Ventral Tegmental Area Dopamine Neuron Firing Research Articles

AST-001 Improves Social Deficits and Restores Dopamine Neuron Activity in a Mouse Model of Autism.

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder characterized by impaired social communication and social interaction, restricted and repetitive behavior, and interests. The core symptoms of ASD are associated with deficits in mesocorticolimbic dopamine pathways that project from the ventral tegmental area (VTA) to the nucleus accumbens (NAc) and medial prefrontal cortex (mPFC). AST-001 is an investigational product currently in a phase 3 clinical trial for treating the core symptoms of ASD, with L-serine as the API (active pharmaceutical ingredient). Because the causes of ASD are extremely heterogeneous, a single genetic ASD model cannot represent all autism models. In this paper, we used the VPA-exposed model, which is more general and widely used than a single genetic model, but this is also one of the animal models of autism. Herein, we conducted experiments to demonstrate the efficacy of AST-001 as L-Serine that alters the regulation of the firing rate in dopamine neurons by inhibiting small conductance Ca2+-activated K+ channels (SK channels). Through these actions, AST-001 improved sociability and social novelty by rescuing the intrinsic excitabilities of dopamine neurons in VPA-exposed ASD mouse models that showed ASD-related behavioral abnormalities. It is thought that this effect of improving social deficits in VPA-exposed ASD mouse models is due to AST-001 normalizing aberrant SK channel activities that slowed VTA dopamine neuron firing. Overall, these findings suggest that AST-001 may be a potential therapeutic agent for ASD patients, and that its mechanism of action may involve the regulation of dopamine neuron activity and the improvement of social interaction.

Nicotine modulation of the lateral habenula/ventral tegmental area circuit dynamics: An electrophysiological study in rats

Nicotine, the addictive component of tobacco, has bivalent rewarding and aversive properties. Recently, the lateral habenula (LHb), a structure that controls ventral tegmental area (VTA) dopamine (DA) function, has attracted attention as it is potentially involved in the aversive properties of drugs of abuse. Hitherto, the LHb-modulation of nicotine-induced VTA neuronal activity in vivo is unknown. Using standard single-extracellular recording in anesthetized rats, we observed that intravenous administration of nicotine hydrogen tartrate (25–800 μg/kg i.v.) caused a dose-dependent increase in the basal firing rate of the LHb neurons of nicotine-naïve rats. This effect underwent complete desensitization in chronic nicotine (6 mg/kg/day for 14 days)-treated animals. As previously reported, acute nicotine induced an increase in the VTA DA neuronal firing rate. Interestingly, only neurons located medially (mVTA) but not laterally (latVTA) within the VTA were responsive to acute nicotine. This pattern of activation was reversed by chronic nicotine exposure which produced the selective increase of latVTA neuronal activity. Acute lesion of the LHb, similarly to chronic nicotine treatment, reversed the pattern of DA cell activation induced by acute nicotine increasing latVTA but not mVTA neuronal activity. Our evidence indicates that LHb plays an important role in mediating the effects of acute and chronic nicotine within the VTA by activating distinct subregional responses of DA neurons. The LHb/VTA modulation might be part of the neural substrate of nicotine aversive properties. By silencing the LHb chronic nicotine could shift the balance of motivational states toward the reward.This article is part of the special Issue on ‘Neurocircuitry Modulating Drug and Alcohol Abuse'.

Dual Effect of 5-HT1B/1D Receptors on Dopamine Neurons in Ventral Tegmental Area: Implication for the Functional Switch After Chronic Cocaine Exposure

