Nociceptive Stimuli Activate the Hypothalamus-Habenula Circuit to Inhibit the Mesolimbic Reward System and Cocaine-Seeking Behaviors.

Nociceptive signals interact with various regions of the brain, including those involved in physical sensation, reward, cognition, and emotion. Emerging evidence points to a role of nociception in the modulation of the mesolimbic reward system. The mechanism by which nociception affects dopamine (DA) signaling and reward is unclear. The lateral hypothalamus (LH) and the lateral habenula (LHb) receive somatosensory inputs and are structurally connected with the mesolimbic DA system. Here, we show that the LH-LHb pathway is necessary for nociceptive modulation of this system using male Sprague Dawley rats. Our extracellular single-unit recordings and head-mounted microendoscopic calcium imaging revealed that nociceptive stimulation by tail pinch excited LHb and LH neurons, which was inhibited by chemical lesion of the LH. Tail pinch increased activity of GABA neurons in ventral tegmental area, decreased the extracellular DA level in the nucleus accumbens ventrolateral shell in intact rats, and reduced cocaine-increased DA concentration, which was blocked by disruption of the LH. Furthermore, tail pinch attenuated cocaine-induced locomotor activity, 22 and 50 kHz ultrasonic vocalizations, and reinstatement of cocaine-seeking behavior, which was inhibited by chemogenetic silencing of the LH-LHb pathway. Our findings suggest that nociceptive stimulation recruits the LH-LHb pathway to inhibit mesolimbic DA system and drug reinstatement.SIGNIFICANCE STATEMENT The LHb and the LH have been implicated in processing nociceptive signals and modulating DA release in the mesolimbic DA system. Here, we show that the LH-LHb pathway is critical for nociception-induced modulation of mesolimbic DA release and cocaine reinstatement. Nociceptive stimulation alleviates extracellular DA release in the mesolimbic DA system, cocaine-induced psychomotor activities, and reinstatement of cocaine-seeking behaviors through the LH-LHb pathway. These findings provide novel evidence for sensory modulation of the mesolimbic DA system and drug addiction.

Interleukin-10 enhances activity of ventral tegmental area dopamine neurons resulting in increased dopamine release.

Dopamine transmission from the ventral tegmental area (VTA) to the nucleus accumbens (NAc) regulates important aspects of motivation and is influenced by the neuroimmune system. The neuroimmune system is a complex network of leukocytes, microglia and astrocytes that detect and remove foreign threats like bacteria or viruses and communicate with each other to regulate non-immune (e.g neuronal) cell activity through cytokine signaling. Inflammation is a key regulator of motivational states, though the effects of specific cytokines on VTA circuitry and motivation are largely unknown. Therefore, electrophysiology, neurochemical, immunohistochemical and behavioral studies were performed to determine the effects of the anti-inflammatory cytokine interleukin-10 (IL-10) on mesolimbic activity, dopamine transmission and conditioned behavior. IL-10 enhanced VTA dopamine firing and NAc dopamine levels via decreased VTA GABA currents in dopamine neurons. The IL-10 receptor was localized on VTA dopamine and non-dopamine cells. The IL-10 effects on dopamine neurons required post-synaptic phosphoinositide 3-kinase activity, and IL-10 appeared to have little-to-no efficacy on presynaptic GABA terminals. Intracranial IL-10 enhanced NAc dopamine levels in vivo and produced conditioned place aversion. Together, these studies identify the IL-10R on VTA dopamine neurons as a potential regulator of motivational states.

Activation of a hypothalamus-habenula circuit by mechanical stimulation inhibits cocaine addiction-like behaviors.

