Brigham Young University Brigham Young University BYU ScholarsArchive BYU ScholarsArchive Theses and Dissertations 2009-12-10 Cocaine and Mefloquine-induced Acute Effects in Ventral Cocaine and Mefloquine-induced Acute Effects in Ventral Tegmental Area Dopamine and GABA Neurons Tegmental Area Dopamine and GABA Neurons David Wilbanks Allison Brigham Young University - Provo Follow this and additional works at: https://scholarsarchive.byu.edu/etd Part of the Neuroscience and Neurobiology Commons BYU ScholarsArchive Citation BYU ScholarsArchive Citation Allison, David Wilbanks, "Cocaine and Mefloquine-induced Acute Effects in Ventral Tegmental Area Dopamine and GABA Neurons" (2009). Theses and Dissertations. 2362. https://scholarsarchive.byu.edu/etd/2362 This Dissertation is brought to you for free and open access by BYU ScholarsArchive. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of BYU ScholarsArchive. For more information, please contact [email protected], [email protected].
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Brigham Young University Brigham Young University
BYU ScholarsArchive BYU ScholarsArchive
Theses and Dissertations
2009-12-10
Cocaine and Mefloquine-induced Acute Effects in Ventral Cocaine and Mefloquine-induced Acute Effects in Ventral
Tegmental Area Dopamine and GABA Neurons Tegmental Area Dopamine and GABA Neurons
David Wilbanks Allison Brigham Young University - Provo
Follow this and additional works at: https://scholarsarchive.byu.edu/etd
Part of the Neuroscience and Neurobiology Commons
BYU ScholarsArchive Citation BYU ScholarsArchive Citation Allison, David Wilbanks, "Cocaine and Mefloquine-induced Acute Effects in Ventral Tegmental Area Dopamine and GABA Neurons" (2009). Theses and Dissertations. 2362. https://scholarsarchive.byu.edu/etd/2362
This Dissertation is brought to you for free and open access by BYU ScholarsArchive. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of BYU ScholarsArchive. For more information, please contact [email protected], [email protected].
COCAINE AND MEFLOQUINE-INDUCED ACUTE EFFECTS IN VENTRAL
TEGMENTAL AREA DOPAMINE AND GABA NEURONS
David W. Allison
Department Physiology and Developmental Biology
Doctor of Philosophy
The aim of the two studies presented here was to evaluate the effects of cocaine and mefloquine (MFQ) on γ-aminobutyric acid (GABA) and dopamine (DA) neurons in the ventral tegmental area (VTA). Cocaine: In vivo, lower doses of intravenous cocaine (0.25-0.5 mg/kg), or methamphetamine (METH), enhanced VTA GABA neuron firing rate via D2/D5 receptor activation. Higher cocaine doses (1.0-2.0 mg/kg) inhibited their firing rate. Cocaine and lidocaine inhibited the firing rate and spike discharges induced by stimulation of the internal capsule (ICPSDs) at dose levels 0.25-2 mg/kg (IC50 1.2 mg/kg), but neither DA nor METH reduced ICPSDs. In VTA GABA neurons in vitro, cocaine reduced (IC50 13 µM) current-evoked spikes and sodium currents in a use-dependent manner. In VTA DA neurons, cocaine reduced IPSCs (IC50 13 µM), increased IPSC paired-pulse facilitation, and decreased sIPSC frequency, without affecting mIPSC frequency or amplitude. These findings suggest cocaine reduces activity-dependent GABA release on DA neurons in the VTA, and that cocaine’s use-dependent blockade of VTA GABA neuron voltage-sensitive sodium channels (VSSCs) may synergize with its DAT inhibiting properties to enhance mesolimbic DA transmission implicated in cocaine reinforcement. Mefloquine: Mefloquine (MFQ) is an anti-malarial agent, Connexin-36 (Cx36) gap junction blocker, 5-HT3 antagonist, and calcium ionophore. Mounting evidence of a Cx36-mediated VTA GABA neuron syncytium suggests MFQ-related dysphoria may attribute to its gap junction blocking effects on VTA synaptic homeostasis. We observed that MFQ (25 µM) increased DA neuron spontaneous IPSC frequency 6 fold, and mIPSC 3 fold. Carbenoxolone (CBX, 100 µM) only increased sIPSC frequency 2 fold, and did not affect DA mIPSC frequency. Ondansetron did not mimic MFQ. Additionally, MFQ did not affect VTA DA evoked IPSC paired pulse ratio (PPR). However, Mefloquine did induce a 3.5 fold increase in bath-applied GABA current. Remarkably, MFQ did not affect VTA GABA neuron inhibition. At VTA DA neuron excitatory synapses MFQ increased sEPSC frequency in-part due to an increase in the AMPA/NMDA ratio. These finding suggest MFQ alters VTA synapses differentially depending on neuron and synapse type, and that these alterations appear to involve MFQ’s gap junction blocking and calcium ionophore actions.
I begin as my life did, with my parents, Judge W. and Frieda H. Allison. They gave me the gift
of life, love, and the desire to live up to my potential. What has appeared to be a good set of
genes hasn’t hurt either.
My loving wife Linda gave me the time, encouragement, and never-ending supply of
forgiveness needed to make mistakes and prove to myself I could do this. This work is no
exception to all the good things in my life; it found its beginning in her.
In many ways I will always owe a debt of gratitude to Scott C. Steffensen for his unyielding
fortitude as a mentor and friend. His ability to transcend the X, Y, and Z axes has made all the
difference.