BackgroundSerotonin (5-HT) 1B/1D receptor (5-HT1B/1DR) agonists undergo an abstinence-induced switch in their effects on cocaine-related behaviors, which may involve changes in modulation of dopamine (DA) neurons in the ventral tegmental area (VTA). However, it is unclear how 5-HT1B/1DRs affect VTA DA neuronal function and whether modulation of these neurons mediates the abstinence-induced switch after chronic cocaine exposure. MethodsWe examined the ability of 5-HT1B/1DRs to modulate D2 autoreceptors (D2ARs) and synaptic transmission in the VTA by slice recording and single unit recording in vivo in naïve mice and in mice with chronic cocaine treatment. ResultsWe report a bidirectional modulation of VTA DA neuronal firing through the interaction of VTA 5-HT1B/1DRs and D2ARs. In both VTA slices and the VTA of anesthetized mice, the 5-HT1B/1DR agonist CP94253 decreased DA neuronal firing rate and evoked excitatory postsynaptic currents to DA neurons in slice. Paradoxically, CP94253 decreased quinpirole-induced inhibition of DA neurons by reducing D2AR-mediated G protein–gated inwardly rectifying potassium current. This manifested decreased GABAA (gamma-aminobutyric acid A) receptor–mediated evoked inhibitory postsynaptic currents in slice, resulting in disinhibition of DA neurons, in opposition to the 5-HT1B/1DR-induced inhibition. This dual effect was verified in chronic cocaine-treated and mild stress-treated, male mice on days 1 and 20 posttreatment. ConclusionsThis study revealed dual effects of CP94253 on VTA DA neurons that are dependent on D2AR sensitivity, with anti-inhibition under normal D2AR sensitivity and inhibition under low D2AR sensitivity. These dual effects may underlie the ability of CP94253 to both enhance and inhibit cocaine-induced behaviors.

Neurotensin receptor 1 deletion decreases methamphetamine self-administration and the associated reduction in dopamine cell firing.

We previously reported that a non-selective pharmacological blockade of neurotensin receptors in the ventral tegmental area (VTA) decreases methamphetamine (METH) self-administration in mice. Here, we explored the consequences of genetic deletion of neurotensin receptor 1 (NtsR1) on METH self-administration and VTA dopamine neuron firing activity. We implanted mice with an indwelling jugular catheter and trained them to nose-poke for intravenous infusions of METH. Mice with NtsR1 deletion (KO) acquired self-administration similar to wildtype (WT) and heterozygous (HET) littermates. However, in NtsR1 KO and HET mice, METH intake and motivated METH seeking decreased when the response requirement was increased to a fixed ratio 3 and when mice were tested on a progressive ratio protocol. After completion of METH self-administration, single cell in vivo extracellular recordings of dopamine firing activity in the VTA were obtained in anesthetized mice. Non-bursting dopamine neurons from KO mice fired at slower rates than those from WT mice, supporting an excitatory role for NtsR1 on VTA dopamine neuronal activity. In WT mice, a history of METH self-administration decreased dopamine cell firing frequency compared with cells from drug-naïve controls. NtsR1 KO and HET mice did not exhibit this decline in dopamine cell firing activity after METH experience. We also observed an increase in population activity following METH self-administration that was strongest in the WT group. Our results suggest a role for NtsR1 in METH-seeking behavior and indicate that ablation of NtsR1 prevents the detrimental effects of prolonged METH self-administration on VTA dopamine cell firing frequency.

Mechanisms of Cannabinoid CB2 Receptor-Mediated Reduction of Dopamine Neuronal Excitability in Mouse Ventral Tegmental Area

We have recently reported that activation of cannabinoid type 2 receptors (CB2Rs) reduces dopamine (DA) neuron excitability in mouse ventral tegmental area (VTA). Here, we elucidate the underlying mechanisms. Using cell-attached recording in VTA slices, bath-application of CB2R agonists (JWH133 or five other CB2R agonists) significantly reduced VTA DA neuron action potential (AP) firing rate. Under patch-clamp whole-cell recording model, JWH133 (10 µM) mildly reduced the frequency of miniature excitatory postsynaptic currents (mEPSCs) but not miniature inhibitory postsynaptic currents (mIPSCs). JWH133 also did not alter evoked EPSCs or IPSCs. In freshly dissociated VTA DA neurons, JWH133 reduced AP firing rate, delayed AP initiation and enhanced AP after-hyperpolarization. In voltage-clamp recordings, JWH133 enhanced M-type K+ currents and this effect was absent in CB2-/- mice and abolished by co-administration of a selective CB2R antagonist (AM630). CB2R-mediated inhibition in VTA DA neuron firing can be mimicked by M-current opener and blocked by M-current blocker. In addition, enhancement of neuronal cAMP by forskolin reduced M-current and increased DA neuron firing rate. Finally, pharmacological block of synaptic transmission by NBQX, D-APV and picrotoxin in VTA slices failed to prevent CB2R-mediated inhibition, while intracellular infusion of guanosine 5'-O-2-thiodiphosphate (GDP-β-S) through recording electrode to block postsynaptic G-protein function prevented JWH133-induced reduction in AP firing. Collectively, our results suggest that CB2Rs modulate VTA DA neuron excitability mainly through an intrinsic mechanism, including a CB2R-mediated reduction of intracellular cAMP, and in turn enhancement of M-type K+ currents. Funding Statement: This research was supported by the Barrow Neuroscience Foundation, the BNI-BMS Seed Fund, the CNSF (81771437), and the National Institute on Drug Abuse, Intramural Research Program. Declaration of Interests: Dr. Ma, ZG reports no disclosures, Dr. Gao, FF reports no disclosures, Dr. Larsen, B. reports no disclosures, Dr. Gao M reports no disclosures, D. Chen, DJ reports no disclosures, Xiaokuang Ma reports no disclosures, Dr. Qiu, SF reports no disclosures, Dr. Zhou Y reports no disclosures, Dr. Xie, JX reports no disclosures, Dr. Xi, ZX reports no disclosures, Dr. Wu J reports no disclosures. Ethics Approval Statement: All experimental procedures were approved by the Institutional Animal Care and Use Committee at the Barrow Neurological Institute.