BACKGROUND: Mechanoreceptor activation modulates GABA neuron firing and dopamine (DA) release in the mesolimbic DA system, an area implicated in reward and substance abuse. The lateral habenula (LHb), the lateral hypothalamus (LH), and the mesolimbic DA system are not only reciprocally connected, but also involved in drug reward. We explored the effects of mechanical stimulation (MS) on cocaine addiction-like behaviors and the role of the LH-LHb circuit in the MS effects. MS was performed over ulnar nerve and the effects were evaluated by using drug seeking behaviors, optogenetics, chemogenetics, electrophysiology and immunohistochemistry. RESULTS: Mechanical stimulation attenuated locomotor activity in a nerve-dependent manner and 50-kHz ultrasonic vocalizations (USVs) and DA release in nucleus accumbens (NAc) following cocaine injection. The MS effects were ablated by electrolytic lesion or optogenetic inhibition of LHb. Optogenetic activation of LHb suppressed cocaine-enhanced 50 kHz USVs and locomotion. MS reversed cocaine suppression of neuronal activity of LHb. MS also inhibited cocaine-primed reinstatement of drug-seeking behavior, which was blocked by chemogenetic inhibition of an LH-LHb circuit. CONCLUSION: These findings suggest that peripheral mechanical stimulation activates LH-LHb pathways to attenuate cocaine-induced psychomotor responses and seeking behaviors.

Opioid-Induced Reductions in Amygdala Lateral Paracapsular GABA Neuron Circuit Activity.

Opioid use and withdrawal evokes behavioral adaptations such as drug seeking and anxiety, though the underlying neurocircuitry changes are unknown. The basolateral amygdala (BLA) regulates these behaviors through principal neuron activation. Excitatory BLA pyramidal neuron activity is controlled by feedforward inhibition provided, in part, by lateral paracapsular (LPC) GABAergic inhibitory neurons, residing along the BLA/external capsule border. LPC neurons express µ-opioid receptors (MORs) and are potential targets of opioids in the etiology of opioid-use disorders and anxiety-like behaviors. Here, we investigated the effects of opioid exposure on LPC neuron activity using immunohistochemical and electrophysiological approaches. We show that LPC neurons, and other nearby BLA GABA and non-GABA neurons, express MORs and δ-opioid receptors. Additionally, DAMGO, a selective MOR agonist, reduced GABA but not glutamate-mediated spontaneous postsynaptic currents in LPC neurons. Furthermore, in LPC neurons, abstinence from repeated morphine-exposure in vivo (10 mg/kg/day, 5 days, 2 days off) decrease the intrinsic membrane excitability, with a ~75% increase in afterhyperpolarization and ~40-50% enhanced adenylyl cyclase-dependent activity in LPC neurons. These data show that MORs in the BLA are a highly sensitive targets for opioid-induced inhibition and that repeated opioid exposure results in impaired LPC neuron excitability.

Whole-Body Vibration Prevents Neuronal, Neurochemical, and Behavioral Effects of Morphine Withdrawal in a Rat Model.

Peripheral mechanoreceptor-based treatments such as acupuncture and chiropractic manipulation have shown success in modulating the mesolimbic dopamine (DA) system originating in the ventral tegmental area (VTA) of the midbrain and projecting to the nucleus accumbens (NAc) of the striatum. We have previously shown that mechanoreceptor activation via whole-body vibration (WBV) ameliorates neuronal and behavioral effects of chronic ethanol exposure. In this study, we employ a similar paradigm to assess the efficacy of WBV as a preventative measure of neuronal and behavioral effects of morphine withdrawal in a Wistar rat model. We demonstrate that concurrent administration of WBV at 80 Hz with morphine over a 5-day period significantly reduced adaptations in VTA GABA neuronal activity and NAc DA release and modulated expression of δ-opioid receptors (DORs) on NAc cholinergic interneurons (CINs) during withdrawal. We also observed a reduction in behavior typically associated with opioid withdrawal. WBV represents a promising adjunct to current intervention for opioid use disorder (OUD) and should be examined translationally in humans.

Teaching Synaptic Transmission Using Primary Literature: A Skills-Focused Pedagogical Approach.

Neuroscience is a burgeoning and intensive undergraduate major at many institutions of higher education and several areas in neuroscience education need further development. One such needed development is an increased focus on the procurement of career-relevant skills in addition to the traditional acquisition of subject knowledge. Skill development is particularly challenging in neuroscience education as the subject's interdisciplinary nature provides an atypically broad range of potential careers for graduates. Skills common to many careers in neuroscience include the ability to understand and analyze quantitative data and to draw conclusions based on those analyses. Here is presented an active learning pedagogical approach involving the analysis of seminal articles in the primary scientific literature to provide practice in analyzing data and drawing conclusions from those data while at the same time learning the fundamental tenets of synaptic transmission. Articles were selected that highlight principles such as the role of Ca2+ in synaptic release, exocytosis, quantal release, and synaptic delay. Figures from these articles that can readily be used to teach these principles were selected, and questions that can help to guide students' analysis of the data are also suggested. Activities like this are needed in greater numbers to facilitate the process of helping students gain skills relevant to a productive career in neuroscience.

