Mouse Ventral Tegmental Area Research Articles

Erratum: Spencer et al., "Regulation of the Mouse Ventral Tegmental Area by Melanin-Concentrating Hormone".

Erratum: Spencer et al., "Regulation of the Mouse Ventral Tegmental Area by Melanin-Concentrating Hormone".

Chronic ethanol exposure in mice evokes pre- and postsynaptic deficits in GABAergic transmission in ventral tegmental area GABA neurons.

GABAergic neurons in mouse ventral tegmental area (VTA) exhibit elevated activity during withdrawal following chronic ethanol exposure. While increased glutamatergic input and decreased GABAA receptor sensitivity have been implicated, the impact of inhibitory signaling in VTA GABA neurons has not been fully addressed. We used electrophysiological and ultrastructural approaches to assess the impact of chronic intermittent ethanol vapour exposure in mice on GABAergic transmission in VTA GABA neurons during withdrawal. We used CRISPR/Cas9 ablation to mimic a somatodendritic adaptation involving the GABAB receptor (GABABR) in ethanol-naïve mice to investigate its impact on anxiety-related behaviour. The frequency of spontaneous inhibitory postsynaptic currents was reduced in VTA GABA neurons following chronic ethanol treatment and this was reversed by GABABR inhibition, suggesting chronic ethanol strengthens the GABABR-dependent suppression of GABAergic input to VTA GABA neurons. Similarly, paired-pulse depression of GABAA receptor-dependent responses evoked by optogenetic stimulation of nucleus accumbens inputs from ethanol-treated mice was reversed by GABABR inhibition. Somatodendritic currents evoked in VTA GABA neurons by GABABR activation were reduced following ethanol exposure, attributable to the suppression of GIRK (Kir3) channel activity. Mimicking this adaptation enhanced anxiety-related behaviour in ethanol-naïve mice. Chronic ethanol weakens the GABAergic regulation of VTA GABA neurons in mice via pre- and postsynaptic mechanisms, likely contributing to their elevated activity during withdrawal and expression of anxiety-related behaviour. As anxiety can promote relapse during abstinence, interventions targeting VTA GABA neuron excitability could represent new therapeutic strategies for treatment of alcohol use disorder.

Sex-Specific ADNP/NAP (Davunetide) Regulation of Cocaine-Induced Plasticity

Cocaine use disorder (CUD) is a chronic neuropsychiatric disorder estimated to effect 1–3% of the population. Activity-dependent neuroprotective protein (ADNP) is essential for brain development and functioning, shown to be protective in fetal alcohol syndrome and to regulate alcohol consumption in adult mice. The goal of this study was to characterize the role of ADNP, and its active peptide NAP (NAPVSIPQ), which is also known as davunetide (investigational drug) in mediating cocaine-induced neuroadaptations. Real time PCR was used to test levels of Adnp and Adnp2 in the nucleus accumbens (NAc), ventral tegmental area (VTA), and dorsal hippocampus (DH) of cocaine-treated mice (15 mg/kg). Adnp heterozygous (Adnp+/−)and wild-type (Adnp+/−) mice were further tagged with excitatory neuronal membrane-expressing green fluorescent protein (GFP) that allowed for in vivo synaptic quantification. The mice were treated with cocaine (5 injections; 15 mg/kg once every other day) with or without NAP daily injections (0.4 µg/0.1 ml) and sacrificed following the last treatment. We analyzed hippocampal CA1 pyramidal cells from 3D confocal images using the Imaris x64.8.1.2 (Oxford Instruments) software to measure changes in dendritic spine density and morphology. In silico ADNP/NAP/cocaine structural modeling was performed as before. Cocaine decreased Adnp and Adnp2 expression 2 h after injection in the NAc and VTA of male mice, with mRNA levels returning to baseline levels after 24 h. Cocaine further reduced hippocampal spine density, particularly synaptically weaker immature thin and stubby spines, in male Adnp+/+) mice while increasing synaptically stronger mature (mushroom) spines in Adnp+/−) male mice and thin and stubby spines in females. Lastly, we showed that cocaine interacts with ADNP on a zinc finger domain identical to ketamine and adjacent to a NAP-zinc finger interaction site. Our results implicate ADNP in cocaine abuse, further placing the ADNP gene as a key regulator in neuropsychiatric disorders. Ketamine/cocaine and NAP treatment may be interchangeable to some degree, implicating an interaction with adjacent zinc finger motifs on ADNP and suggestive of a potential sex-dependent, non-addictive NAP treatment for CUD.