Mechanisms of cannabinoid CB2 receptor-mediated reduction of dopamine neuronal excitability in mouse ventral tegmental area.

BackgroundWe have recently reported that activation of cannabinoid type 2 receptors (CB2Rs) reduces dopamine (DA) neuron excitability in mouse ventral tegmental area (VTA). Here, we elucidate the underlying mechanisms.MethodsPatch-clamp recordings were performed in mouse VTA slices and dissociated single VTA DA neurons.FindingsUsing cell-attached recording in VTA slices, bath-application of CB2R agonists (JWH133 or five other CB2R agonists) significantly reduced VTA DA neuron action potential (AP) firing rate. Under the patch-clamp whole-cell recording model, JWH133 (10 μM) mildly reduced the frequency of miniature excitatory postsynaptic currents (mEPSCs) but not miniature inhibitory postsynaptic currents (mIPSCs). JWH133 also did not alter evoked EPSCs or IPSCs. In freshly dissociated VTA DA neurons, JWH133 reduced AP firing rate, delayed AP initiation and enhanced AP after-hyperpolarization. In voltage-clamp recordings, JWH133 (1 μM) enhanced M-type K+ currents and this effect was absent in CB2−/− mice and abolished by co-administration of a selective CB2R antagonist (10 μM, AM630). CB2R-mediated inhibition in VTA DA neuron firing can be mimicked by M-current opener (10 μM retigabine) and blocked by M-current blocker (30 μM XE991). In addition, enhancement of neuronal cAMP by forskolin (10 μM) reduced M-current and increased DA neuron firing rate. Finally, pharmacological block of synaptic transmission by NBQX (10 μM), D-APV (50 μM) and picrotoxin (100 μM) in VTA slices failed to prevent CB2R-mediated inhibition, while intracellular infusion of guanosine 5'-O-2-thiodiphosphate (600 μM, GDP-β-S) through recording electrode to block postsynaptic G-protein function prevented JWH133-induced reduction in AP firing.InterpretationOur results suggest that CB2Rs modulate VTA DA neuron excitability mainly through an intrinsic mechanism, including a CB2R-mediated reduction of intracellular cAMP, and in turn enhancement of M-type K+ currents.FundThis research was supported by the Barrow Neuroscience Foundation, the BNI-BMS Seed Fund, and CNSF (81771437).

HCN2 channels in the ventral tegmental area regulate behavioral responses to chronic stress.

Dopamine neurons in the ventral tegmental area (VTA) are powerful regulators of depression-related behavior. Dopamine neuron activity is altered in chronic stress-based models of depression, but the underlying mechanisms remain incompletely understood. Here, we show that mice subject to chronic mild unpredictable stress (CMS) exhibit anxiety- and depressive-like behavior, which was associated with decreased VTA dopamine neuron firing in vivo and ex vivo. Dopamine neuron firing is governed by voltage-gated ion channels, in particular hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. Following CMS, HCN-mediated currents were decreased in nucleus accumbens-projecting VTA dopamine neurons. Furthermore, shRNA-mediated HCN2 knockdown in the VTA was sufficient to recapitulate CMS-induced depressive- and anxiety-like behavior in stress-naïve mice, whereas VTA HCN2 overexpression largely prevented CMS-induced behavioral deficits. Together, these results reveal a critical role for HCN2 in regulating VTA dopamine neuronal activity and depressive-related behaviors.