Regional and sex differences in spontaneous striatal dopamine transmission.

Striatal dopamine release is key for learning and motivation and is composed of subregions including the dorsal striatum (DS), nucleus accumbens core, and the nucleus accumbens shell. Spontaneously occurring dopamine release was compared across these subregions. Dopamine release/uptake dynamics differ across striatal subregions, with dopamine transient release amplitude and release frequency greatest in male mice, and the largest signals observed in the DS. Surprisingly, female mice exhibited little regional differences in dopamine release for DS and nucleus accumbens core regions, but lower release in the nucleus accumbens shell. Blocking voltage-gated K+ channel (Kv channels) with 4-aminopyridine enhanced dopamine detection without affecting reuptake. The 4-aminopyridine effects were greatest in ventral regions of female mice, suggesting regional differences in Kv channel expression. The dopamine transporter blocker cocaine also enhanced detection across subregions in both sexes, with greater overall increased release in females than males. Thus, sex differences in dopamine transmission are apparent and likely include differences in the Kv channel and dopamine transporter function. The lack of regional differences in dopamine release observed in females indicates differential regulation of spontaneous and evoked dopamine release.

The peripheral dopamine 2 receptor antagonist domperidone attenuates ethanol enhancement of dopamine levels in the nucleus accumbens.

BACKGROUND: Dopamine neuron firing in the ventral tegmental area (VTA) and dopamine release in the nucleus accumbens have been implicated in reward learning. Ethanol is known to increase both dopamine neuron firing in the VTA and dopamine levels in the nucleus accumbens. Despite this, some discrepancies exist between the dose of ethanol required to enhance firing in vivo and ex vivo. In the present study we investigated the effects of peripheral dopamine 2 subtype receptor antagonism on ethanol's effects on dopamine neurotransmission. METHODS: Plasma catecholamine levels were assessed following ethanol administration across four different doses of EtOH. Microdialysis and voltammetry were used to assess the effects of domperidone pretreatment on ethanol-mediated increases in dopamine release in the nucleus accumbens. A place conditioning paradigm was used to assess conditioned preference for ethanol and whether domperidone pretreatment altered this preference. Open-field and loss-of-righting reflex paradigms were used to assess the effects of domperidone on ethanol-induced sedation. A rotarod apparatus was used to assess the effects of domperidone on ethanol-induced motor impairment. RESULTS: Domperidone attenuated ethanol's enhancement of mesolimbic dopamine release under non-physiological conditions at intermediate (1.0 and 2.0 g/kg) doses of ethanol. Domperidone also decreased EtOH-induced sedation at 2.0 g/kg. Domperidone did not alter ethanol conditioned place preference nor did it affect ethanol-induced motor impairment. CONCLUSIONS: These results show that peripheral dopamine 2 receptors mediate some of the effects of ethanol on nonphysiological dopamine neurotransmission, although these effects are not related to the rewarding properties of ethanol.

Modulation of dopamine release by ethanol is mediated by atypical GABAA receptors on cholinergic interneurons in the nucleus accumbens.