In addition I thank the members of my committee, Connie Provost, and C. Fernando Valenzuela
(University of New Mexico) for providing technical and spiritual guidance. I also thank the
Board of Directors of Brigham Young University, the tithe-payers of the Church of Jesus Christ
of Latter-day Saints, and the many donors to Brigham Young University for providing the
platform to conduct this research. I acknowledge the financial support of the United States
Government through the National Institute on Alcohol Abuse and Alcoholism (NIAAA).
iv
TABLE OF CONTENTS List of Figures ............................................................................................................................. vi
Chapter 2. Cocaine disinhibits dopamine neurons in the VTA via blockade of GABA neuron voltage-sensitive sodium channels .................................................................................................7
Effects of Systemic Cocaine, Cocaine Methiodide, Dopamine, Lidocaine and Methamphetamine on VTA GABA Neuron Firing Rate in vivo ..................................19
Dopamine Pharmacology of Cocaine Effects on VTA GABA Neuron Firing Rate in vivo ................................................................................................................................19
Effects of Systemic Cocaine, Cocaine Methiodide, Dopamine, Lidocaine and Methamphetamine on VTA GABA Neuron Evoked Spikes in vivo.............................20
Dopamine Pharmacology of Cocaine Effects on VTA GABA Neuron Evoked Spikes in vivo ............................................................................................................................22
Expression of Select Genes in the Dopamine vs GABA Neurons in Mature Rats: Quantitative Single-cell RT-PCR..................................................................................22
Effects of Cocaine on Current-Evoked VTA GABA Neuron Spikes in vitro...............23
Effects of Cocaine on VTA GABA Neuron Sodium Currents in vitro.........................23
Effects of Cocaine on VTA DA Neuron Evoked and Spontaneous IPSCs in vitro ......25
Chapter 3. Mefloquine disrupts VTA synaptic activity via blockade of Cx36 gap junctions and disruption of calcium homeostasis ...............................................................................................41
Time Course of Mefloquine Effects on VTA DA sIPSC Frequency ............................48
Mefloquine Effects on VTA DA sIPSC Frequency, Amplitude, and Inter-event Interval in WT and Cx36 Mice .....................................................................................49
Mefloquine Effects on VTA DA mIPSC Frequency, Amplitude, and Inter-event Interval in WT and Cx36 Mice......................................................................................51
Neither Presoaking nor Bath-application of Mefloquine Alters VTA DA Neuron Evoked IPSC Paired-pulse Ratio or Primary Current Amplitude .................................53
vi
Mefloquine Increases VTA DA Neuron GABA(A) Receptor Current .........................55
Mefloquine Does Not Affect VTA GABA Neuron sIPSC Frequency or Paired-pulse Ratio ..............................................................................................................................55
Mefloquine Increases VTA DA Neuron sEPSC Frequency in Wildtype and Cx36 KO Mice...............................................................................................................................56
Mefloquine Increases VTA DA Neuron mEPSC Frequency in WT Mice ...................57
Mefloquine Effects on VTA DA and GABA Neuron EPSC PPR and AMPAR to NMDAR Current ratios .................................................................................................58
MFQ Reduces VTA DA Neuron Firing Rate and Current-evoked Spiking .................59
Figure 1: The Mesolimbic Dopamine Pathway ............................................................................6
Chapter 2
Figure 1: DA pharmacology of cocaine effects on VTA GABA neuron firing rate in vivo .....34
Figure 2: Cocaine inhibition of VTA GABA neuron ICPSDs in vivo: Time course and comparison to dopamine agonists, VSSC blockers and DAT inhibitors ..........................................35
Figure 3: Expression of TH and D2 receptors in dopamine versus GABA neurons...................36
Figure 4: Cocaine reduces current-evoked spiking of VTA GABA neurons in vitro .................37
Figure 5: Concentration-dependent and use-dependent effects of cocaine on sodium currents in VTA GABA neurons in vitro .......................................................................................................38
Figure 6: Cocaine reduces evoked and spontaneous GABA inhibitory synaptic transmission to VTA dopamine (DA) neurons in vitro .........................................................................................39
Chapter 3 Figure 1: Time course of Mefloquine effects VTA DA sIPSC frequency ..................................66
Figure 2: Mefloquine effects on VTA DA neuron sIPSC frequency, amplitude, and inter-event interval in WT and Cx36 KO mice ..............................................................................................67
Figure 3: Mefloquine effects on VTA DA mIPSC frequency, amplitude, and inter-event interval in WT and Cx36 KO mice ..............................................................................................68
Figure 4: Neither presoaking nor bath-application of Mefloquine alters VTA DA neuron evoked IPSC paired-pulse ratio or IPSC primary current amplitude ...........................................69
Figure 5: Mefloquine increases VTA DA neuron GABA(A) receptor current...........................70
viii
Figure 6: Mefloquine does not affect VTA GABA neuron sIPSC frequency or paired-pulse ratio...............................................................................................................................................71
Figure 7: Mefloquine increases VTA DA neuron sEPSC frequency in wildtype and Cx36 KO mice ..............................................................................................................................................72
Figure 8: Mefloquine increases VTA DA mEPSC frequency in WT mice ................................73
Figure 9: Mefloquine effects on VTA DA and GABA neuron EPSC PPR and AMPAR to NMDAR current ratio ..................................................................................................................74
Figure 10: MFQ reduces VTA DA neuron firing rate and current-evoked spiking....................75
1
CHAPTER 1
INTRODUCTION
The rationale for the cocaine and mefloquine research presented here is predicated on the
belief that advancement in the understanding of the brain mechanisms underlying the
recreational use and abuse potential of cocaine and other drugs of abuse will pave the way for
more effective treatment strategies that would save lives and resources throughout the world.