α6 subunit-containing nicotinic receptors mediate low-dose ethanol effects on ventral tegmental area neurons and ethanol reward.

Dopamine (DA) neuron excitability is regulated by inhibitory GABAergic synaptic transmission and modulated by nicotinic acetylcholine receptors (nAChRs). The aim of this study was to evaluate the role of α6 subunit-containing nAChRs (α6*-nAChRs) in acute ethanol effects on ventral tegmental area (VTA) GABA and DA neurons. α6*-nAChRs were visualized on GABA terminals on VTA GABA neurons, and α6*-nAChR transcripts were expressed in most DA neurons, but only a minority of VTA GABA neurons from GAD67 GFP mice. Low concentrations of ethanol (1-10mM) enhanced GABAA receptor (GABAA R)-mediated spontaneous and evoked inhibition with blockade by selective α6*-nAChR antagonist α-conotoxins (α-Ctxs) and lowered sensitivity in α6 knock-out (KO) mice. Ethanol suppression of VTA GABA neuron firing rate in wild-type mice in vivo was significantly reduced in α6 KO mice. Ethanol (5-100mM) had no effect on optically evoked GABAA R-mediated inhibition of DA neurons, and ethanol enhancement of VTA DA neuron firing rate at high concentrations was not affected by α-Ctxs. Ethanol conditioned place preference was reduced in α6 KO mice compared with wild-type controls. Taken together, these studies indicate that relatively low concentrations of ethanol act through α6*-nAChRs on GABA terminals to enhance GABA release onto VTA GABA neurons, in turn to reduce GABA neuron firing, which may lead to VTA DA neuron disinhibition, suggesting a possible mechanism of action of alcohol and nicotine co-abuse.

Cocaine Exposure Enhances the Activity of Ventral Tegmental Area Dopamine Neurons via Calcium-Impermeable NMDARs.

We show that cocaine-evoked synaptic changes onto ventral tegmental area (VTA) dopamine (DA) neurons leads to long-lasting increases in their burst firing. This increase is due to impaired function of Ca2+-activated small-conductance calcium-dependent potassium (SK) channels; SK channels regulate firing of VTA DA neurons, but this regulation was absent after cocaine. Cocaine exposure drives the insertion of GluN3A-containing NMDARs onto VTA DA neurons. These receptors are Ca2+-impermeable, and thus SK channels are not efficiently activated by synaptic activity. In GluN3A knock-out mice, cocaine did not alter SK channel function or VTA DA neuron firing. This study directly links synaptic changes to increased intrinsic excitability of VTA DA neurons after cocaine, and explains how acute cocaine induces long-lasting remodeling of the mesolimbic DA system.

The Nucleus Reuniens of the Midline Thalamus Gates Prefrontal-Hippocampal Modulation of Ventral Tegmental Area Dopamine Neuron Activity.

The circuitry mediating top-down control of dopamine (DA) neurons in the ventral tegmental area (VTA) is exceedingly complex. Characterizing these networks will be critical to our understanding of fundamental behaviors, such as motivation and reward processing, as well as several disease states. Previous work suggests that the medial prefrontal cortex (mPFC) exerts a profound influence on VTA DA neuron firing. Recently, our group reported that inhibition of the infralimbic subdivision of the medial prefrontal cortex (ilPFC) increases the proportion of VTA DA neurons that are spontaneously active (i.e., "population activity") and that this effect depends on activity in the ventral subiculum of the hippocampus (vSub). However, there is no direct projection from the mPFC to the vSub. Anatomical evidence suggests that communication between the two structures is mediated by the nucleus reuniens of the midline thalamus (RE). Here, we used in vivo electrophysiological and behavioral approaches in rats to explore the role of the RE in the circuitry governing VTA DA neuron firing. We show that pharmacological stimulation of the RE enhances VTA DA neuron population activity and amphetamine-induced hyperlocomotion, a behavioral indicator of an over-responsive DA system. Furthermore, the effect of RE stimulation on population activity is prevented if vSub is also inhibited. Finally, pharmacological inhibition of ilPFC enhances VTA DA neuron population activity, but this effect does not occur if RE is also inhibited. These findings suggest that disruption of ilPFC-RE-vSub communication could lead to a dysregulated, hyperdopaminergic state, and may play a role in psychiatric disorders. Dopamine (DA) neurons in the ventral tegmental area (VTA) are involved in a variety of fundamental brain functions. To understand the neurobiological basis for these functions it is essential to identify regions controlling DA neuron activity. The medial prefrontal cortex (mPFC) is emerging as a key regulator of DA neuron activity, but the circuitry by which it exerts its influence remains poorly described. Here, we show that the nucleus reuniens of the midline thalamus gates mPFC control of VTA DA neuron firing by the hippocampus. These data identify a unique role for this corticothalamic-hippocampal circuit, and suggest that dysfunction in these regions likely influences the pathophysiology of psychiatric disorders.