Voluntary exercise suppresses inflammation and improves insulin resistance in the arcuate nucleus and ventral tegmental area in mice on a high-fat diet

A high-fat diet (HFD) causes inflammation with an increase in microglial activity in the hypothalamic arcuate nucleus (ARC) and ventral tegmental area (VTA), resulting in insulin resistance in both regions. This leads to a deterioration in glucose and energy metabolism. The effect of voluntary exercise on HFD-induced inflammation in the central nervous system (CNS) remains unclear. To clarify the effects of voluntary exercise on the CNS, 8-week-old male C57BL6 mice were fed a chow diet (CHD) or HFD for 4 weeks; each group was further divided into running exercise (EX+) on a wheel and no exercise (EX-) groups. The expression of the inflammatory cytokine, tumor necrosis factor alpha (TNFα), in the ARC and VTA was significantly increased in the HFD/EX- group, with an increase of microglial activity noted, compared to the CHD/EX- group. The expression of TNFα was significantly suppressed, with a decrease of microglial activity, in the HFD/EX+ compared to HFD/EX- group. Insulin resistance in the ARC and VTA was improved with the suppression of TNFα expression. The HFD/EX- group showed significant weight gain and impaired glucose metabolism compared to the CHD/EX- group. The HFD/EX+ group showed an improvement in glucose and energy metabolism compared to the HFD/EX- group. In addition, voluntary wheel running suppressed HFD-induced inflammation in the ARC, with a decrease in microglial activity observed independently of weight changes. Our data suggest that voluntary exercise prevents obesity and improves glucose metabolism by suppressing inflammation in the ARC and VTA under HFD conditions.

Impact of Serotonin Deficiency on Circadian Dopaminergic Rhythms.

Physiology and behavior are structured temporally to anticipate daily cycles of light and dark, ensuring fitness and survival. Neuromodulatory systems in the brain-including those involving serotonin and dopamine-exhibit daily oscillations in neural activity and help shape circadian rhythms. Disrupted neuromodulation can cause circadian abnormalities that are thought to underlie several neuropsychiatric disorders, including bipolar mania and schizophrenia, for which a mechanistic understanding is still lacking. Here, we show that genetically depleting serotonin in Tph2 knockout mice promotes manic-like behaviors and disrupts daily oscillations of the dopamine biosynthetic enzyme tyrosine hydroxylase (TH) in midbrain dopaminergic nuclei. Specifically, while TH mRNA and protein levels in the Substantia Nigra (SN) and Ventral Tegmental Area (VTA) of wild-type mice doubled between the light and dark phase, TH levels were high throughout the day in Tph2 knockout mice, suggesting a hyperdopaminergic state. Analysis of TH expression in striatal terminal fields also showed blunted rhythms. Additionally, we found low abundance and blunted rhythmicity of the neuropeptide cholecystokinin (Cck) in the VTA of knockout mice, a neuropeptide whose downregulation has been implicated in manic-like states in both rodents and humans. Altogether, our results point to a previously unappreciated serotonergic control of circadian dopamine signaling and propose serotonergic dysfunction as an upstream mechanism underlying dopaminergic deregulation and ultimately maladaptive behaviors.

Regulation of the Mouse Ventral Tegmental Area by Melanin-Concentrating Hormone.

Melanin-concentrating hormone (MCH) acts via its sole receptor MCHR1 in rodents and is an important regulator of homeostatic behaviors like feeding, sleep, and mood to impact overall energy balance. The loss of MCH signaling by MCH or MCHR1 deletion produces hyperactive mice with increased energy expenditure, and these effects are consistently associated with a hyperdopaminergic state. We recently showed that MCH suppresses dopamine release in the nucleus accumbens, which principally receives dopaminergic projections from the ventral tegmental area (VTA), but the mechanisms underlying MCH-regulated dopamine release are not clearly defined. MCHR1 expression is widespread and includes dopaminergic VTA cells. However, as the VTA is a neurochemically diverse structure, we assessed Mchr1 gene expression at glutamatergic, GABAergic, and dopaminergic VTA cells and determined if MCH inhibited the activity of VTA cells and/or their local microcircuit. Mchr1 expression was robust in major VTA cell types, including most dopaminergic (78%) or glutamatergic cells (52%) and some GABAergic cells (38%). Interestingly, MCH directly inhibited dopaminergic and GABAergic cells but did not regulate the activity of glutamatergic cells. Rather, MCH produced a delayed increase in excitatory input to dopamine cells and a corresponding decrease in GABAergic input to glutamatergic VTA cells. Our findings suggested that MCH may acutely suppress dopamine release while disinhibiting local glutamatergic signaling to restore dopamine levels. This indicated that the VTA is a target of MCH action, which may provide bidirectional regulation of energy balance.