Previous studies indicate that moderate-to-high ethanol (EtOH) concentrations enhance dopamine (DA) neurotransmission in the mesolimbic DA system from the ventral tegmental area (VTA) and projecting to the nucleus accumbens core (NAc). However, voltammetry studies demonstrate that moderate-to-high EtOH concentrations decrease evoked DA release at NAc terminals. The involvement of γ-aminobutyric acid (GABA) receptors (GABAA Rs), glycine (GLY) receptors (GLYRs) and cholinergic interneurons (CINs) in mediating EtOH inhibition of evoked NAc DA release were examined. Fast scan cyclic voltammetry, electrophysiology, optogenetics and immunohistochemistry techniques were used to evaluate the effects of acute and chronic EtOH exposure on DA release and CIN activity in C57/BL6, CD-1, transgenic mice and δ-subunit knockout (KO) mice (δ-/-). Ethanol decreased DA release in mice with an IC50 of 80 mM ex vivo and 2.0 g/kg in vivo. GABA and GLY decreased evoked DA release at 1-10 mM. Typical GABAA R agonists inhibited DA release at high concentrations. Typical GABAA R antagonists had minimal effects on EtOH inhibition of evoked DA release. However, EtOH inhibition of DA release was blocked by the α4 ß3 δ GABAA R antagonist Ro15-4513, the GLYR antagonist strychnine and by the GABA ρ1 (Rho-1) antagonist TPMPA (10 µM) and reduced significantly in GABAA R δ-/- mice. Rho-1 expression was observed in CINs. Ethanol inhibited GABAergic synaptic input to CINs from the VTA and enhanced firing rate, both of which were blocked by TPMPA. Results herein suggest that EtOH inhibition of DA release in the NAc is modulated by GLYRs and atypical GABAA Rs on CINs containing δ- and Rho-subunits.

Mechanical Stimulation Alters Chronic Ethanol-Induced Changes to VTA GABA Neurons, NAc DA Release and Measures of Withdrawal.

Therapeutic activation of mechanoreceptors (MStim) in osteopathy, chiropractic and acupuncture has been in use for hundreds of years with a myriad of positive outcomes. It has been previously shown to modulate the firing rate of neurons in the ventral tegmental area (VTA) and dopamine (DA) release in the nucleus accumbens (NAc), an area of interest in alcohol-use disorder (AUD). In this study, we examined the effects of MStim on VTA GABA neuron firing rate, DA release in the NAc, and behavior during withdrawal from chronic EtOH exposure in a rat model. We demonstrate that concurrent administration of MStim and EtOH significantly reduced adaptations in VTA GABA neurons and DA release in response to a reinstatement dose of EtOH (2.5 g/kg). Behavioral indices of EtOH withdrawal (rearing, open-field crosses, tail stiffness, gait, and anxiety) were substantively ameliorated with concurrent application of MStim. Additionally, MStim significantly increased the overall frequency of ultrasonic vocalizations, suggesting an increased positive affective state.

The role of substance P in acupuncture signal transduction and effects.

BACKGROUND: Acupuncture has been used to treat a wide variety of diseases, disorders, and conditions for more than 2500 years. While the anatomical structures of acupuncture points (or acupoints) are largely unknown, our previous studies have suggested that many acupoints can be identified as cutaneous neurogenic inflammatory spots (neurogenic spots or Neuro-Sps), arising from the release of neuropeptides from activated small diameter sensory afferents at topographically distinct body surfaces due to the convergence of visceral and somatic afferents. In turn, the neuropeptides released during neurogenic inflammation may play important roles in the effects of acupuncture as well as the formation of active acupoints. Thus, the present study has focused on the role of substance P (SP) in acupuncture signal transduction and effects. METHODS: Neuro-Sps were detected by using in vivo fluorescence imaging after intravenous injection of Evans blue dye (EBD) and compared with traditional acupoints. Stimulatory effects of the Neuro-Sps were examined in a rat model of immobilization-induced hypertension (IMH). The roles of increased SP in Neuro-Sps were also investigated by using immunohistochemistry, in vivo single-fiber peripheral nerve recordings, and in vivo midbrain extracellular recordings. RESULTS: Neurogenic inflammation quickly appeared at acupoints on the wrist and was fully developed within 15 min in IMH model. The Neuro-Sps showed an increased release of SP from afferent nerve terminals. Mechanical stimulation of these Neuro-Sps increased cell excitability in the midbrain (rostral ventrolateral medulla) and alleviated the development of hypertension, which was blocked by the local injection of the SP receptor antagonist CP-99994 into Neuro-Sps prior to acupuncture and mimicked by the local injection of capsaicin. Single fiber recordings of peripheral nerves showed that increased SP into the Neuro-Sps elevated the sensitivity of A- and C-fibers in response to acupuncture stimulation. In addition, the discharge rates of spinal wide dynamic response (WDR) neurons significantly increased following SP or acupuncture treatment in Neuro-Sps in normal rats, but decreased following the injection of CP-99994 into Neuro-Sps in IMH rats. CONCLUSIONS: Our findings suggest that SP released during neurogenic inflammation enhances the responses of sensory afferents to the needling of acupoints and triggers acupuncture signaling to generate acupuncture effects.