PPARα modulation of mesolimbic dopamine transmission rescues depression-related behaviors

Depressive disorders cause a substantial burden for the individual and the society. Key depressive symptoms can be modeled in animals and enable the development of novel therapeutic interventions. Chronic unavoidable stress disrupts rats' competence to escape noxious stimuli and self-administer sucrose, configuring a depression model characterized by escape deficit and motivational anhedonia associated to impaired dopaminergic responses to sucrose in the nucleus accumbens shell (NAcS). Repeated treatments that restore these responses also relieve behavioral symptoms. Ventral tegmental area (VTA) dopamine neurons encode reward and motivation and are implicated in the neuropathology of depressive-like behaviors. Peroxisome proliferator-activated receptors type-α (PPARα) acutely regulate VTA dopamine neuron firing via β2 subunit-containing nicotinic acetylcholine receptors (β2*nAChRs) through phosphorylation and this effect is predictive of antidepressant-like effects.Here, by combining behavioral, electrophysiological and biochemical techniques, we studied the effects of repeated PPARα stimulation by fenofibrate on mesolimbic dopamine system. We found decreased β2*nAChRs phosphorylation levels and a switch from tonic to phasic activity of dopamine cells in the VTA, and increased phosphorylation of dopamine and cAMP-regulated phosphoprotein Mr 32,000 (DARPP-32) in the NAcS. We then investigated whether long-term fenofibrate administration to stressed rats reinstated the decreased DARPP-32 response to sucrose and whether this effect translated into antidepressant-like properties. Fenofibrate restored dopaminergic responses to appetitive stimuli, reactivity to aversive stimuli and motivation to self-administer sucrose. Overall, this study suggests PPARα as new targets for antidepressant therapies endowed with motivational anti-anhedonic properties, further supporting the role of an unbalanced mesolimbic dopamine system in pathophysiology of depressive disorders.

Expression of functional cannabinoid CB2 receptor in VTA dopamine neurons in rats.

We have recently reported the expression of functional cannabinoid CB2 receptors (CB2 Rs) in midbrain dopamine (DA) neurons in mice. However, little is known whether CB2 Rs are similarly expressed in rat brain because significant species differences in CB2 R structures and expression are found. In situ hybridization and immunohistochemical assays detected CB2 gene and receptors in DA neurons of the ventral tegmental area (VTA), which was up-regulated in cocaine self-administration rats. Electrophysiological studies demonstrated that activation of CB2 Rs by JWH133 inhibited VTA DA neuronal firing in single dissociated neurons. Systemic administration of JWH133 failed to alter, while local administration of JWH133 into the nucleus accumbens inhibited cocaine-enhanced extracellular DA and i.v. cocaine self-administration. This effect was blocked by AM630, a selective CB2 R antagonist. These data suggest that CB2 Rs are expressed in VTA DA neurons and functionally modulate DA neuronal activities and cocaine self-administration behavior in rats.

Hippocampal cannabinoid transmission modulates dopamine neuron activity: impact on rewarding memory formation and social interaction.

