Nicotinic Cholinergic Receptors in VTA Glutamate Neurons Modulate Excitatory Transmission.
ABSTRACT: Ventral tegmental area (VTA) glutamate neurons are important components of reward circuitry, but whether they are subject to cholinergic modulation is unknown. To study this, we used molecular, physiological, and photostimulation techniques to examine nicotinic acetylcholine receptors (nAChRs) in VTA glutamate neurons. Cells in the medial VTA, where glutamate neurons are enriched, are responsive to acetylcholine (ACh) released from cholinergic axons. VTA VGLUT2+ neurons express mRNA and protein subunits known to comprise heteromeric nAChRs. Electrophysiology, coupled with two-photon microscopy and laser flash photolysis of photoactivatable nicotine, was used to demonstrate nAChR functional activity in the somatodendritic subcellular compartment of VTA VGLUT2+ neurons. Finally, optogenetic isolation of intrinsic VTA glutamatergic microcircuits along with gene-editing techniques demonstrated that nicotine potently modulates excitatory transmission within the VTA via heteromeric nAChRs. These results indicate that VTA glutamate neurons are modulated by cholinergic mechanisms and participate in the cascade of physiological responses to nicotine exposure.
Project description:Evidence shows that the neurotransmitter dopamine mediates the rewarding effects of nicotine and other drugs of abuse, while nondopaminergic neural substrates mediate the negative motivational effects. β2* nicotinic acetylcholine receptors (nAChR) are necessary and sufficient for the experience of both nicotine reward and aversion in an intra-VTA (ventral tegmental area) self-administration paradigm. We selectively reexpressed β2* nAChRs in VTA dopamine or VTA γ-amino-butyric acid (GABA) neurons in β2-/- mice to double-dissociate the aversive and rewarding conditioned responses to nicotine in nondependent mice, revealing that β2* nAChRs on VTA dopamine neurons mediate nicotine's conditioned aversive effects, while β2* nAChRs on VTA GABA neurons mediate the conditioned rewarding effects in place-conditioning paradigms. These results stand in contrast to a purely dopaminergic reward theory, leading to a better understanding of the neurobiology of nicotine motivation and possibly to improved therapeutic treatments for smoking cessation.
Project description:High-affinity, beta2-subunit-containing (beta2*) nicotinic acetylcholine receptors (nAChRs) are essential for nicotine reinforcement; however, these nAChRs are found on both gamma-aminobutyric acid (GABA) and dopaminergic (DA) neurons in the ventral tegmental area (VTA) and also on terminals of glutamatergic and cholinergic neurons projecting from the pedunculopontine tegmental area and the laterodorsal tegmental nucleus. Thus, systemic nicotine administration stimulates many different neuronal subtypes in various brain nuclei. To identify neurons in which nAChRs must be expressed to mediate effects of systemic nicotine, we investigated responses in mice with low-level, localized expression of beta2* nAChRs in the midbrain/VTA. Nicotine-induced GABA and DA release were partially rescued in striatal synaptosomes from transgenic mice compared with tissue from beta2 knockout mice. Nicotine-induced locomotor activation, but not place preference, was rescued in mice with low-level VTA expression, suggesting that low-level expression of beta2* nAChRs in DA neurons is not sufficient to support nicotine reward. In contrast to control mice, transgenic mice with low-level beta2* nAChR expression in the VTA showed no increase in overall levels of cyclic AMP response element-binding protein (CREB) but did show an increase in CREB phosphorylation in response to exposure to a nicotine-paired chamber. Thus, CREB activation in the absence of regulation of total CREB levels during place preference testing was not sufficient to support nicotine place preference in beta2 trangenic mice. This suggests that partial activation of high-affinity nAChRs in VTA might block the rewarding effects of nicotine, providing a potential mechanism for the ability of nicotinic partial agonists to aid in smoking cessation.