The mesolimbic dopamine system
The mesolimbic dopamine (DA) system (Figure 1) consists of projections from the
ventral tegmental area (VTA) to structures associated with the limbic system, primarily the
nucleus accumbens (NAcc). The NAcc (part of the ventral striatum) located in the ventral
forebrain. The can be divided anatomically and by the input it receives into the nucleus
accumbens core and the nucleus accumbens shell. This system has been implicated in the
rewarding effects of drugs of abuse (J. R. Blackburn et al., 1986; R. A. Wise and M. A. Bozarth,
1987; G. F. Koob, 1992; R. A. Wise, 1996), (D. L. McKinzie et al., 1999; R. C. Pierce and V.
Kumaresan, 2006). The VTA is a relatively amorphous midbrain structure that contains at least
three neuron types: the primary type or DA neurons that project to the NAcc, the secondary type
or γ-aminobutyric acid (GABA) neurons that may participate in local circuitry (acting to inhibit
DA neurons) or project to other brain regions, and a population of glutamatergic neurons (T.
Yamaguchi et al., 2007). The medial VTA running rostral to caudal seems hold the greatest
concentration of DA neurons. These DA neurons project to shell region of the nucleus
accumbens The shell portion of the nucleus accumbens appears to be more linked to drug
reward than the core (S. Ikemoto, 2007). Many drugs of abuse act in both the VTA and the
2
NAcc. Most rats and mice will self-administer (SA) cocaine (David et al., 2004, Rodd et al.,
2005), ethanol (E) (Gatto et al., 1994, Rodd-Henricks et al., 2000), nicotine (Museo and Wise,
1981, Laviolette and van der Kooy, 2003), cannabinoids (Zangen et al., 2006), and opiates (M.
A. Bozarth and R. A. Wise, 1981; H. Welzl et al., 1989; V. David and P. Cazala, 1994; D. P.
Devine and R. A. Wise, 1994) into the VTA. Taken together, these data suggest that DA
neurons in the VTA that project to the shell of the NAcc, and the GABA neurons that may
inhibit these DA neurons locally in the VTA, play an important role in mediating addiction to
various drugs of abuse.
GABAergic transmission: A non-dopamine-dependent pathway of addiction
Due to the evidence pointing to DA’s involvement in most drugs of abuse, a “Dopamine
Hypothesis” for reward and addiction has developed on the tenet that DA might be crucial for all
drug reward. The emerging view, however, is that DA is crucial for the rewarding effects of the
psychomotor stimulants such as cocaine and methamphetamine, and is important, but perhaps
not crucial, for the rewarding effects of benzodiazepines, opiates, nicotine, cannabis and ethanol.
The notion that DA-dependent mechanisms are the final common pathway in the processes
mediating drug or natural reward is perhaps too restrictive. It has been theorized that a non-DA-
dependent pathway may function in tandem or independently of this DA-dependent pathway.
We have previously identified a homogeneous population of GABA neurons in the VTA that are
linked by Connexin 36 (Cx36) gap junctions (GJs) (S. H. Stobbs et al., 2004; D. W. Allison et
al., 2006), and form part of a larger syncytium of GABA neurons in the reticular formation (M.
B. Lassen et al., 2007). We theorize that given their anatomical location, and previously
demonstrated role in cocaine (J. H. Ye et al., 1999; W. X. Shi et al., 2004), ethanol (M. Melis et
al., 2002; S. H. Stobbs et al., 2004; J. W. Theile et al., 2009) and opiate reward (H. Vargas-Perez
3
et al., 2009) that VTA GABA neurons are a possible candidate for a non-DA-dependent
addiction pathway.
Plasticity and the process of addiction
An important conceptual advance in understanding addiction is that the process of
addiction shares striking similarities with natural reward plasticity or reward learning and
memory. The newly emerging thought is that the basic mechanisms of reward and learning are
hijacked by drugs of abuse. The two neurotransmitter systems hijacked by drugs of abuse are
the DA system (previously summarized) and the glutamate (GLU) transmitter system, including
their intracellular and genomic targets (A. E. Kelley, 2004). Briefly put, maladaptive changes
such as drug-induced long-term potentiation (LTP) at GLU synapses on VTA DA neurons
translate into the long-term cellular and molecular alterations that form the physiological basis,
or substrates of addiction and addiction-related maladaptive behaviors (G. F. Koob and M. Le
Moal, 1997; J. D. Berke and S. E. Hyman, 2000; S. E. Hyman and R. C. Malenka, 2001; A. E.
Kelley and K. C. Berridge, 2002; S. E. Hyman et al., 2006). In addition to plasticity being
exhibited in the GLUergic and DAergic transmitter systems, plasticity at GABAergic synapses
has now been demonstrated in many brain areas including the VTA. Evidence suggests the
forms of plasticity in the GABAergic transmitter system are similar to those forms expressed in
the DA and GLU transmitter systems (for review F. S. Nugent and J. A. Kauer, 2008). The
adaptability of the GABAergic transmitter system, with its unique characteristic of being partly
regulated through gap junctions, could make it an especially vulnerable target for drugs of
abuse. Dividing these systems into completely separate and distinct pathways however, is
probably an oversimplification. Together they form an integrated system. The
interconnectedness of these transmitter systems underscores the vulnerability of the brain as a
4
whole to pharmacological insult. So while we refer to separate DA-dependent and non-DA-
dependent pathways for the sake of simplicity, they are interconnected, and share points of
convergence and divergence. It is the unique characteristic of its individual parts, and the
interconnected nature of the reward system that makes it such an important focus of addiction
researchers.
