regulate
addiction circuitry by negatively regulating D1 receptor-dependent signals. It remains an
interesting possibility that the D2-like component antagonizing the D1 receptor increase of
GABAA-IPSCs in our study is in fact the D3 receptor subtype, upregulated along with the D1
receptors as a compensatory mechanism to down-regulate excessive transmission by postsynaptic
D1 receptors. However, due to the absence of selective D3 and D2 receptor subtype antagonists
that can be used at relevant electrophysiological concentrations, currently this hypothesis is not
testable.
In conclusion, this study identified robust alterations in the effects of dopamine on
inhibitory synaptic transmission following chronic, voluntary cocaine intake in rats in a largely
neglected DA receptive region of the mesolimbic system. The effect of DA switched through the
combination of impaired pre-synaptic D2-like component with de novo addition of post-synaptic
D1- and D2-like effects. Furthermore, these alterations were not observed with passive drug
exposure or self-administration of a natural reward, suggesting a specific role in voluntary drug
intake within the BST. Finally, we demonstrated that the D1 induced increase in GABAA-IPSC
involved Src-tyrosine kinases in a G-protein-independent, highlighting a novel signal
transduction pathway through which D1 receptors modulate inhibitory transmission, which may
possibly serve as a potential target for pharmacological treatment of addiction in the future.
51
Chapter 5 Future Directions
Future studies should be directed towards further characterizing the D1Rmediated increase in inhibitory transmission at the molecular, cellular, and neural network levels.
We have shown that the D1-mediated signaling pathway was mediated through c-Src in a Gprotein independent way, and likely involves a retrograde messenger that travels backward to
increase GABA release at pre-synaptic terminals. There are probably multiple molecular
intermediates that are involved in this signaling pathway, and these results raise interesting
questions such as: What are the molecular components that allow D1R to activate c-Src tyrosine
kinases? What are the signaling pathways engaged by c-Src that result in the release of a
retrograde messenger? What is the identity of the retrograde messenger?
As mentioned above, currently in the literature there is an absence of information regarding
D1R signaling independent of G-proteins. However there is a plethora of proteins that may act as
intermediates between D1R and c-Src independent of G-proteins. The most likely candidates are
adaptor proteins termed arrestins, since they bind to activated D1R and they have been shown to
link ligand-activated metabotropic receptors to c-Src (Heuss et al., 1999, Heuss and Gerber,
2000). Other adaptor or scaffolding proteins interacting with D1R include postsynaptic density 95
(PSD95) or neurofilament-M (NF-M) (Wang et al., 2008). It would be an interesting avenue of
further study to determine if these specific molecular intermediates are involved in the D1R-c-Src
increase in inhibitory transmission.
With regards to the identity of the retrograde messenger, recent evidence demonstrated
that nitric oxide (NO) is involved in a form of activity-dependent plasticity of GABA synapses
52
(Nugent et al., 2007). Given that the BST has high level of NO synthase (NOS), it is thus possible
that NO contributes to our D1R increase in inhibitory transmission (Vincent and Kimura, 1992).
Since the ovBST is also rich in CRF and neurotensin (NT) positive neurons, these neuropeptides
may also act as retrograde messengers (Shimada et al., 1989). Both CRF and NT were shown to
facilitate GABA transmission in the CNS (Kash and Winder, 2006; Tanganelli et al., 1994). In
fact, CRF increases GABA-IPSC in the ventral BST (Kash and Winder, 2006). The possibility of
these molecules acting as the retrograde messenger activated by D1R-cSrc should be investigated
in the near future.
All our experiments were conducted using whole-cell patch clamp in brain slices, and
there are two inherent limitations associated with this assay. First this assay is used to study the
neurophysiology at the cellular level, therefore the effects of D1R signaling on inhibitory
transmission cannot be extrapolated to the neuronal network level. Secondly, using isolated
preparations devoid of any external inputs raises questions as to how closely this assay resembles
physiological conditions in live animals. Therefore a future study should evaluate the
consequence of D1R signaling on the local neuronal network activity through the means of in
vivo recordings in anesthetized animals.
Finally, intra-BST pharmacological or gene silencing interventions of these molecular
intermediates will tell us whether the observed neural mechanisms effectively contribute to
specific aspects of addictive behaviors. These results will help identify potential therapeutic
targets and may help in controlling compulsive drug use.
53
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