Transcriptional signatures of fentanyl use in the mouse ventral tegmental area.

Synthetic opioids such as fentanyl contribute to the vast majority of opioid-related overdose deaths, but fentanyl use remains broadly understudied. Like other substances with misuse potential, opioids cause lasting molecular adaptations to brain reward circuits, including neurons in the ventral tegmental area (VTA). The VTA contains numerous cell types that play diverse roles in opioid use and relapse; however, it is unknown how fentanyl experience alters the transcriptional landscape in specific subtypes. Here, we performed single nuclei RNA sequencing to study transcriptional programs in fentanyl-experienced mice. Male and female C57/BL6 mice self-administered intravenous fentanyl (1.5μg/kg/infusion) or saline for 10days. After 24 h abstinence, VTA nuclei were isolated and prepared for sequencing on the 10× platform. We identified different patterns of gene expression across cell types. In dopamine neurons, we found enrichment of genes involved in growth hormone signalling. In dopamine-glutamate-GABA combinatorial neurons, and some GABA neurons, we found enrichment of genes involved in Pi3k-Akt signalling. In glutamate neurons, we found enrichment of genes involved in cholinergic signalling. We identified transcriptional regulators for the differentially expressed genes in each neuron cluster, including downregulated transcriptional repressor Bcl6, and upregulated transcription factor Tcf4. We also compared the fentanyl-induced gene expression changes identified in mouse VTA with a published rat dataset in bulk VTA, and found overlap in genes related to GABAergic signalling and extracellular matrix interaction. Together, we provide a comprehensive picture of how fentanyl self-administration alters the transcriptional landscape of the mouse VTA that serves as the foundation for future mechanistic studies.

Control of polymers’ amorphous-crystalline transition enables miniaturization and multifunctional integration for hydrogel bioelectronics

Soft bioelectronic devices exhibit motion-adaptive properties for neural interfaces to investigate complex neural circuits. Here, we develop a fabrication approach through the control of metamorphic polymers’ amorphous-crystalline transition to miniaturize and integrate multiple components into hydrogel bioelectronics. We attain an about 80% diameter reduction in chemically cross-linked polyvinyl alcohol hydrogel fibers in a fully hydrated state. This strategy allows regulation of hydrogel properties, including refractive index (1.37-1.40 at 480 nm), light transmission (>96%), stretchability (139-169%), bending stiffness (4.6 ± 1.4 N/m), and elastic modulus (2.8-9.3 MPa). To exploit the applications, we apply step-index hydrogel optical probes in the mouse ventral tegmental area, coupled with fiber photometry recordings and social behavioral assays. Additionally, we fabricate carbon nanotubes-PVA hydrogel microelectrodes by incorporating conductive nanomaterials in hydrogel for spontaneous neural activities recording. We enable simultaneous optogenetic stimulation and electrophysiological recordings of light-triggered neural activities in Channelrhodopsin-2 transgenic mice.

Liver kinase B-1 modulates the activity of dopamine neurons in the ventral tegmental area and regulates social memory formation.

Social memory is the ability to discriminate between familiar and unknown conspecifics. It is an important component of social cognition and is therefore essential for the establishment of social relationships. Although the neural circuit mechanisms underlying social memory encoding have been well investigated, little focus has been placed on the regulatory mechanisms of social memory processing. The dopaminergic system, originating from the midbrain ventral tegmental area (VTA), is a key modulator of cognitive function. This study aimed to illustrate its role in modulating social memory and explore the possible molecular mechanisms. Here, we show that the activation of VTA dopamine (DA) neurons is required for the formation, but not the retrieval, of social memory. Inhibition of VTA DA neurons before social interaction, but not 24 h after social interaction, significantly impaired social discrimination the following day. In addition, we showed that the activation of VTA DA neurons was regulated by the serine/threonine protein kinase liver kinase B1 (Lkb1). Deletion of Lkb1 in VTA DA neurons reduced the frequency of burst firing of dopaminergic neurons. Furthermore, Lkb1 plays an important role in regulating social behaviors. Both genetic and virus-mediated deletions of Lkb1 in the VTA of adult mice impaired social memory and subsequently attenuated social familiarization. Altogether, our results provide direct evidence linking social memory formation to the activation of VTA DA neurons in mice and illustrate the crucial role of Lkb1 in regulating VTA DA neuron function.