Cocaine may act via a dopamine-dependent and non-dopamine-dependent pathway
Cocaine, known as a dopamine transporter (DAT) blocker and local anesthetic, is widely
believed to exert its addictive influence via the DA-dependent pathway, by blocking DAT in the
NAcc leading to an increase in synaptic DA. Little attention has been paid to the question of
whether cocaine’s anesthetic properties contribute to its addictive liability as well. The few
studies that have examined this question have been unable to conclusively demonstrate that
lidocaine, an anesthetic similar to cocaine but without its DAT blocking properties, has any
addictive liability. For this reason, many researchers have ignored the anesthetic properties of
cocaine to the point of not controlling for this property in addiction-related studies. In the
following study we examined the possibility that cocaine’s anesthetic properties may bridge the
gap between the DA-dependent and non-DA-dependent pathways of addiction. This may occur
through cocaine decreasing VTA GABA neuron inhibition of VTA DA neurons via its anesthetic
actions which may lead to an effectual synergizing of the two properties to increase cocaine’s
addictive liability.
Mefloquine as a pharmacological tool
Mefloquine (MFQ) is a selective Cx36 GJ blocker commonly used to study electrical
synapses. MFQ is also used as an anti-malaria agent and reportedly has many adverse side
effects in humans ranging in severity from mild dysphoria to severe psychotic episodes or
5
seizure. In addition to data demonstrating the MFQ blockade of gap junctions, recent studies
have also observed non-specific MFQ effects not usually associated with gap junction blockade.
For us, the most interesting of these effects are the increase in inhibitory and excitatory synaptic
activity (S. J. Cruikshank et al., 2004; C. Zhou et al., 2006), which may stem in part from MFQ’s
ability to disrupt intracellular calcium (G. S. Dow et al., 2003; D. Caridha et al., 2008). In spite
of its many non-specific effects that raise questions regarding its suitability as an effective
pharmacological tool to study gap junctions, we reasoned that some of the heretofore labeled
“non-specific” effects of MFQ might actually be the result of blockade of electrical synapses.
The study of these “non-specific” effects in the VTA may shed light on the physiological
relevance of an electrically coupled network of VTA GABA neurons. Indeed, the research
presented here appears to show that MFQ is the pharmacological discrimination tool needed to
demonstrate the possible mechanism whereby Cx36 gap junctions facilitate VTA GABA neuron
inhibition of mesolimbic DA transmission.
Objectives
The overall objectives of the two studies detailed in this dissertation were: 1) Evaluate the
role of a specific class of VTA GABA neurons in mediating the rewarding properties of cocaine;
2) Determine the role Cx36 gap junction connected VTA GABA neurons play in regulating VTA
DA neuron activity, and how MFQ affects VTA synaptic activity.
6
Figure 1: The Mesolimbic Dopamine Pathway
This diagram depicts the mesolimbic dopamine pathway. The VTA DA neuron and receptors are beige. The
VTA GABA neurons, GABA(A) receptors, and inhibitory input are red. Excitatory input and glutamate receptors
are green. The DA neuron projection target neuron located in the nucleus accumbens is blue. Connexin 36 gap
junctions between VTA GABA neurons are the small purple lines. The dopamine transporters located
presynaptically on DA neurons are black. The two studies described in this dissertation involve the circuitry in
this diagram.
7
CHAPTER 2
Cocaine Disinhibits Dopamine Neurons in the Ventral Tegmental Area via Use-
Dependent Blockade of GABA Neuron Voltage-Sensitive Sodium Channels
(The work presented in this chapter has been published under the same title in the
European Journal of Neuroscience 2008 Nov; 28(10):2028-40. The research diagramed in
Figures 1 and 2 of this chapter are not the work of the author, but have been included to maintain
continuity and context.)
INTRODUCTION
The mesocorticolimbic dopamine (DA) system originating in the ventral tegmental area
(VTA) and projecting to the nucleus accumbens (NAcc) has been implicated in motivated
behaviors, various types of reward, and in the habit-forming actions of addictive drugs including
cocaine (for review see (R. A. Wise, 2004)). The prevailing view is that cocaine’s locomotor
and reinforcing properties (D. C. S. Roberts et al., 1980; G. F. Koob et al., 1994) are mediated
primarily by enhancement of extracellular DA release (Y. L. Hurd et al., 1989; H. O. Pettit and J.
B. Justice, Jr., 1989, 1991; R. A. Wise et al., 1995; S. E. Hemby et al., 1997) via inhibition of the
DA transporter (DAT; (M. J. Kuhar et al., 1991; M. J. Kuhar, 1992; W. L. Woolverton and K. M.
Johnson, 1992)). Cocaine-induced cellular and molecular reshaping of this system may
contribute to learned reinforcement (for review see (S. Jones and A. Bonci, 2005)). The potency
of psychostimulants as positive reinforcers being correlated to their DAT binding affinity (M. C.
Ritz et al., 1987; J. Bergman et al., 1989; K. M. Wilcox et al., 1999), and cocaine’s high affinity
for the DAT (IC50 = 0.3-0.8 µM; (R. B. Rothman et al., 2001)), support this view.