Project description:Diverse nicotinic acetylcholine receptor (nAChR) subtypes containing different subunit combinations can be placed on nerve terminals or soma/dendrites in the ventral tegmental area (VTA). nAChR ?6 subunit message is abundant in the VTA, but ?6*-nAChR cellular localization, function, pharmacology, and roles in cholinergic modulation of dopaminergic (DA) neurons within the VTA are not well understood. Here, we report evidence for ?6?2*-nAChR expression on GABA neuronal boutons terminating on VTA DA neurons. ?-Conotoxin (?-Ctx) MII labeling coupled with immunocytochemical staining localizes putative ?6*-nAChRs to presynaptic GABAergic boutons on acutely dissociated, rat VTA DA neurons. Functionally, acetylcholine (ACh) induces increases in the frequency of bicuculline-, picrotoxin-, and 4-aminopyridine-sensitive miniature IPSCs (mIPSCs) mediated by GABA(A) receptors. These increases are abolished by ?6*-nAChR-selective ?-Ctx MII or ?-Ctx PIA (1 nm) but not by ?7 (10 nm methyllycaconitine) or ?4* (1 ?m dihydro-?-erythroidine)-nAChR-selective antagonists. ACh also fails to increase mIPSC frequency in VTA DA neurons prepared from nAChR ?2 knock-out mice. Moreover, ACh induces an ?-Ctx PIA-sensitive elevation in intraterminal Ca(2+) in synaptosomes prepared from the rat VTA. Subchronic exposure to 500 nm nicotine reduces ACh-induced GABA release onto the VTA DA neurons, as does 10 d of systemic nicotine exposure. Collectively, these results indicate that ?6?2*-nAChRs are located on presynaptic GABAergic boutons within the VTA and modulate GABA release onto DA neurons. These presynaptic ?6?2*-nAChRs likely play important roles in nicotinic modulation of DA neuronal activity.
Project description:Dopamine (DA) neurons of the ventral tegmental area (VTA) integrate cholinergic inputs to regulate key functions such as motivation and goal-directed behaviors. Yet the temporal dynamic range and mechanism of action of acetylcholine (ACh) on the modulation of VTA circuits and reward-related behaviors are not known. Here, we used a chemical-genetic approach for rapid and precise optical manipulation of nicotinic neurotransmission in VTA neurons in living mice. We provide direct evidence that the ACh tone fine-tunes the firing properties of VTA DA neurons through ?2-containing (?2*) nicotinic ACh receptors (nAChRs). Furthermore, locally photo-antagonizing these receptors in the VTA was sufficient to reversibly switch nicotine reinforcement on and off. By enabling control of nicotinic transmission in targeted brain circuits, this technology will help unravel the various physiological functions of nAChRs and may assist in the design of novel therapies relevant to neuropsychiatric disorders.
Project description:Nicotine activation of nicotinic acetylcholine receptors (nAChRs) within the dopaminergic (DAergic) neuron-rich ventral tegmental area (VTA) is necessary and sufficient for nicotine reinforcement. In this study, we show that rewarding doses of nicotine activated VTA DAergic neurons in a region-selective manner, preferentially activating neurons in the posterior VTA (pVTA) but not in the anterior VTA (aVTA) or in the tail VTA (tVTA). Nicotine (1??M) directly activated pVTA DAergic neurons in adult mouse midbrain slices, but had little effect on DAergic neurons within the aVTA. Quantification of nAChR subunit gene expression revealed that pVTA DAergic neurons expressed higher levels of ?4, ?6, and ?3 transcripts than did aVTA DAergic neurons. Activation of nAChRs containing the ?4 subunit (?4(*) nAChRs) was necessary and sufficient for activation of pVTA DAergic neurons: nicotine failed to activate pVTA DAergic neurons in ?4 knockout animals; in contrast, pVTA ?4(*) nAChRs were selectively activated by nicotine in mutant mice expressing agonist-hypersensitive ?4(*) nAChRs (Leu9'Ala mice). In addition, whole-cell currents induced by nicotine in DAergic neurons were mediated by ?4(*) nAChRs and were significantly larger in pVTA neurons than in aVTA neurons. Infusion of an ?6(*) nAChR antagonist into the VTA blocked activation of pVTA DAergic neurons in WT mice and in Leu9'Ala mice at nicotine doses, which only activate the mutant receptor indicating that ?4 and ?6 subunits coassemble to form functional receptors in these neurons. Thus, nicotine selectively activates DAergic neurons within the pVTA through ?4?6(*) nAChRs. These receptors represent novel targets for smoking-cessation therapies.