A machine-learning tool to identify bistable states from calcium imaging data.

Mapping neuronal activation using calcium imaging in vivo during behavioural tasks has advanced our understanding of nervous system function. In almost all of these studies, calcium imaging is used to infer spike probabilities because action potentials activate voltage-gated calcium channels and increase intracellular calcium levels. However, neurons not only fire action potentials, but also convey information via intrinsic dynamics such as by generating bistable membrane potential states. Although a number of tools for spike inference have been developed and are currently being used, no tool exists for converting calcium imaging signals to maps of cellular state in bistable neurons. Purkinje neurons in the larval zebrafish cerebellum exhibit membrane potential bistability, firing either tonically or in bursts. Several studies have implicated the role of a population code in cerebellar function, with bistability adding an extra layer of complexity to this code. In the present study, we develop a tool, CaMLSort, which uses convolutional recurrent neural networks to classify calcium imaging traces as arising from either tonic or bursting cells. We validate this classifier using a number of different methods and find that it performs well on simulated event rasters as well as real biological data that it had not previously seen. Moreover, we find that CaMLsort generalizes to other bistable neurons, such as dopaminergic neurons in the ventral tegmental area of mice. Thus, this tool offers a new way of analysing calcium imaging data from bistable neurons to understand how they participate in network computation and natural behaviours. KEY POINTS: Calcium imaging, compriising the gold standard of inferring neuronal activity, does not report cellular state in neurons that are bistable, such as Purkinje neurons in the cerebellum of larval zebrafish. We model the relationship between Purkinje neuron electrical activity and its corresponding calcium signal to compile a dataset of state-labelled simulated calcium signals. We apply machine-learning methods to this dataset to develop a tool that can classify the state of a Purkinje neuron using only its calcium signal, which works well on real data even though it was trained only on simulated data. CaMLsort (Calcium imaging and Machine Learning based tool to sort intracellular state) also generalizes well to bistable neurons in a different brain region (ventral tegmental area) in a different model organism (mouse). This tool can facilitate our understanding of how these neurons carry out their functions in a circuit.

Restoring GABAB receptor expression in the ventral tegmental area of methamphetamine addicted mice inhibits locomotor sensitization and drug seeking behavior.

Repeated exposure to psychostimulants such as methamphetamine (METH) induces neuronal adaptations in the mesocorticolimbic dopamine system, including the ventral tegmental area (VTA). These changes lead to persistently enhanced neuronal activity causing increased dopamine release and addictive phenotypes. A factor contributing to increased dopaminergic activity in this system appears to be reduced GABAB receptor-mediated neuronal inhibition in the VTA. Dephosphorylation of serine 783 (Ser783) of the GABAB2 subunit by protein phosphatase 2A (PP2A) appears to trigger the downregulation GABAB receptors in psychostimulant-addicted rodents. Therefore, preventing the interaction of GABAB receptors with PP2A using an interfering peptide is a promising strategy to restore GABAB receptor-mediated neuronal inhibition. We have previously developed an interfering peptide (PP2A-Pep) that inhibits the GABAB receptors/PP2A interaction and thereby restores receptor expression under pathological conditions. Here, we tested the hypothesis that restoration of GABAB receptor expression in the VTA of METH addicted mice reduce addictive phenotypes. We found that the expression of GABAB receptors was significantly reduced in the VTA and nucleus accumbens but not in the hippocampus and somatosensory cortex of METH-addicted mice. Infusion of PP2A-Pep into the VTA of METH-addicted mice restored GABAB receptor expression in the VTA and inhibited METH-induced locomotor sensitization as assessed in the open field test. Moreover, administration of PP2A-Pep into the VTA also reduced drug seeking behavior in the conditioned place preference test. These observations underscore the importance of VTA GABAB receptors in controlling addictive phenotypes. Furthermore, this study illustrates the value of interfering peptides targeting diseases-related protein-protein interactions as an alternative approach for a potential development of selective therapeutic interventions.

