Kelly R. Tan

GABA Neurons of the VTA Drive Conditioned Place Aversion

Salient but aversive stimuli inhibit the majority of dopamine (DA) neurons in the ventral tegmental area (VTA) and cause conditioned place aversion (CPA). The cellular mechanism underlying DA neuron inhibition has not been investigated and the causal link to behavior remains elusive. Here, we show that GABA neurons of the VTA inhibit DA neurons through neurotransmission at GABA(A) receptors. We also observe that GABA neurons increase their firing in response to a footshock and provide evidence that driving GABA neurons with optogenetic effectors is sufficient to affect behavior. Taken together, our data demonstrate that synaptic inhibition of DA neurons drives place aversion.

Neural bases for addictive properties of benzodiazepines

Benzodiazepines are widely used in clinics and for recreational purposes, but will lead to addiction in vulnerable individuals. Addictive drugs increase the levels of dopamine and also trigger long-lasting synaptic adaptations in the mesolimbic reward system that ultimately may induce the pathological behaviour. The neural basis for the addictive nature of benzodiazepines, however, remains elusive. Here we show that benzodiazepines increase firing of dopamine neurons of the ventral tegmental area through the positive modulation of GABAA (γ-aminobutyric acid type A) receptors in nearby interneurons. Such disinhibition, which relies on α1-containing GABAA receptors expressed in these cells, triggers drug-evoked synaptic plasticity in excitatory afferents onto dopamine neurons and underlies drug reinforcement. Taken together, our data provide evidence that benzodiazepines share defining pharmacological features of addictive drugs through cell-type-specific expression of α1-containing GABAA receptors in the ventral tegmental area. The data also indicate that subunit-selective benzodiazepines sparing α1 may be devoid of addiction liability.

Ventral tegmental area GABA projections pause accumbal cholinergic interneurons to enhance associative learning

The ventral tegmental area (VTA) and nucleus accumbens (NAc) are essential for learning about environmental stimuli associated with motivationally relevant outcomes. The task of signalling such events, both rewarding and aversive, from the VTA to the NAc has largely been ascribed to dopamine neurons. The VTA also contains GABA (γ-aminobutyric acid)-releasing neurons, which provide local inhibition and also project to the NAc. However, the cellular targets and functional importance of this long-range inhibitory projection have not been ascertained. Here we show that GABA-releasing neurons of the VTA that project to the NAc (VTA GABA projection neurons) inhibit accumbal cholinergic interneurons (CINs) to enhance stimulus–outcome learning. Combining optogenetics with structural imaging and electrophysiology, we found that VTA GABA projection neurons selectively target NAc CINs, forming multiple symmetrical synaptic contacts that generated inhibitory postsynaptic currents. This is remarkable considering that CINs represent a very small population of all accumbal neurons, and provide the primary source of cholinergic tone in the NAc. Brief activation of this projection was sufficient to halt the spontaneous activity of NAc CINs, resembling the pause recorded in animals learning stimulus–outcome associations. Indeed, we found that forcing CINs to pause in behaving mice enhanced discrimination of a motivationally important stimulus that had been associated with an aversive outcome. Our results demonstrate that VTA GABA projection neurons, through their selective targeting of accumbal CINs, provide a novel route through which the VTA communicates saliency to the NAc. VTA GABA projection neurons thus emerge as orchestrators of dopaminergic and cholinergic modulation in the NAc.

Cocaine Disinhibits Dopamine Neurons by Potentiation of GABA Transmission in the Ventral Tegmental Area