Project description:There are about twice as many women as men who experience depression during their lifetime. Although life circumstances and especially exposure to stressful situations constitute a major risk factor to develop depression, the underlying mechanisms have yet to be unraveled. We employed the chronic social defeat procedure to elicit depressive-like symptoms in females and ketamine to validate the model. We performed ex-vivo patch clamp recordings to assess cellular adaptations and used pharmacological agents to dissect these deregulations. Chronic social defeat exposure triggers a hyperactivity of VTA putative dopamine (DA) neurons in females susceptible to stress but not resilient ones. This hyperactivity was fully reversed by a single administration of ketamine. In virally-identified brain circuits of both susceptible and resilient females, we found a hypercholinergic tone to the VTA arising from the laterodorsal tegmentum. Application of puffs of nicotine revealed a decreased sensitivity of DA neurons in resilient mice when compared to naive or susceptible ones. The in vivo acute administration of the positive allosteric modulator for α7 nicotinic acetylcholine receptors (nAChRs) not only increased susceptibility to stress by enhancing activity of VTA DA neurons, but also triggered a switch in phenotype from resilient to susceptible. Our data unravel dysregulations of VTA DA neurons activity exclusively in females exhibiting depressive-like symptoms and identify VTA nAChRs as key molecular substrates that exacerbate susceptibility to stress.
Project description:Nicotine is the primary psychoactive component of tobacco. Its reinforcing and addictive properties depend on nicotinic acetylcholine receptors (nAChRs) located within the mesolimbic axis originating in the ventral tegmental area (VTA). The roles and oligomeric assembly of subunit ?4- and subunit ?6-containing nAChRs in dopaminergic (DAergic) neurons are much debated. Using subunit-specific knockout mice and targeted lentiviral re-expression, we have determined the subunit dependence of intracranial nicotine self-administration (ICSA) into the VTA and the effects of nicotine on dopamine (DA) neuron excitability in the VTA and on DA transmission in the nucleus accumbens (NAc). We show that the ?4 subunit, but not the ?6 subunit, is necessary for ICSA and nicotine-induced bursting of VTA DAergic neurons, whereas subunits ?4 and ?6 together regulate the activity dependence of DA transmission in the NAc. These data suggest that ?4-dominated enhancement of burst firing in DA neurons, relayed by DA transmission in NAc that is gated by nAChRs containing ?4 and ?6 subunits, underlies nicotine self-administration and its long-term maintenance.