Ventral tegmental area dopaminergic circuits participates in stress-induced chronic postsurgical pain in male mice

BackgroundChronic postsurgical pain (CPP) markedly impairs patients’ quality of life. Research has shown that chronic stress may extend incisional nociception in male mice. Dopaminergic (DAergic) neurons in the ventral tegmental area (VTA) are integral to stress-related mental disorders (including major depressive disorder, anxiety disorders, and PTSD) and pain. However, the impact of chronic social defeat stress (CSDS) on mesolimbic dopamine (DA) transmission in the development of CPP is yet to be established. It remains uncertain whether the dopamine signals in the rostral anterior cingulate cortex (rACC), which regulate pain, derive from the VTA. This study aims to explore the role of VTA-rACC dopaminergic circuits in a mouse model of CPP induced by CSDS.MethodsWe conducted CSDS on C57BL/6 J wild-type male mice (n = 12–16 mice/group) and DAT-cre male mice (n = 10–12 mice/group). After 10 days of CSDS, a left posterior plantar incision was made to establish a mouse model of CPP. Paw withdrawal thresholds (PWTs) were evaluated using Von-Frey fibre stimulation. The open field test (OFT) and elevated plus maze test (EPM) were used to assess pain-related negative emotions. We used immunofluorescence staining and Western Blot to analyse D1, D2, c-Fos, and TH expression. DAergic fibre projections in the VTA-rACC neural pathway were traced using retrograde tracing and immunofluorescence staining. Optogenetics and Chemogenetics were employed to manipulate DAergic neurons in the VTA and their axons in the rACC.ResultsThe ipsilateral PWTs in male C57BL/6 J mice significantly decreased after surgery, returning to baseline after seven days. Conversely, in CSDS mice, ipsilateral PWTs remained reduced for at least 30 days post-incision. A significant reduction in TH-positive neurons expressing c-Fos in the VTA of CPP mice was observed 15 days post-incision. Activating DAergic neurons significantly improved ipsilateral PWTs and locomotor performance in the OFT and EPM in CPP mice post-incision. Additionally, D1 expression in the rACC was found to decrease in CPP mice, and this reduction counteracted the increase in PWTs caused by activating DAergic neuron axon terminals in the rACC.ConclusionCSDS results in chronicity of postsurgical nociception and anxiety-like negative emotions, with alterations in DA transmission playing a role in CPP. Specific activation of DAergic neurons mitigates nociceptive responses and anxiety-like bahaviors, possibly mediated by D1 receptors in the rACC.

Morphine- and foot shock-responsive neuronal ensembles in the VTA possess different connectivity and biased GPCR signaling pathway.

Background: Neurons in the ventral tegmental area (VTA) are sensitive to stress and their maladaptation have been implicated in the psychiatric disorders such as anxiety and addiction, etc. The cellular properties of the VTA neurons in response to different stressors related to different emotional processing remain to be investigated. Methods: By combining immediate early gene (IEG)-dependent labeling, rabies virus tracing, ensemble-specific transcriptomic analysis and fiber photometry recording in the VTA of male mice, the spatial distribution, brain-wide connectivity and cellular signaling pathways in the VTA neuronal ensembles in response to morphine (Mor-Ens) or foot shock (Shock-Ens) stimuli were investigated. Results: Optogenetic activation of the Mor-Ens drove approach behavior, whereas chemogenetic activation of the Shock-Ens increased the anxiety level in mice. Mor-Ens were clustered and enriched in the ventral VTA, contained a higher proportion of dopaminergic neurons, received more inputs from the dorsal medial striatum and the medial hypothalamic zone, and exhibited greater axonal arborization in the zona incerta and ventral pallidum. Whereas Shock-Ens were more dispersed, contained a higher proportion of GABAergic neurons, and received more inputs from the ventral pallidum and the lateral hypothalamic area. The downstream targets of the G protein and β-arrestin pathways, PLCβ3 and phosphorylated AKT1Thr308, were relatively enriched in the Mor-Ens and Shock-Ens, respectively. Cariprazine, the G-protein-biased agonist for the dopamine D2 receptor, increased the response of Mor-Ens to sucrose water and decreased the anxiety-like behavior during morphine withdrawal, whereas the β-arrestin-biased agonist UNC9994 decreased the response of Shock-Ens to tail suspension. Conclusions: Taken together, these findings reveal the heterogeneous connectivity and signaling pathways of the VTA neurons in response to morphine and foot shock, providing new insights for development of specific interventions for psychiatric disorders caused by various stressors associated with different VTA neuronal functions.