8
Other high-affinity targets for cocaine include voltage-sensitive sodium channels
(VSSCs; (F. H. Gawin and E. H. Ellinwood, Jr., 1988)). It is well established that local
anesthetics, including cocaine, are use-dependent blockers of VSSCs (G. Strichartz, 1976; B. P.
Bean et al., 1983; S. W. Postma and W. A. Catterall, 1984; M. E. O'Leary and M. Chahine,
2002). Although cocaine’s affinity for VSSCs is lower than that for monoamine transporters
(IC50=14-17 µM (M. C. Ritz et al., 1987; A. N. Gifford and K. M. Johnson, 1992)), peak brain
(2-6 min) cocaine levels of 2, 6, 9, and 26 µM can be obtained from single intravenous
reinforcing doses of 0.1, 0.25, 0.5, and 1 mg/kg, respectively (J. S. Fowler et al., 1998). Indeed,
much higher levels of cocaine would be obtained by self-administration, given that response
intervals at these doses are typically shorter than the elimination kinetics of cocaine (H. O. Pettit
et al., 1990; H. T. Pan et al., 1991). The studies demonstrating acute and chronic cocaine-
induced synaptic plasticity in rodent VTA DA neurons utilize a 15 mg/kg intraperitoneal dose
(M. A. Ungless et al., 2001; Q. S. Liu et al., 2005), corresponding to peak brain concentrations of
at least 15 and 25 µM, respectively (H. T. Pan et al., 1991). Recently, it has been hypothesized
that the reinforcing properties of cocaine might involve combined or opposing effects at both the
DAT and VSSCs (E. A. Kiyatkin and P. Leon Brown, 2006).
Since repeated high-dose cocaine exposure induces LTP in VTA DA neurons via a
reduction of GABA-mediated inhibition, a possible role for VTA GABA neurons in cocaine
induced plasticity has emerged (Q. S. Liu et al., 2005). We have identified a homogeneous
population of GABA neurons in the VTA which may serve to inhibit DA neurons (S. C.
Steffensen et al., 1998), and whose firing rate and afferent-evoked responses are enhanced by
DA (S. H. Stobbs et al., 2004; M. B. Lassen et al., 2007). We hypothesized that cocaine would
enhance GABA neuron firing rate and evoked discharges at low reinforcing doses due to its
9
DAT inhibiting properties, but at higher reinforcing doses its use-dependent VSSC blocking
effect would inhibit VTA GABA neurons leading to DA neuron disinhibition.
METHODS
Animal Subjects
Rats were housed two to a cage from the time of weaning (P25), with ad libitum access to
food and water. The room temperature was controlled (22-25 oC) and maintained on a reverse
12 hr light/dark cycle (OFF 08:00 hrs, ON 20:00 hrs). Animal care, maintenance and
experimental procedures were in accordance with the Brigham Young University Animal
Research Committee and met or exceeded National Institutes of Health guidelines for the care
and use of laboratory animals.
Single-unit Recordings in Anesthetized Rats
Extracellular potentials in Isoflurane (1%) anesthetized adult 250-400 g male Wistar rats
(Charles River Laboratory, Hollister, CA) were recorded by a single 3.0 M NaCl filled
micropipette (1-3 MΩ; 1-2 µm inside diameter), cemented 10-20 µm distal to a 4-barrel
micropipette (20-60 MΩ resistance), and amplified and filtered with a MultiClamp 700A
programmable amplifier (Axon Instruments, Union City, CA). Microelectrode assemblies were
oriented into the VTA [from bregma: 5.6-6.5 posterior (P), 0.5-1.0 lateral (L), 7.0-8.5 ventral
(V)] with a piezoelectric inchworm microdrive (Burleigh, Fishers, NY). Single-unit activity was
filtered at 0.3-10 kHz (-3dB) and displayed on Tektronix 2200 digital oscilloscopes. Square-
wave constant current pulses (50-1000 µA; 0.15 msec duration; average frequency, 0.1Hz) were
generated by an IsoFlex constant current isolation unit controlled by a MASTER-8 Pulse
Generator (AMPI, Israel), or by computer. The internal capsule (IC; from bregma: -1.5 AP, 2.5-
10
3.0 ML, 5.0-6.5 V) was stimulated with insulated, bipolar stainless steel electrodes.
Extracellularly recorded action potentials (min 5:1 signal-to-noise ratio) were discriminated with
WPI-121 (Sarasota, Fl) spike analyzers and converted to computer-level pulses.
Characterization of VTA GABA Neurons in vivo
All neurons classified as VTA GABA neurons in vivo were located in the VTA, met the
criteria established in previous studies for spike waveform characteristics and response to IC
stimulation (S. C. Steffensen et al., 1998; S. H. Stobbs et al., 2004; D. W. Allison et al., 2006),
and often were activated and spike-coupled by microelectrophoretic dopamine (DA; (S. H.
Stobbs et al., 2004)). Presumed VTA GABA neurons were characterized by short-duration
(<200 µsec; measured at half-peak amplitude of the spike), initially negative-going, non-bursting
spikes, and were identified by the following IC stimulation criteria (S. C. Steffensen et al., 1998):
Short latency (i.e., 2-5 msec) antidromic or orthodromic activation via single stimulation of the
IC; and multiple spiking following high-frequency (10 pulses, 200 Hz) stimulation of the IC
(ICPSDs; (S. C. Steffensen et al., 1998; S. H. Stobbs et al., 2004; D. W. Allison et al., 2006; M.