Project description:Alpha6-containing nicotinic acetylcholine receptors are primarily found in neurons of the midbrain dopaminergic (DA) system, suggesting these receptors are potentially involved in drug reward and dependence. Here, we report a novel effect that cocaine directly inhibits ?6N/?3C?2?3-nAChR (?6*-nAChRs) function. Human ?6*-nAChRs were heterologously expressed within cells of the SH-EP1 cell line for functional characterization. Mechanically dissociated DA neurons from mouse ventral tegmental area (VTA) were used as a model of presynaptic ?6*-nAChR activation since this method preserves terminal boutons. Patch-clamp recordings in whole-cell configuration were used to measure ?6*-nAChR function as well as evaluate the effects of cocaine. In SH-EP1 cells containing heterologously expressed human ?6*-nAChRs, cocaine inhibits nicotine-induced inward currents in a concentration-dependent manner with an IC50 value of 30 ?M. Interestingly, in the presence of 30 ?M cocaine, the maximal current response of the nicotine concentration-response curve is reduced without changing nicotine's EC50 value, suggesting a noncompetitive mechanism. Furthermore, analysis of whole-cell current kinetics demonstrated that cocaine slows nAChR channel activation but accelerates whole-cell current decay time. Our findings demonstrate that cocaine-induced inhibition occurs solely with bath application, but not during intracellular administration, and this inhibition is not use-dependent. Additionally, in Xenopus oocytes, cocaine inhibits both ?6N/?3C?2?3-nAChRs and ?6M211L/?3IC?2?3-nCAhRs similarly, suggesting that cocaine may not act on the ?3 transmembrane domain of chimeric ?6N/?3C?2?3-nAChR. In mechanically isolated VTA DA neurons, cocaine abolishes ?6*-nAChR-mediated enhancement of spontaneous inhibitory postsynaptic currents (sIPSCs). Collectively, these studies provide the first evidence that cocaine directly inhibits the function of both heterologously and naturally expressed ?6*-nAChRs. These findings suggest that ?6*-nAChRs may provide a novel pharmacological target mediating the effects of cocaine and may underlie a novel mechanism of cocaine reward and dependence.
Project description:Cholinergic neurons in the medial habenula (MHb) modulate anxiety during nicotine withdrawal although the molecular neuroadaptation(s) within the MHb that induce affective behaviors during nicotine cessation is largely unknown. MHb cholinergic neurons are unique in that they robustly express neuronal nicotinic acetylcholine receptors (nAChRs), although their behavioral role as autoreceptors in these neurons has not been described. To test the hypothesis that nAChR signaling in MHb cholinergic neurons could modulate anxiety, we expressed novel "gain of function" nAChR subunits selectively in MHb cholinergic neurons of adult mice. Mice expressing these mutant nAChRs exhibited increased anxiety-like behavior that was alleviated by blockade with a nAChR antagonist. To test the hypothesis that anxiety induced by nicotine withdrawal may be mediated by increased MHb nicotinic receptor signaling, we infused nAChR subtype selective antagonists into the MHb of nicotine naïve and withdrawn mice. While antagonists had little effect on nicotine naïve mice, blocking ?4?2 or ?6?2, but not ?3?4 nAChRs in the MHb alleviated anxiety in mice undergoing nicotine withdrawal. Consistent with behavioral results, there was increased functional expression of nAChRs containing the ?6 subunit in MHb neurons that also expressed the ?4 subunit. Together, these data indicate that MHb cholinergic neurons regulate nicotine withdrawal-induced anxiety via increased signaling through nicotinic receptors containing the ?6 subunit and point toward nAChRs in MHb cholinergic neurons as molecular targets for smoking cessation therapeutics.
Project description:Midbrain dopaminergic (DA) neurons are governed by an endogenous cholinergic system, originated in the mesopontine nuclei. Nicotine hijacks nicotinic acetylcholine receptors (nAChRs) and interferes with physiological function of the cholinergic system. In this review, we describe the anatomical organization of the cholinergic system and the key nAChR subtypes mediating cholinergic regulation of DA transmission and nicotine reward and dependence, in an effort to identify potential targets for smoking intervention. Cholinergic modulation of midbrain DA systems relies on topographic organization of mesopontine cholinergic projections, and activation of nAChRs in midbrain DA neurons. Previous studies have revealed that α4, α6, and β2 subunit-containing nAChRs expressed in midbrain DA neurons and their terminals in the striatum regulate firings of midbrain DA neurons and activity-dependent dopamine release in the striatum. These nAChRs undergo modification upon chronic nicotine exposure. Clinical investigation has demonstrated that partial agonists of these receptors elevate the success rate of smoking cessation relative to placebo. However, further investigations are required to refine the drug targets to mitigate unpleasant side-effects.