Somatostatin-expressing neurons in the ventral tegmental area innervate specific forebrain regions and are involved in stress response.

Expanding knowledge about the cellular composition of subcortical brain regions demonstrates large heterogeneity and differences from the cortical architecture. Recently, we described three subtypes of somatostatin-expressing (Sst) neurons in the mouse ventral tegmental area (VTA) and showed their local inhibitory action on the neighbouring dopaminergic neurons (Nagaeva et al., 2020). Here, we report that mouse Sst+ neurons especially from the anterolateral part of the VTA also project far outside the VTA and innervate forebrain regions that are mainly involved in the regulation of emotional behaviour, including the ventral pallidum (VP), lateral hypothalamus (LH), the medial part of the central amygdala (CeM), anterolateral division of the bed nucleus of stria terminalis (alBNST), and paraventricular thalamic nucleus (PVT). Deletion of these VTASst neurons in mice affected several behaviours, such as home cage activity, sensitization of locomotor activity to morphine, fear conditioning responses and reactions to the inescapable stress of forced swimming, often in a sex-dependent manner. Together, these data demonstrate that VTASst neurons have selective projection targets distinct from the main targets of VTA dopamine neurons. VTASst neurons are involved in the regulation of behaviours primarily associated with the stress response, making them a relevant addition to the efferent VTA pathways and stress-related neuronal network.Significance StatementThe ventral tegmental area (VTA) is involved in many reward- and aversion-related processes contributing to brain disorders, but the cellular composition and brain-wide connections of this subcortical brain region are still poorly known. We recently described a heterogeneous somatostatin-expressing (Sst) neuron population in the mouse VTA, and, here, we revealed that, in addition to being local interneurons, VTA Sst neurons send long-distance projections to specific forebrain regions. Deletion of these neurons altered behavioural reactions to acute stressors and to repeated morphine administration. The results add the VTA Sst neurons to the stress-related network of the brain and establish a possibility to find out the exact roles of these neurons acting as behaviourally relevant interneurons and/or projection neurons.

Spatial Distribution of Neurons Expressing Single, Double, and Triple Molecular Characteristics of Glutamatergic, Dopaminergic, or GABAergic Neurons in the Mouse Ventral Tegmental Area.

The ventral tegmental area (VTA) is a heterogeneous midbrain area that plays a significant role in diverse neural processes such as reward, aversion, and motivation. The VTA contains three main neuronal populations, namely, dopamine (DA), γ-aminobutyric acid (GABA), and glutamate neurons, but some neurons exhibit combinatorial molecular characteristics of dopaminergic, GABAergic, or glutamatergic neurons. However, little information is available regarding detailed distribution of neurons with single, double, and triple molecular characteristics of glutamatergic, dopaminergic, or GABAergic neurons in mice. We present a topographical distribution map of three main neuronal populations expressing a single molecular characteristic of dopaminergic, GABAergic, or glutamatergic neurons, and four neuronal populations co-expressing double or triple molecular characteristics in combinatorial manners, in the mouse VTA, following analysis of triple fluorescent in situ hybridization for the simultaneous detection of tyrosine hydroxylase (TH, marker for dopaminergic neurons), vesicular glutamate transporter 2 (VGLUT2, marker for glutamatergic neurons), and glutamic acid decarboxylase 2 (GAD2, marker for GABAergic neurons) mRNA. We found that the vast majority of neurons expressed a single type of mRNA, and these neurons were intermingled with neurons co-expressing double or triple combinations of VGLUT2, TH, or GAD2 in the VTA. These seven neuronal populations were differentially distributed in the VTA sub-nuclei across the rostro-caudal and latero-medial axes. This histochemical study will lead to a deeper understanding of the complexity of neuronal molecular characteristics in different VTA sub-nuclei, and potentially facilitate clarification of diverse functions of the VTA.