B. Lassen et al., 2007)). In all studies, stimulation was performed at a level that produced 50%
maximum VTA GABA neuron ICPSDs. This was accomplished by determining the current
needed to produce the maximum number of ICPSDs at 200 Hz and 10 pulses, and then adjusting
the stimulus intensity until 50% ICPSDs were achieved.
Single-unit Recordings in vivo
Single-unit potentials, discriminated spikes, and stimulation events in vivo were captured
by National Instrument’s NB-MIO-16 digital I/O and counter/timer data acquisition boards
(Austin, TX) and processed by customized National Instruments LabVIEW software in
Macintosh-type computers. Potentials were digitized at 20 kHz and 12-bit voltage resolution.
11
For single-unit activity, all spikes were captured by computer and time stamped. Spontaneous
firing rates were determined on- and off-line by calculating the number of events over a 5 min
epoch, typically 5 min before and at specific intervals after drug injection. Peri-stimulus and
interval-spike histograms were generated off-line using IGOR Pro (WaveMetrics, Lake Oswego,
OR) analysis of the time-stamped data. The duration (msec) and extent (#events/bin) of post-
stimulus permutation of ICPSDs was determined by rectangular integration at specific time
points on the peri-stimulus spike histogram using IGOR Pro analysis software. The minimum
bin width for peri-stimulus spike histograms was 1.0 msec and the number of bins was 1000.
These parameters allow for detection of all phases of pre- and post-stimulus spike activity.
Drug Preparation and Administration in vivo
Cocaine hydrochloride, cocaine methiodide, DA, lidocaine hydrochloride, and
methamphetamine hydrochloride were dissolved in 0.9% saline and administered intravenously
through an indwelling jugular catheter. Given the transient duration of effect of cocaine and
lidocaine on VTA GABA neuron firing rate and ICPSDs, dose-response studies were performed
for these two drugs, as well as for cocaine methiodide, in the same rats with a 40 min interval
between each dose and by randomizing the sequence of dose levels, while dose-response studies
for amphetamine, whose effects on firing rate were more prolonged, were performed in separate
rats. For in situ microelectrophoretic application of drugs in the VTA, DA (10 mM), eticlopride
(ICPSDs), showing cocaine’s ability to block a putative measure of electrical coupling. In vitro,
cocaine reduced current-driven spikes in VTA GABA neurons via its block of VSSCs leading to
a marked decreased VTA GABA neuron inhibition of VTA DA neurons; demonstrating
77
cocaine’s addictive liability may be greatly increased if its DAT blocking and anesthetic actions
can combine to simultaneously increase DA in the NAcc by blocking DAT and increase DA
release by acting to disinhibit DA neurons in the VTA. This study underscores cocaine’s
dangers as an illicit compound, provides direct evidence of VTA GABA neuron regulation of
mesolimbic DA, and highlights the important role electrical connectivity of VTA GABA neurons
plays in regulating VTA DA neurons.
It should be noted that any research examining the role that electrically-coupled VTA
GABA neurons play in addiction and regulation of the mesolimbic DA system encounters the
same stumbling blocks: the lack of specific GJ blockers and the inability to definitively indentify
GABA neurons in vitro. The study presented in Chapter 3 attempts to overcome these stumbling
blocks by combining the most selective, albeit non-specific, GJ blocking drugs, mefloquine
(MFQ) and carbenoxolone (CBX), with the use of GAD67-GFP and Cx36 KO mouse models.
Some studies have relied exclusively on the use of Cx36 KO mice to study electrical synapses.
By utilizing wildtype and KO mice in conjunction with more than one GJ blocking drug we
avoid many of the pitfalls associated with using only KO mice.
The integral role that electrically-connected VTA GABA neurons play in regulating VTA
DA neurons was addressed in Chapter 3. This research demonstrated that inhibition of VTA DA
neurons could be increased significantly by decreasing the electrical connectivity of VTA GABA
neurons. To our knowledge we are the first to demonstrate GJs playing a critical role in
regulating a well-established physiologically relevant activity in the brain such as excitatory and
inhibitory synaptic activity. With this data, and other evidence suggesting electrical coupling
may play a role in many phenomena such as: rhythmic activity (N. Berretta et al., 2001; M. A.
Long et al., 2002; M. A. Long et al., 2005); propagation of slow calcium waves (L. R. Wolszon
78
et al., 1994); learning and memory; epilepsy (V. M. Nemani and D. K. Binder, 2005); etc (for
review, B. W. Connors and M. A. Long, 2004), we are loath to envision GJs as anything less
than critical brain machinery. This work supports the belief that by virtue of their dynamic range
(high discharge rate and short refractory period), widespread distribution to structures implicated
in drug reward, and their electrical network properties, VTA GABA neurons are a prime target
and focal point for addiction research.