Role of the central amygdala in acupuncture inhibition of methamphetamine-induced behaviors in rats.

Methamphetamine (METH) enhances dopamine (DA) transmission in the mesolimbic system implicated in its reinforcing effects. Our previous studies have shown that acupuncture attenuates drug-seeking behaviors by modulating GABAergic transmission in the ventral tegmental area and DA release in the nucleus accumbens (NAc) of the striatum. The effects of acupuncture on METH-induced behaviors and its mediation by neural pathways remain a relatively understudied area of research. The central amygdala (CeA) plays a critical role in physiological and behavioral responses to somatosensory and drug stimuli and has been implicated in negative reinforcement. Thus, we evaluated the role of the CeA in acupuncture effects on locomotor activity, positive affective states, and DA release in the NAc following acute administration of METH. Acupuncture at acupoint HT7 reduced locomotor activity, 50-kHz ultrasonic vocalizations (USVs), and NAc DA release following systemic injection of METH, which was prevented by electrolytic lesions or optogenetic inhibition of the CeA. Acupuncture alone excited CeA neurons and reversed the suppression of CeA neurons induced by METH. These results suggest that acupuncture can relieve psychomotor responses and positive affective states following METH by inhibiting NAc DA release and this effect is mediated by activation of CeA neurons.

Acupuncture reduces cocaine psychomotor responses by activating the rostromedial tegmental nucleus.

The rostromedial tegmental nucleus (RMTg), a GABAergic afferent to midbrain dopamine (DA) neurons, has emerged as an integral player in both rewarding and nociceptive responses. While previous studies have demonstrated that acupuncture modulates DA transmission in the mesolimbic reward system originating in the ventral tegmental area (VTA) and projecting to the nucleus accumbens (NAc) and can reduce drug self-administration, the central links between peripheral acupuncture signals and brain reward systems are not well-characterized. Thus, we hypothesised that acupuncture would elicit inhibitory signals from RMTg neurons to brain reward systems. Acupuncture reduced acute cocaine-induced locomotor activity and DA release in a point-specific manner, which was blocked by optogenetic silencing or chemical lesion of the RMTg. The acupuncture effect was mimicked by chemical activation of the RMTg. Acupuncture activated RMTg GABA neurons. In addition, the inhibitory effects of acupuncture on acute cocaine-induced locomotor activity were prevented by electrolytic lesions of the lateral habenula (LHb) or fasciculus retroflexus (FR), areas known to project to the RMTg. These findings suggest that acupuncture recruits the RMTg to reduce the psychomotor responses enhanced by acute cocaine.

Role of Lateral Hypothalamus in Acupuncture Inhibition of Cocaine Psychomotor Activity.

Acupuncture modulates the mesolimbic dopamine (DA) system; an area implicated in drug abuse. However, the mechanism by which peripheral sensory afferents, during acupuncture stimulation, modulate this system needs further investigation. The lateral hypothalamus (LH) has been implicated in reward processing and addictive behaviors. To investigate the role of the LH in mediating acupuncture effects, we evaluated the role of LH and spinohypothalamic neurons on cocaine-induced psychomotor activity and NAc DA release. Systemic injection of cocaine increased locomotor activity and 50 kHz ultrasonic vocalizations (USVs), which were attenuated by mechanical stimulation of needles inserted into HT7 but neither ST36 nor LI5. The acupuncture effects were blocked by chemical lesions of the LH or mimicked by activation of LH neurons. Single-unit extracellular recordings showed excitation of LH and spinohypothalamic neurons following acupuncture. Our results suggest that acupuncture recruits the LH to suppress the mesolimbic DA system and psychomotor responses following cocaine injection.

The potent psychomotor, rewarding and reinforcing properties of 3-fluoromethamphetamine in rodents.