Sex differences in VTA GABA transmission and plasticity during opioid withdrawal

The effectiveness of current treatments for opioid use disorder (OUD) varies by sex. Our understanding of the neurobiological mechanisms mediating negative states during withdrawal is lacking, particularly with regard to sex differences. Based on preclinical research in male subjects, opioid withdrawal is accompanied by increased gamma-aminobutyric acid (GABA) release probability at synapses onto dopamine neurons in the ventral tegmental area (VTA). It is unclear, however, if the physiological consequences of morphine that were originally elucidated in male rodents extend to females. The effects of morphine on the induction of future synaptic plasticity are also unknown. Here, we show that inhibitory synaptic long-term potentiation (LTPGABA) is occluded in the VTA in male mice after repeated morphine injections and 1 day of withdrawal, while morphine-treated female mice maintain the ability to evoke LTPGABA and have basal GABA activity similar to controls. Our observation of this physiological difference between male and female mice connects previous reports of sex differences in areas upstream and downstream of the GABA-dopamine synapse in the VTA during opioid withdrawal. The sex differences highlight the mechanistic distinctions between males and females that can be targeted when designing and implementing treatments for OUD.

RGS6 negatively regulates inhibitory G protein signaling in dopamine neurons and positively regulates binge-like alcohol consumption in mice.

Drugs of abuse, including alcohol, increase dopamine in the mesocorticolimbic system via actions on dopamine neurons in the ventral tegmental area (VTA). Increased dopamine transmission can activate inhibitory G protein signalling pathways in VTA dopamine neurons, including those controlled by GABAB and D2 receptors. Members of the R7 subfamily of regulator of G protein signalling (RGS) proteins can regulate inhibitory G protein signalling, but their influence on VTA dopamine neurons is unclear. Here, we investigated the influence of RGS6, an R7 RGS family memberthat has been implicated in the regulation of alcohol consumption in mice, on inhibitory G protein signalling in VTA dopamine neurons. We used molecular, electrophysiological and genetic approaches to probe the impact of RGS6 on inhibitory G protein signalling in VTA dopamine neurons and on binge-like alcohol consumption in mice. RGS6 is expressed in adult mouse VTA dopamine neurons and it modulates inhibitory G protein signalling in a receptor-dependent manner, tempering D2 receptor-induced somatodendritic currents and accelerating deactivation of synaptically evoked GABAB receptor-dependent responses. RGS6-/- mice exhibit diminished binge-like alcohol consumption, a phenotype replicated in female (but not male) mice lacking RGS6 selectively in VTA dopamine neurons. RGS6 negatively regulates GABAB - and D2 receptor-dependent inhibitory G protein signalling pathways in mouse VTA dopamine neurons and exerts a sex-dependent positive influence on binge-like alcohol consumption in adult mice. As such, RGS6 may represent a new diagnostic and/or therapeutic target for alcohol use disorder.

Neurotensin receptor 1-biased ligand attenuates neurotensin-mediated excitation of ventral tegmental area dopamine neurons and dopamine release in the nucleus accumbens

Strong expression of the G protein-coupled receptor (GPCR) neurotensin receptor 1 (NTR1) in ventral tegmental area (VTA) dopamine (DA) neurons and terminals makes it an attractive target to modulate DA neuron activity and normalize DA-related pathologies. Recent studies have identified a novel class of NTR1 ligand that shows promising effects in preclinical models of addiction. A lead molecule, SBI-0654553 (SBI-553), can act as a positive allosteric modulator of NTR1 β-arrestin recruitment while simultaneously antagonizing NTR1 Gq protein signaling. Using cell-attached recordings from mouse VTA DA neurons we discovered that, unlike neurotensin (NT), SBI-553 did not independently increase spontaneous firing. Instead, SBI-553 blocked the NT-mediated increase in firing. SBI-553 also antagonized the effects of NT on dopamine D2 auto-receptor signaling, potentially through its inhibitory effects on G-protein signaling. We also measured DA release directly, using fast-scan cyclic voltammetry in the nucleus accumbens and observed antagonist effects of SBI-553 on an NT-induced increase in DA release. Further, in vivo administration of SBI-553 did not notably change basal or cocaine-evoked DA release measured in NAc using fiber photometry. Overall, these results indicate that SBI-553 blunts NT's effects on spontaneous DA neuron firing, D2 auto-receptor function, and DA release, without independently affecting these measures. In the presence of NT, SBI-553 has an inhibitory effect on mesolimbic DA activity, which could contribute to its efficacy in animal models of psychostimulant use.