79
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Curriculum vitae
David W. Allison
Personal Data Graduate Student, PhD Candidate (Neuroscience)
Department of Psychology and Neuroscience Center Brigham Young University, 1290 SWKT Provo, Utah 84602 (801) 422-6089 [email protected]
Education
2010 1997
PhD Neuroscience, Brigham Young University, Utah BS in Biochemistry/ Cell Biology, University of California San Diego
Professional Experience
2006-2010 2001-2006 1998-2001
Awards
Graduate Student, (PhD candidate- Neuroscience) Faculty Research Associate, Brigham Young University Research Technician II, The Scripps Research Institute 2009/2010 Brigham Young University Graduate Student Fellowship Award- “Cocaine disinhibits VTA dopamine neurons via its anesthetic effects”
Publications Refereed Journal Articles
Allison, D.W., Ellefsen, K.L., Askew, C.E., Wilcox, B.S., Sandoval, S.S., Hansen, D.M., Yanagawa, Y., Kaneke, T., Steffensen, S.C. Cocaine and Mefloquine-induced Acute Effects in Ventral Tegmental Area Dopamine and GABA Neurons. J Neurosci (in submission). Vargas-Perez H., Ting-A-Kee R., Walton C.H., Hansen D.M., Razavi R., Clarke L., Bufalino M.R., Allison D.W., Steffensen S.C., van der Kooy D. Ventral Tegmental Aream BDNF Induces Opiate-Dependent-Like Reward in Naïve Rats. Science. 2009 May 28. Hales, K.H., Bradley, K.D., Allison, D.W., Taylor S.R., Hansen, D.M., Yorgason, J.T., Walton, C.H., Sudweeks, S.N. and Steffensen, S.C. Physiological and molecular adaptation of the GABA neurons in the ventral tegmental area to chronic alcohol consumption. Alcohol Clin Exp Res. 2008 Dec (in press) Steffensen, S.C., Taylor, S.R., Horton, M.L., Barber, E.N., Lyle, L.T., Stobbs, S.H. and Allison, D.W. Cocaine disinhibits dopamine neurons in the ventral tegmental area via use-
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dependent blockade of GABA neuron voltage-sensitive sodium channels. Eur J Neurosci. 2008 Nov; 28(10):2028-40. Steffensen, S.C., Hales, K., Jones, M., Allison, D.W. Dehydroepiandrosterone sulfate and estrone sulfate reduce GABA recurrent inhibition in the hippocampus via muscarinic receptors. Hippocampus (2006) 16(12):1080-90 Allison, D.W., Ohran, A.L., Stobbs, S.H., Mameli, M., Valenzuela, F., Sudweeks, S.N., Ray, A.P., Henriksen, S.J. and Steffensen, S.C. Involvement of Connexin-36 Gap Junctions in Ventral Tegmental Area GABA neuron excitability and electrical coupling. Synapse, In Press (2006) Stobbs, S.H., Ohran, A.L., Lassen, M.B., Allison, D.W., Brown, J.E. and Steffensen, S.C., Ethanol suppression of ventral tegmental area GABA neuron electrical transmission involves NMDA receptors. J Pharmacol Exp Ther. 311 (2004) 282-289 Krucker T., Siggins G.R., McNamara R.K., Lindsley K.A., Dao A., Allison D.W., De Lecea L, Lovenberg TW, Sutcliffe JG, Gerendasy D.D. Targeted disruption of RC3 reveals a calmodulin-based mechanism for regulating metaplasticity in the hippocampus. Journal of Neuroscience. (2002) Jul 1; 22(13): 5525-35 Dissertation (in preparation) Allison, D.W. Cocaine and Mefloquine-induced Acute Effects in Ventral Tegmental Area Dopamine and GABA Neurons. Submitted to the faculty of Brigham Young University, Provo Utah 2009.
Abstracts Allison, D. W., Askew, C. E., Ellefsen, K. L., Hamaker, A. N., Steffensen S. C. Ethanol effects on midbrain dopamine neurons in connexin 36 knock out mice. Soc. Neurosci. Absts 2009 233.50 Steffensen, S. C., Bradley, K. D., Allison, D. W., Hansen, D. M., Wilcox, J. D., Foote, M., Hoyt, B., Yorgason, J. T. The role of connexin-36 gap junctions in alcohol intoxication and reward. Soc. Neurosci. Absts 2009 233.60 Allison, D.W., Hansen, D.M., Wilcox, J.D., Yorgason, J.T., Steffensen, S.C. Effects of dopamine receptor antagonists on ethanol-induced modulation of dopamine release in the nucleus accumbens. Alcoholism: Clin. Exp. Res. (2009) 33(6) 037 Steffensen, S. C., Flemming, D. E., Taylor, S. R., Hansen, D.M., Walton, C.H., Allison, D. W. Cocaine disinhibits dopamine neurons in the ventral tegmental area via use-dependent blockade of GABA neuron voltage-sensitive sodium channels. Soc. Neurosci. Absts 2008 661.20
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Allison, D.W., Taylor, S.R., Askew, C.E., Anderson, E., Partridge, L.D., Valenzuela, C.F., Steffensen, S. C. Pregnenolone sulfate enhances GABA inhibition in the CA1 subfield of the hippocampus. Soc. Neurosci. Absts 2008 589.1 Steffensen, S. C., Allison, D.W., Yorgason, J.T., Hansen, D.M. The retina as a window on alcohol effects on mesolimbic dopamine neurotransmission. Alcoholism: Clin. Exp. Res. (2008) 32(6) 076 Steffensen, S. C., Hales, K., Bradely, K., Trika, A., Hansen, D.M., Yorgason, J.T., Walton, C.H., Thomas, S. J., Taylor, S. R., Askew, C.E., Allison, D.W. Physiological and molecular adaptation of ventral tegmental area GABA neurons to chronic alcohol. Alcoholism: Clin. Exp. Res. (2008) 32(6) 064 Criado, J.R., Lassen, M.B., Allison, D.W., Hansen, D.M., Layton, S.P., Steffensen, S. C. Opiod effects on the dentate gyrus