3-fluoromethamphetamine (3-FMA), a derivative of methamphetamine (METH), produces behavioral impairment and deficits in dopaminergic transmission in the striatum of mice. The abuse potential of 3-FMA has not been fully characterized. The aim of this study was to evaluate the effects of 3-FMA on locomotor activity as well as its rewarding and reinforcing properties in the conditioned place preference (CPP) and self-administration procedures. Intravenous (i.v.) administration of 3-FMA (0.5 and 1.0 mg/kg) significantly increased locomotor activity in a dose-dependent manner in rats. In the CPP procedure, intraperitoneal administration of 3-FMA (10 and 30 mg/kg) produced a significant alteration in place preference in mice. In the self-administration paradigms, 3-FMA showed drug-taking behavior at the dose of 0.1 mg/kg/infusion (i.v.) during 2 hr sessions under fixed ratio schedules and high breakpoints at the dose of 0.3 and 1.0 mg/kg/infusion (i.v.) during 6 hr sessions under progressive ratio schedule of reinforcement in rats. A priming injection of 3-FMA (0.4 mg/kg, i.v.), METH (0.2 mg/kg, i.v.), or cocaine (2.0 mg/kg, i.v.) reinstated 3-FMA-seeking behavior after an extinction period in 3-FMA-trained rats during 2 hr session. Taken together, these findings demonstrate robust psychomotor, rewarding and reinforcing properties of 3-FMA, which may underlie its potential for compulsive use in humans.

The Abuse Potential of Novel Synthetic Phencyclidine Derivative 1-(1-(4-Fluorophenyl)Cyclohexyl)Piperidine (4'-F-PCP) in Rodents.

The dissociative anesthetic phencyclidine (PCP) and PCP derivatives, including 4'-F-PCP, are illegally sold and abused worldwide for recreational and non-medical uses. The psychopharmacological properties and abuse potential of 4'-F-PCP have not been fully characterized. In this study, we evaluated the psychomotor, rewarding, and reinforcing properties of 4'-F-PCP using the open-field test, conditioned place preference (CPP), and self-administration paradigms in rodents. Using Western immunoblotting, we also investigated the expression of dopamine (DA)-related proteins and DA-receptor-mediated downstream signaling cascades in the nucleus accumbens (NAc) of 4'-F-PCP-self-administering rats. Intraperitoneal administration of 10 mg/kg 4'-F-PCP significantly increased locomotor and rearing activities and increased CPP in mice. Intravenous administration of 1.0 mg/kg/infusion of 4'-F-PCP significantly enhanced self-administration during a 2 h session under fixed ratio schedules, showed a higher breakpoint during a 6 h session under progressive ratio schedules of reinforcement, and significantly altered the expression of DA transporter and DA D1 receptor in the NAc of rats self-administering 1.0 mg/kg 4'-F-PCP. Additionally, the expression of phosphorylated (p) ERK, pCREB, c-Fos, and FosB/ΔFosB in the NAc was significantly enhanced by 1.0 mg/kg 4'-F-PCP self-administration. Taken together, these findings suggest that 4'-F-PCP has a high potential for abuse, given its robust psychomotor, rewarding, and reinforcing properties via activation of DAergic neurotransmission and the downstream signaling pathways in the NAc.

Acupuncture inhibition of methamphetamine-induced behaviors, dopamine release and hyperthermia in the nucleus accumbens: mediation of group II mGluR.

Methamphetamine (METH) increases metabolic neuronal activity in the mesolimbic dopamine (DA) system and mediates the reinforcing effect. To explore the underlying mechanism of acupuncture intervention in reducing METH-induced behaviors, we investigated the effect of acupuncture on locomotor activity, ultrasonic vocalizations, extracellular DA release in the nucleus accumbens (NAcs) using fast-scan cyclic voltammetry and alterations of brain temperature (an indicator of local brain metabolic activity) produced by METH administration. When acupuncture was applied to HT7, but not TE4, both locomotor activity and 50-kHz ultrasonic vocalizations were suppressed in METH-treated rats. Acupuncture at HT7 attenuated the enhancement of electrically stimulated DA release in the NAc of METH-treated rats. Systemic injection of METH produced a sustained increase in NAc temperature, which was reversed by the DA D1 receptor antagonist SCH 23390 or acupuncture at HT7. Acupuncture inhibition of METH-induced NAc temperature was prevented by pre-treatment with a group II metabotropic glutamate receptors (mGluR2/3) antagonist EGLU into the NAc or mimicked by injection of an mGluR2/3 agonist DCG-IV into the NAc. These results suggest that acupuncture reduces extracellular DA release and metabolic neuronal activity in the NAc through activation of mGluR2/3 and suppresses METH-induced affective states and locomotor behavior.