Disturbances in cannabinoid type 1 receptor (CB1R) signaling have been linked to emotional and cognitive deficits characterizing neuropsychiatric disorders, including schizophrenia. Thus, there is growing interest in characterizing the relationship between cannabinoid transmission, emotional processing, and dopamine (DA)-dependent behavioral deficits. The CB1R is highly expressed in the mammalian nervous system, particularly in the hippocampus. Activation of the ventral hippocampal subregion (vHipp) is known to increase both the activity of DAergic neurons located in the ventral tegmental area (VTA) and DA levels in reward-related brain regions, particularly the nucleus accumbens (NAc). However, the possible functional relationship between hippocampal CB1R transmission and VTA DA neuronal activity is not currently understood. In this study, using in vivo neuronal recordings in rats, we demonstrate that activation of CB1R in the vHipp strongly increases VTA DA neuronal firing and bursting activity, while simultaneously decreasing the activity of VTA non-DA neurons. Furthermore, using a conditioned place preference procedure and a social interaction test, we report that intra-vHipp CB1R activation potentiates the reward salience of normally sub-threshold conditioning doses of opiates and induces deficits in natural sociability and social recognition behaviors. Finally, these behavioral effects were prevented by directly blocking NAc DAergic transmission. Collectively, these findings identify hippocampal CB1R transmission as a critical modulator of the mesolimbic DA pathway and in the processing of reward and social-related behavioral phenomena.

Cannabinoid CB2 receptors modulate midbrain dopamine neuronal activity and dopamine-related behavior in mice.

Cannabinoid CB2 receptors (CB2Rs) have been recently reported to modulate brain dopamine (DA)-related behaviors; however, the cellular mechanisms underlying these actions are unclear. Here we report that CB2Rs are expressed in ventral tegmental area (VTA) DA neurons and functionally modulate DA neuronal excitability and DA-related behavior. In situ hybridization and immunohistochemical assays detected CB2 mRNA and CB2R immunostaining in VTA DA neurons. Electrophysiological studies demonstrated that activation of CB2Rs by JWH133 or other CB2R agonists inhibited VTA DA neuronal firing in vivo and ex vivo, whereas microinjections of JWH133 into the VTA inhibited cocaine self-administration. Importantly, all of the above findings observed in WT or CB1(-/-) mice are blocked by CB2R antagonist and absent in CB2(-/-) mice. These data suggest that CB2R-mediated reduction of VTA DA neuronal activity may underlie JWH133's modulation of DA-regulated behaviors.

Electrophysiological characterization of dopamine neuronal activity in the ventral tegmental area across the light-dark cycle.

Direct evidence that dopamine (DA) neurotransmission varies during the 24 h of the day is lacking. Here, we have characterized the firing activity of DA neurons located in the ventral tegmental area (VTA) using single-unit extracellular recordings in anesthetized rats kept on a standard light-dark cycle. DA neuronal firing activity was measured under basal conditions and in response to intravenous administration of increasing doses of amphetamine (AMPH: 0.5, 1, 2, 5 mg/kg), apomorphine (APO: 25, 50, 100, 200 µg/kg) and melatonin (MLT: 0.1, 1, 10 mg/kg) at different time intervals of the light-dark cycle. DA firing activity peaked between 07:00 and 11:00 h (3.5 ± 0.3 Hz) and between 19:00 and 23:00 h (4.1 ± 0.7 Hz), with lowest activity occurring between 11:00 and 15:00 h (2.4 ± 0.2 Hz) and between 23:00 and 03:00 h (2.6 ± 0.2 Hz). The highest number of spontaneously active neurons was observed between 03:00 and 06:00 h (2.5 ± 0.3 neurons/track), whereas the lowest was between 19:00 and 23:00 h (1.5 ± 0.2 neurons/track). The inhibitory effect of AMPH on DA firing rate was similar in both phases. The inhibitory effect of low dose of APO (25 μg/kg, dose selective for D2 autoreceptor) was more potent in the dark phase, whereas APO effects at higher doses were similar in both phases. Finally, MLT administration (1 mg/kg) produced a moderate inhibition of DA cell firing in both phases. These experiments demonstrate the existence of an intradiurnal rhythmic pattern of VTA DA neuronal firing activity and a higher pharmacological response of D2 autoreceptors in the dark phase.