Drugs, Dopamine, and Disinhibition Drugs often change the neuronal circuitry in the brain and thereby cause a long-lasting change in behavior. Using a wide range of in vivo and in vitro techniques in mice, Bocklisch et al. (p. 1521) observed that cocaine profoundly altered dopamine neuron function and that drug-evoked synaptic plasticity in a specific set of neurons represents a crucial step in circuit remodeling. Changes in specific neuronal circuits suggest that drug-evoked synaptic plasticity facilitates drug-adaptive behavior. Drug-evoked synaptic plasticity in the mesolimbic system reshapes circuit function and drives drug-adaptive behavior. Much research has focused on excitatory transmission in the ventral tegmental area (VTA) and the nucleus accumbens (NAc). How drug-evoked synaptic plasticity of inhibitory transmission affects circuit adaptations remains unknown. We found that medium spiny neurons expressing dopamine (DA) receptor type 1 (D1R-MSNs) of the NAc project to the VTA, strongly preferring the GABA neurons of the VTA. Repeated in vivo exposure to cocaine evoked synaptic potentiation at this synapse, occluding homosynaptic inhibitory long-term potentiation. The activity of the VTA GABA neurons was thus reduced and DA neurons were disinhibited. Cocaine-evoked potentiation of GABA release from D1R-MSNs affected drug-adaptive behavior, which identifies these neurons as a promising target for novel addiction treatments.

Hooked on benzodiazepines: GABAA receptor subtypes and addiction

Benzodiazepines are widely used clinically to treat anxiety and insomnia. They also induce muscle relaxation, control epileptic seizures, and can produce amnesia. Moreover, benzodiazepines are often abused after chronic clinical treatment and also for recreational purposes. Within weeks, tolerance to the pharmacological effects can develop as a sign of dependence. In vulnerable individuals with compulsive drug use, addiction will be diagnosed. Here we review recent observations from animal models regarding the cellular and molecular basis that might underlie the addictive properties of benzodiazepines. These data reveal how benzodiazepines, acting through specific GABA(A) receptor subtypes, activate midbrain dopamine neurons, and how this could hijack the mesolimbic reward system. Such findings have important implications for the future design of benzodiazepines with reduced or even absent addiction liability.

Methamphetamine-evoked depression of GABA(B) receptor signaling in GABA neurons of the VTA.

Psychostimulants induce neuroadaptations in excitatory and fast inhibitory transmission in the ventral tegmental area (VTA). Mechanisms underlying drug-evoked synaptic plasticity of slow inhibitory transmission mediated by GABA(B) receptors and G protein-gated inwardly rectifying potassium (GIRK/Kir(3)) channels, however, are poorly understood. Here, we show that 1 day after methamphetamine (METH) or cocaine exposure both synaptically evoked and baclofen-activated GABA(B)R-GIRK currents were significantly depressed in VTA GABA neurons and remained depressed for 7 days. Presynaptic inhibition mediated by GABA(B)Rs on GABA terminals was also weakened. Quantitative immunoelectron microscopy revealed internalization of GABA(B1) and GIRK2, which occurred coincident with dephosphorylation of serine 783 (S783) in GABA(B2), a site implicated in regulating GABA(B)R surface expression. Inhibition of protein phosphatases recovered GABA(B)R-GIRK currents in VTA GABA neurons of METH-injected mice. This psychostimulant-evoked impairment in GABA(B)R signaling removes an intrinsic brake on GABA neuron spiking, which may augment GABA transmission in the mesocorticolimbic system.

VTA GABA neurons modulate specific learning behaviors through the control of dopamine and cholinergic systems

The mesolimbic reward system is primarily comprised of the ventral tegmental area (VTA) and the nucleus accumbens (NAc) as well as their afferent and efferent connections. This circuitry is essential for learning about stimuli associated with motivationally-relevant outcomes. Moreover, addictive drugs affect and remodel this system, which may underlie their addictive properties. In addition to dopamine (DA) neurons, the VTA also contains approximately 30% γ-aminobutyric acid (GABA) neurons. The task of signaling both rewarding and aversive events from the VTA to the NAc has mostly been ascribed to DA neurons and the role of GABA neurons has been largely neglected until recently. GABA neurons provide local inhibition of DA neurons and also long-range inhibition of projection regions, including the NAc. Here we review studies using a combination of in vivo and ex vivo electrophysiology, pharmacogenetic and optogenetic manipulations that have characterized the functional neuroanatomy of inhibitory circuits in the mesolimbic system, and describe how GABA neurons of the VTA regulate reward and aversion-related learning. We also discuss pharmacogenetic manipulation of this system with benzodiazepines (BDZs), a class of addictive drugs, which act directly on GABAA receptors located on GABA neurons of the VTA. The results gathered with each of these approaches suggest that VTA GABA neurons bi-directionally modulate activity of local DA neurons, underlying reward or aversion at the behavioral level. Conversely, long-range GABA projections from the VTA to the NAc selectively target cholinergic interneurons (CINs) to pause their firing and temporarily reduce cholinergic tone in the NAc, which modulates associative learning. Further characterization of inhibitory circuit function within and beyond the VTA is needed in order to fully understand the function of the mesolimbic system under normal and pathological conditions.