Glutamate Transmission to Ventral Tegmental Area GABA Neurons Is Altered by Acute and Chronic Ethanol.

BACKGROUND: Ventral tegmental area (VTA) GABA neurons have been heavily implicated in alcohol reinforcement and reward. In animals that self-administer alcohol, VTA GABA neurons exhibit increased excitability that may contribute to alcohol's rewarding effects. The present study investigated the effects of acute and chronic ethanol exposure on glutamate (GLU) synaptic transmission to VTA GABA neurons. METHODS: Whole-cell recordings of evoked, spontaneous, and miniature excitatory postsynaptic currents (eEPSCs, sEPSCs, and mEPSCs, respectively) were performed on identified GABA neurons in the VTA of GAD67-GFP+ transgenic mice. Three ethanol exposure paradigms were used: acute ethanol superfusion; a single ethanol injection; and chronic vapor exposure. RESULTS: Acute ethanol superfusion increased the frequency of EPSCs but inhibited mEPSC frequency and amplitude. During withdrawal from a single injection of ethanol, the frequency of sEPSCs was lower than saline controls. There was no difference in α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/N-methyl-d-aspartate (NMDA) ratio between neurons following withdrawal from a single exposure to ethanol. However, following withdrawal from chronic ethanol, sEPSCs and mEPSCs had a greater frequency than air controls. There was no difference in AMPA/NMDA ratio following chronic ethanol. CONCLUSIONS: These results suggest that presynaptic mechanisms involving local circuit GLU neurons, and not GLU receptors, contribute to adaptations in VTA GABA neuron excitability that accrue to ethanol exposure, which may contribute to the rewarding properties of alcohol via their regulation of mesolimbic dopamine transmission.

Pharmacological and functional comparisons of α6/α3ß2ß3-nAChRs and α4ß2-nAChRs heterologously expressed in the human epithelial SH-EP1 cell line.

Neuronal nicotinic acetylcholine receptors containing α6 subunits (α6*-nAChRs) show highly restricted distribution in midbrain neurons associated with pleasure, reward, and mood control, suggesting an important impact of α6*-nAChRs in modulating mesolimbic functions. However, the function and pharmacology of α6*-nAChRs remain poorly understood because of the lack of selective agonists for α6*-nAChRs and the challenging heterologous expression of functional α6*-nAChRs in mammalian cell lines. In particular, the α6 subunit is commonly co-expressed with α4*-nAChRs in the midbrain, which masks α6*-nAChR (without α4) function and pharmacology. In this study, we systematically profiled the pharmacology and function of α6*-nAChRs and compared these properties with those of α4ß2 nAChRs expressed in the same cell line. Heterologously expressed human α6/α3 chimeric subunits (α6 N-terminal domain joined with α3 trans-membrane domains and intracellular loops) with ß2 and ß3 subunits in the human SH-EP1 cell line (α6*-nAChRs) were used. Patch-clamp whole-cell recordings were performed to measure these receptor-mediated currents. Functionally, the heterologously expressed α6*-nAChRs exhibited excellent function and showed distinct nicotine-induced current responses, such as kinetics, inward rectification and recovery from desensitization, compared with α4ß2-nAChRs. Pharmacologically, α6*-nAChR was highly sensitive to the α6 subunit-selective antagonist α-conotoxin MII but had lower sensitivity to mecamylamine and dihydro-ß-erythroidine. Nicotine and acetylcholine were found to be full agonists for α6*-nAChRs, whereas epibatidine and cytisine were determined to be partial agonists. Heterologously expressed α6*-nAChRs exhibited pharmacology and function distinct from those of α4ß2-nAChRs, suggesting that α6*-nAChRs may mediate different cholinergic signals. Our α6*-nAChR expression system can be used as an excellent cell model for future investigations of α6*-nAChR function and pharmacology.

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 (1-10 mM) 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 (5-100 mM) 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.