Coupled intracerebral microdialysis and electrophysiology for the assessment of dopamine neuron function in vivo

Introduction: The central dopaminergic system is involved in the pathophysiology of several neuropsychiatric disorders. Intracerebral microdialysis and electrophysiology provide two powerful techniques to investigate dopamine (DA) function and the mechanism of action of psychotropic drugs in vivo. Methods: Here, we developed a protocol allowing the combined measurement of neurochemical and electrical activities of the nigrostriatal and mesoaccumbens DA pathways, by coupling in vivo microdialysis and electrophysiology in the same isoflurane-anesthetized animal. DA neuron firing rate and burst firing were measured in the substantia nigra pars compacta (SNc) and the ventral tegmental area (VTA), whereas extracellular levels of DA and its main metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) were monitored in the striatum and the nucleus accumbens (NAc). The validity of the protocol was assessed using various drugs known to modify DA neuron activity in vivo. Results: The peripheral administration of the DA-D2 agonist quinpirole decreased SNc DA neuron firing rate and burst firing, as well as DA and DOPAC outflow in the rat striatum. Opposite effects were observed after the peripheral administration of the DA-D2 antagonist haloperidol. In rats and mice, the peripheral administration of cocaine elicited a decrease in VTA DA neuron firing rate and burst firing, and an increase in accumbal DA outflow, paralleled by a reduction in DOPAC outflow. Discussion: The obtained results, confirming previous electrophysiological and microdialysis studies, demonstrate that this protocol provides a suitable method for the study of DA neuron function and the mechanism of action of psychotropic drugs in the living brain of both rats and mice.

Mefloquine effects on ventral tegmental area dopamine and GABA neuron inhibition: A physiologic role for connexin‐36 GAP junctions

Connexin-36 (Cx36) gap junctions (GJs) appear to be involved in the synchronization of GABA interneurons in many brain areas. We have previously identified a population of Cx36-connected ventral tegmental area (VTA) GABA neurons that may regulate mesolimbic dopamine (DA) neurotransmission, a system implicated in reward from both natural behaviors and drugs of abuse. The aim of this study was to determine the effect mefloquine (MFQ) has on midbrain DA and GABA neuron inhibition, and the role Cx36 GJs play in regulating midbrain VTA DA neuron activity in mice. In brain slices from adolescent wild-type (WT) mice the Cx36-selective GJ blocker mefloquine (MFQ, 25 μM) increased VTA DA neuron sIPSC frequency sixfold, and mIPSC frequency threefold. However, in Cx36 KO mice, MFQ only increased sIPSC and mIPSC frequency threefold. The nonselective GJ blocker carbenoxolone (CBX, 100 μM) increased DA neuron sIPSC frequency twofold in WT mice, did not affect Cx36 KO mouse sIPSCs, and did not affect mIPSCs in WT or Cx36 KO mice. Interestingly, MFQ had no effect on VTA GABA neuron sIPSC frequency. We also examined MFQ effects on VTA DA neuron firing rate and current-evoked spiking in WT and Cx36 KO mice, and found that MFQ decreased WT DA neuron firing rate and current-evoked spiking, but did not alter these measures in Cx36 KO mice. Taken together these findings suggest that blocking Cx36 GJs increases VTA DA neuron inhibition, and that GJs play in key role in regulating inhibition of VTA DA neurons. Synapse, 2011. © 2011 Wiley-Liss, Inc.

Specific Role of VTA Dopamine Neuronal Firing Rates and Morphology in the Reversal of Anxiety-Related, but not Depression-Related Behavior in the ClockΔ19 Mouse Model of Mania

Lithium has been used extensively for mood stabilization, and it is particularly efficacious in the treatment of bipolar mania. Like other drugs used in the treatment of psychiatric diseases, it has little effect on the mood of healthy individuals. Our previous studies found that mice with a mutation in the Clock gene (ClockΔ19) have a complete behavioral profile that is very similar to human mania, which can be reversed with chronic lithium treatment. However, the cellular and physiological effects that underlie its targeted therapeutic efficacy remain unknown. Here we find that ClockΔ19 mice have an increase in dopaminergic activity in the ventral tegmental area (VTA), and that lithium treatment selectively reduces the firing rate in the mutant mice with no effect on activity in wild-type mice. Furthermore, lithium treatment reduces nucleus accumbens (NAc) dopamine levels selectively in the mutant mice. The increased dopaminergic activity in the Clock mutants is associated with cell volume changes in dopamine neurons, which are also rescued by lithium treatment. To determine the role of dopaminergic activity and morphological changes in dopamine neurons in manic-like behavior, we manipulated the excitability of these neurons by overexpressing an inwardly rectifying potassium channel subunit (Kir2.1) selectively in the VTA of ClockΔ19 mice and wild-type mice using viral-mediated gene transfer. Introduction of this channel mimics the effects of lithium treatment on the firing rate of dopamine neurons in ClockΔ19 mice and leads to a similar change in dopamine cell volume. Furthermore, reduction of dopaminergic firing rates in ClockΔ19 animals results in a normalization of locomotor- and anxiety-related behavior that is very similar to lithium treatment; however, it is not sufficient to reverse depression-related behavior. These results suggest that abnormalities in dopamine cell firing and associated morphology underlie alterations in anxiety-related behavior in bipolar mania, and that the therapeutic effects of lithium come from a reversal of these abnormal phenotypes.