Preclinical Pharmacology of Solriamfetol: Potential Mechanisms for Wake Promotion

AbstractIntroductionSolriamfetol is a wake-promoting agent (WPA) approved for the treatment of excessive daytime sleepiness associated with narcolepsy and obstructive sleep apnea. The wake-promoting mechanism of solriamfetol may result from dopamine and norepinephrine reuptake inhibition. Preclinical pharmacology studies were conducted to further elucidate the molecular targets activated by solriamfetol and compare them to that of known WPAs and traditional stimulants.MethodsIn vitro binding and functional studies were conducted in a panel of cell lines or membrane preparations expressing transmembrane receptors and monoamine transporters to measure the activity of solriamfetol, comparator WPAs, and traditional stimulants. Electrophysiology studies were conducted in slice preparations from mouse ventral tegmental area (VTA). Studies to measure locomotor activity and wake-promoting effects were conducted in mice.ResultsIn vitro functional studies showed agonist activity of solriamfetol at human, mouse, and rat TAAR1 receptors. hTAAR1 EC50 values (10–16 μM) were within the clinically observed therapeutic solriamfetol plasma concentration range and overlapped with the observed DAT/NET inhibitory potencies of solriamfetol in vitro. TAAR1 agonist activity was unique to solriamfetol; neither the WPA modafinil nor the DAT/NET inhibitor bupropion had TAAR1 agonist activity. Solriamfetol (1–10 μM) dose-dependently inhibited the firing frequency of dopaminergic VTA neurons in mouse brain slices, similar to known TAAR1 agonists; however, these effects were inhibited by a D2 antagonist, suggesting a DAT-mediated effect. Unlike traditional stimulants, solriamfetol did not increase locomotor activity in naive mice, but inhibited the increase in locomotor activity in DAT knockout mice.ConclusionsPreclinical studies have identified agonist activity at the TAAR1 receptor and, possibly, lower potency agonist activity at 5-HT1A receptors as potential pharmacological targets for solriamfetol, in addition to its activity as a DAT/NET inhibitor. Given the current understanding of TAAR1 agonists as modulators of monoamine transmission with potential wake-promoting effects in multiple preclinical species, agonist activity at the hTAAR1 receptor may represent an additional pharmacological target underlying the wake-promoting effects for solriamfetol, in addition to its DNRI activity.FundingAxsome Therapeutics, Jazz Pharmaceuticals

Sex Differences in the Impact of Electronic Nicotine Vapor on Corticotropin-Releasing Factor Receptor 1 Neurons in the Mouse Ventral Tegmental Area.

Nicotine engages dopamine neurons in the ventral tegmental area (VTA) to encode reward and drive the development of nicotine addiction, however how nicotine alters a stress associated VTA population remains unclear. Here, we used male and female CRF1-GFP mice and nicotine vapor exposure to examine the effects of nicotine in VTA corticotropin-releasing factor receptor 1 (CRF1) neurons. We use immunohistochemistry and electrophysiology to examine neuronal activity, excitability, and inhibitory signaling. We found that VTA CRF1 neurons are mainly dopaminergic and project to the nucleus accumbens (NAc; VTA-NAcCRF1 neurons). VTA-NAcCRF1 neurons show greater phasic inhibition in naive females and greater focal nicotine-induced increases in firing in naive males. Following acute nicotine vapor exposure, phasic inhibition was not altered, but focal nicotine-induced tonic inhibition was enhanced in females and diminished in males. Acute nicotine vapor exposure did not affect firing in VTA-NAcCRF1 neurons, but females showed lower baseline firing and higher focal nicotine-induced firing. Activity (cFos) was increased in the CRF1 dopaminergic VTA population in both sexes, but with greater increases in females. Following chronic nicotine vapor exposure, both sexes displayed reduced basal phasic inhibition and the sex difference in tonic inhibition following acute vapor exposure was no longer observed. Additionally, activity of the CRF1 dopaminergic VTA population was no longer elevated in either sex. These findings reveal sex-dependent and exposure-dependent changes in mesolimbic VTA-NAc CRF1 neuronal activity, inhibitory signaling, and nicotine sensitivity following nicotine vapor exposure. These changes potentially contribute to nicotine-dependent behaviors and the intersection between stress, anxiety, and addiction.SIGNIFICANCE STATEMENT Nicotine is known to engage reward systems in the brain historically centering the neurotransmitter dopamine however, how nicotine impacts other neurons in the reward pathway is less clear. The current study investigates the impact of acute and chronic electronic nicotine vapor exposure in a genetically-defined cell population containing the stress receptor corticotropin-releasing factor 1 (CRF1) that is located in the reward circuitry. This study employs functional measures of neuronal activity and identifies important sex differences in nicotine's effects across time and exposure.