Covalent modification of GABAA receptor isoforms by a diazepam analogue provides evidence for a novel benzodiazepine binding site that prevents modulation by these drugs

Classical benzodiazepines, for example diazepam, interact with αxβ2γ2 GABAA receptors, x = 1, 2, 3, 5. Little is known about effects of α subunits on the structure of the binding pocket. We studied here the interaction of the covalently reacting diazepam analog 7‐Isothiocyanato‐5‐phenyl‐1,3‐dihydro‐2H‐1,4‐benzodiazepin‐2‐one (NCS compound) with α1H101Cβ2γ2 and with receptors containing the homologous mutation, α2H101Cβ2γ2, α3H126Cβ2γ2 and α5H105Cβ2γ2. This comparison was extended to α6R100Cβ2γ2 receptors as this mutation conveys to these receptors high affinity towards classical benzodiazepines. The interaction was studied at the ligand binding level and at the functional level using electrophysiological techniques. Results indicate that the geometry of α6R100Cβ2γ2 enables best interaction with NCS compound, followed by α3H126Cβ2γ2, α1H101Cβ2γ2 and α2H101Cβ2γ2, while α5H105Cβ2γ2 receptors show little interaction. Our results allow conclusions about the relative apposition of α1H101 and homologous positions in α2, α3, α5 and α6 with the position occupied by –Cl in diazepam. During this study we found evidence for the presence of a novel site for benzodiazepines that prevents modulation of GABAA receptors via the classical benzodiazepine site. The novel site potentially contributes to the high degree of safety to some of these drugs. Our results indicate that this site may be located at the α/β subunit interface pseudo‐symmetrically to the site for classical benzodiazepines located at the α/γ interface.

Dissociation and trafficking of rat GABAB receptor heterodimer upon chronic capsaicin stimulation

Gamma‐aminobutyric acid type B receptors (GABAB) are G‐protein‐coupled receptors that mediate GABAergic inhibition in the brain. Their functional expression is dependent upon the formation of heterodimers between GABAB1 and GABAB2 subunits, a process that occurs within the endoplasmic reticulum. However, the mechanisms that regulate GABAB receptor oligomerization at the plasma membrane remain largely unknown.

Proximity-accelerated chemical coupling reaction in the benzodiazepine-binding site of gamma-aminobutyric acid type A receptors: superposition of different allosteric modulators.

Benzodiazepines are widely used drugs. They exert sedative/hypnotic, anxiolytic, muscle relaxant, and anticonvulsant effects and act through a specific high affinity binding site on the major inhibitory neurotransmitter receptor, the γ-aminobutyric acid type A (GABAA) receptor. Ligands of the benzodiazepine-binding site are classified into three groups depending on their mode of action: positive and negative allosteric modulators and antagonists. To rationally design ligands of the benzodiazepine site in different isoforms of the GABAA receptor, we need to understand the relative positioning and overlap of modulators of different allosteric properties. To solve these questions, we used a proximity-accelerated irreversible chemical coupling reaction. GABAA receptor residues thought to reside in the benzodiazepine-binding site were individually mutated to cysteine and combined with a cysteine-reactive benzodiazepine site ligand. Direct apposition of reaction partners is expected to lead to a covalent reaction. We describe here such a reaction of predominantly α1H101C and also three other mutants (α1G157C, α1V202C, and α1V211C) with an Imid-NCS derivative in which a reactive isothiocyanate group (–NCS) replaces the azide group (–N3) in the partial negative allosteric modulator Ro15-4513. Our results show four contact points of imidazobenzodiazepines with the receptor, α1H101C being shared by classical benzodiazepines. Taken together with previous data, a similar orientation of these ligands within the benzodiazepine-binding pocket may be proposed.