Mefloquine effects on ventral tegmental area dopamine and GABA neuron inhibition: A physiologic role for Connexin-36 gap junctions

Connexin-36 (Cx36) gap junctions (GJs) appear to be involved in the synchronization of GABA interneurons in many brain areas. We have previously identified a population of Cx36-connected ventral tegmental area (VTA) GABA neurons that may regulate mesolimbic dopamine (DA) neurotransmission, a system implicated in reward from both natural behaviors and drugs of abuse. The aim of this study was to determine the effect mefloquine (MFQ) has on midbrain DA and GABA neuron inhibition, and the role Cx36 GJs play in regulating midbrain VTA DA neuron activity in mice. In brain slices from adolescent wild-type (WT) mice the Cx36-selective GJ blocker mefloquine (MFQ, 25 μM) increased VTA DA neuron sIPSC frequency sixfold, and mIPSC frequency threefold. However, in Cx36 KO mice, MFQ only increased sIPSC and mIPSC frequency threefold. The nonselective GJ blocker carbenoxolone (CBX, 100 μM) increased DA neuron sIPSC frequency twofold in WT mice, did not affect Cx36 KO mouse sIPSCs, and did not affect mIPSCs in WT or Cx36 KO mice. Interestingly, MFQ had no effect on VTA GABA neuron sIPSC frequency. We also examined MFQ effects on VTA DA neuron firing rate and current-evoked spiking in WT and Cx36 KO mice, and found that MFQ decreased WT DA neuron firing rate and current-evoked spiking, but did not alter these measures in Cx36 KO mice. Taken together these findings suggest that blocking Cx36 GJs increases VTA DA neuron inhibition, and that GJs play in key role in regulating inhibition of VTA DA neurons. Synapse, 2011. © 2011 Wiley-Liss, Inc.

Corticotropin‐releasing factor increases mouse ventral tegmental area dopamine neuron firing through a protein kinase C‐dependent enhancement of Ih

Stress induces the release of the peptide corticotropin-releasing factor (CRF) into the ventral tegmental area (VTA), and also increases dopamine levels in brain regions receiving dense VTA input. Therefore, stress may activate the mesolimbic dopamine system in part through the actions of CRF in the VTA. Here, we explored the mechanism by which CRF affects VTA dopamine neuron firing. Using patch-clamp recordings from brain slices we first determined that the presence of I(h) is an excellent predictor of dopamine content in mice. We next showed that CRF dose-dependently increased VTA dopamine neuron firing, which was prevented by antagonism of the CRF receptor-1 (CRF-R1), and was mimicked by CRF-R1 agonists. Inhibition of the phospholipase C (PLC)-protein kinase C (PKC) signalling pathway, but not the cAMP-protein kinase A (PKA) signalling pathway, prevented the increase in dopamine neuron firing by CRF. Furthermore, the effect of CRF on VTA dopamine neurons was not attenuated by blockade of I(A), I(K(Ca)) or I(Kir), but was completely eliminated by inhibition of I(h). Although cAMP-dependent modulation of I(h) through changes in the voltage dependence of activation is well established, we surprisingly found that CRF, through a PKC-dependent mechanism, enhanced I(h) independent of changes in the voltage dependence of activation. Thus, our results demonstrated that CRF acted on the CRF-R1 to stimulate the PLC-PKC signalling pathway, which in turn enhanced I(h) to increase VTA dopamine neuron firing. These findings provide a cellular mechanism of the interaction between CRF and dopamine, which can be involved in promoting the avoidance of threatening stimuli, the pursuit of appetitive behaviours, as well as various psychiatric conditions.