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 (1-10 mM) enhanced GABA receptor (GABA 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 (5-100 mM) had no effect on optically evoked GABA 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.

The VTA is necessary for reward behavior with dopamine cells critically involved in reward signaling. Dopamine cells in turn are innervated and regulated by neighboring inhibitory GABA cells. Using whole-cell electrophysiology in juvenile-adolescent GAD67-GFP male mice, we examined excitatory plasticity in fluorescent VTA GABA cells. A novel CB1-dependent LTD was induced in GABA cells that was dependent on metabotropic glutamate receptor 5, and cannabinoid receptor 1 (CB1). LTD was absent in CB1 knock-out mice but preserved in heterozygous littermates. Bath applied ⌬ 9-tetrahydrocannabinol depressed GABA cell activity, therefore downstream dopamine cells will be disinhibited; and thus, this could potentially result in increased reward. Chronic injections of ⌬ 9 -tetrahydrocannabinol occluded LTD compared with vehicle injections; however, a single exposure was insufficient to do so. As synaptic modifications by drugs of abuse are often tied to addiction, these data suggest a possible mechanism for the addictive effects of ⌬ 9 -tetrahydrocannabinol in juvenile-adolescents, by potentially altering reward behavioral outcomes.

The ventral tegmental area (VTA) is involved in adaptive reward and motivation processing and is composed of dopamine (DA) and GABA neurons. Defining the elements regulating activity and synaptic plasticity of these cells is critical to understanding mechanisms of reward and addiction. While endocannabinoids (eCBs) that potentially contribute to addiction are known to be involved in synaptic plasticity mechanisms in the VTA, where they are produced is poorly understood. In this study, DA and GABAergic cells were identified using electrophysiology, cellular markers, and a transgenic mouse model that specifically labels GABA cells. Using single-cell RT-qPCR and immunohistochemistry, we investigated mRNA and proteins involved in eCB signaling such as diacylglycerol lipase α, N-acyl-phosphatidylethanolamine-specific phospholipase D, and 12-lipoxygenase, as well as type I metabotropic glutamate receptors (mGluRs). Our results demonstrate the first molecular evidence of colocalization of eCB biosynthetic enzyme and type I mGluR mRNA in VTA neurons. Further, these data reveal higher expression of mGluR1 in DA neurons, suggesting potential differences in eCB synthesis between DA and GABA neurons. These data collectively suggest that VTA GABAergic and DAergic cells have the potential to produce various eCBs implicated in altering neuronal activity or plasticity in adaptive motivational reward or addiction.

GPR55, an orphan G-protein coupled receptor, is activated by lysophosphatidylinositol (LPI) and the endocannabinoid anandamide, as well as by other compounds including THC. Such signaling molecules are capable of modulating synaptic plasticity. LPI is a potent endogenous ligand of GPR55 and neither GPR55 nor LPIs’ functions in the brain are well understood. While endocannabinoids are well known to modulate brain synaptic plasticity, the potential role LPI could have on brain plasticity has never been demonstrated. Therefore, we examined not only GPR55 expression, but also the role its endogenous ligand could play in long-term potentiation, a common form of synaptic plasticity. Using quantitative RT-PCR, electrophysiology, and behavioral assays, we examined hippocampal GPR55 expression and function. qRT-PCR results indicate that GPR55 is expressed in hippocampi of both rats and mice. Immunohistochemistry and single cell PCR demonstrates GPR55 protein in pyramidal cells of CA1 and CA3 layers in the hippocampus. Application of the GPR55 endogenous agonist LPI to hippocampal slices of GPR55+/+ mice significantly enhanced CA1 LTP. This effect was absent in GPR55−/− mice, and blocked by the GPR55 antagonist CID 16020046. We also examined paired-pulse ratios of GPR55−/− and GPR55+/+ mice with or without LPI and noted significant enhancement in paired-pulse ratios by LPI in GPR55+/+ mice. Behaviorally, GPR55−/− and GPR55+/+ mice did not differ in memory tasks including novel object recognition, radial arm maze, or Morris water maze. However, performance on radial arm maze and elevated plus maze task suggests GPR55−/− mice have a higher frequency of immobile behavior. This is the first demonstration of LPI involvement in hippocampal synaptic plasticity.

The novel nuclear protein nBMP2 is synthesized from the BMP2 gene by translational initiation at an alternative start codon. We generated a targeted mutant mouse, nBmp2NLStm, in which the nuclear localization signal (NLS) was inactivated to prevent nuclear translocation of nBMP2 while still allowing the normal synthesis and secretion of the BMP2 growth factor. These mice exhibit abnormal muscle function due to defective Ca2+ transport in skeletal muscle. We hypothesized that neurological function, which also depends on intracellular Ca2+ transport, could be affected by the loss of nBMP2. Age-matched nBmp2NLStm and wild type mice were analyzed by immunohistochemistry, behavioral tests, and electrophysiology to assess nBMP2 expression and neurological function. Immunohistochemical staining of the hippocampus detected nBMP2 in the nuclei of CA1 neurons in wild type but not mutant mice, consistent with nBMP2 playing a role in the hippocampus. Mutant mice showed deficits in the novel object recognition task, suggesting hippocampal dysfunction. Electrophysiology experiments showed that long-term potentiation (LTP) in the hippocampus, which is dependent on intracellular Ca2+ transport and is thought to be the cellular equivalent of learning and memory, was impaired. Together, these results suggest that nBMP2 in the hippocampus impacts memory formation.