responses are mediated by effects on mu-opioid receptor containing GABA neurons in the ventral tegmental area. Soc. Neurosci. Absts 2007 611.10 Steffensen, S. C., Allison, D.W., Hales, K., Mickelsen, R., Maes, L., Valenzuela, C.F., Effects of acute and chronic ethanol on the excitability and electrical coupling of GABA neurons in the ventral tegmental area. Soc. Neurosci. Absts 2007 611.13 Allison, D.W., Mickelsen, R., Taylor, S. R., Lassen, M. B., Flemming, D. E., Steffensen, S. C., Thomas, S. J. Cocaine reduces the excitability and electrical coupling of VTA GABA neurons via its sodium channel blocking actions. Soc. Neurosci. Absts 2007 912.11 Hales, K., Allison, D.W., Valenzuela, C.F., Maes, L., Steffensen, S. C. Effects of acute and chronic ethanol on the excitability and electrical coupling of GABA neurons in the ventral tegmental area. Alcoholism: Clin. Exp. Res. (2007) 31(6) 538 Allison D.W., Mickelsen, R.S., Steffensen, S. C., Effects of ethanol on the excitability and electrical coupling of GABA neurons in the midbrain. Alcoholism: Clin. Exp. Res. (2007) 31(6) 543 Steffensen, S.C., Barber, E.N., Horton, M.L., Hansen, D.M., Mickelsen, R., Rogers, D.C., Clark, T. and Allison, D.W. Contigent and non-contingent effects of cocaine on the discharge activity and electrical coupling of ventral tegmental area GABA neurons. Soc. Neurosci. Absts 2006 32: 691.2
Allison, D.W., Horton, M.L., Sudweeks, S.N., Valenzuela, F.C. and Steffensen, S.C., Ethanol modulates electrical coupling between GABA neurons in the ventral tegmental area. Soc. Neurosci. Absts (2006) 32: 292.10
Steffensen, S.C., Horton, M.L., and Allison, D.W. Ethanol influences T-type calcium currents and electrical coupling between GABA neurons in the ventral tegmental area. Alcoholism: Clin. Exp. Res. (2006) 30(6) 182A
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Allison, D.W., Hawes, M.E., Horton, M.L., Stobbs, S.H., Sudweeks, S.N., Steffensen, S.C. Ethanol modulates electrical coupling between GABA neurons in the ventral tegmental area. Soc. Neurosci. Absts. (2005) Steffensen S.C., Ohran A.J., Bevan A., Haws M.E., Allison, D.W., and Stobbs S.H. Ethanol modulates electrical coupling between GABA neurons in the ventral tegmental area. Alcoholism. Clin. Exp. Res. (2005) 29(5) 93A Steffensen S.C., Ohran A.L., Stobbs S.H., Allison D.W., Lassen M.B., Ray A.P., Henriksen S.J. Ventral tegmental area GABA neurons form a network of dopamine-sensitive electrical synapses. Soc. Neurosci. Absts. (2004) 30: 272.9 Steffensen, S.C., Stobbs, S.H., Allison, D.W., Lassen, M.B., Brown, J.E., Henriksen, S.J. Ventral tegmental area GABA neurons form a network of dopamine-sensitive electrical synapses: Role in brain stimulation reward. Soc. Neurosci. Absts. (2003) 29: 679.4 Steffensen, S.C., Stobbs, S.H., Allison, D.W., Brown, J.E., Lassen, M.B., Lee, R.S., and Henriksen, S.J. Differential adaptation of ventral tegmental area GABA neurons by ethanol and barbiturates. Alcoholism: Clin. Exp. Res. (2003) 27(5) 56A Krucker, T., Mosley, A., Allison, D.W., Siggins, G.R. Methamphetamine treatment modifies synaptic transmission and plasticity in hippocampal slices from wild type and transgenic mice with cerebral expression of HIV-1 coat protein GP120. Soc. Neurosci. Absts. (2002) 808.18 Meyer, E.P., Allison, D.W., Staufenbiel, M., Siggins, G.R., Krucker T. Three-dimensional reconstruction of early changes in microvascular architecture and morphology in APP23 transgenic mice with neural overexpression of mutant amyloid precursor protein. Soc. Neurosci. Absts. (2001) 321.14 Meyer, E.P., Allison, D.W., Campbell, I.L., Siggins, G.R., Krucker, T. The microvasculature of transgenic mice with glial expression of interleukin 6 shows extensive morphological changes as revealed by vascular corrosion casts. Soc. Neurosci. Absts. (2000) 27.9 Krucker, T., Allison, D.W, Staufenbiel, M., Sommer, B., Sturchler-Pierrat, C., Siggins, G.R. Changes in excitabililty and long-term plasticity in hippocampal CA1 of transgenic mice with neuronal overexpression of mutant amyloid precursor protein. Soc. Neurosci. Absts. (2000) 275.15 Manuscripts in preparation Allison, D.W., Ellefsen, K., Askew, C.L., Steffensen, S.C. Connexin 36 Gap Junctions Play an Integral Role in Regulating Ventral Tegmental Area Synaptic Activity. December 2009 Membership in Professional Societies
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Society for Neuroscience (Member from 2001-present)
Teaching Experience 2001-present Psychology 370: Sensation and Perception
Brigham Young University Department of Psychology
Winter 2009 2001-2003
Neuroscience 205: Neurobiology Brigham Young University Neuroscience Center Neuroscience 480: Advanced Neuroscience Brigham Young University Neuroscience Center
2001-present Neuroscience 481: Advanced Neuroscience Laboratory Brigham Young University Neuroscience Center
2001-present Neuroscience 449R: Research Brigham Young University Neuroscience Center
Licenses held
Amateur radio license, KF7BDU Community professional service