Localized low-level re-expression of high-affinity mesolimbic nicotinic acetylcholine receptors restores nicotine-induced locomotion but not place conditioning

High-affinity, β2-subunit-containing (β2*) 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 β2* nAChRs in the midbrain/VTA. Nicotine-induced GABA and DA release were partially rescued in striatal synaptosomes from transgenic mice compared with tissue from β2 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 β2* nAChRs in DA neurons is not sufficient to support nicotine reward. In contrast to control mice, transgenic mice with low-level β2* 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 β2 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.     

Activation of VTA GABA neurons disrupts reward consumption

The activity of ventral tegmental area (VTA) dopamine (DA) neurons promotes behavioral responses to rewards and environmental stimuli that predict them. VTA GABA inputs synapse directly onto DA neurons and may regulate DA neuronal activity to alter reward-related behaviors; however, the functional consequences of selective activation of VTA GABA neurons remains unknown. Here, we show that in?vivo optogenetic activation of VTA GABA neurons disrupts reward consummatory behavior but not conditioned anticipatory behavior in response to reward-predictive cues. In addition, direct activation of VTA GABA projections to the nucleus accumbens (NAc) resulted in detectable GABA release but did not alter reward consumption. Furthermore, optogenetic stimulation of VTA GABA neurons directly suppressed the activity and excitability of neighboring DA neurons as well as the release of DA in the NAc, suggesting that the dynamic interplay between VTA DA and GABA neurons can control the initiation and termination of reward-related behaviors.     

Nicotinic modulation of synaptic transmission and plasticity in cortico-limbic circuits

Nicotine is the principle addictive agent delivered via cigarette smoking. The addictive activity of nicotine is due to potent interactions with nicotinic acetylcholine receptors (nAChRs) on neurons in the reinforcement and reward circuits of the brain. Beyond its addictive actions, nicotine is thought to have positive effects on performance in working memory and short-term attention-related tasks. The brain areas involved in such behaviors are part of an extensive cortico-limbic network that includes relays between prefrontal cortex (PFC) and cingulate cortex (CC), hippocampus, amygdala, ventral tegmental area (VTA) and the nucleus accumbens (nAcc). Nicotine activates a broad array of nAChRs subtypes that can be targeted to pre- as well as peri- and post-synaptic locations in these areas. Thereby, nicotine not only excites different types of neurons, but it also perturbs baseline neuronal communication, alters synaptic properties and modulates synaptic plasticity.In this review we focus on recent findings on nicotinic modulation of cortical circuits and their targets fields, which show that acute and transient activation of nicotinic receptors in cortico-limbic circuits triggers a series of events that affects cognitive performance in a long lasting manner. Understanding how nicotine induces long-term changes in synapses and alters plasticity in the cortico-limbic circuits is essential to determining how these areas interact in decoding fundamental aspects of cognition and reward.     

A long CAG repeat in the mouse Sca1 locus replicates SCA1 features and reveals the impact of protein solubility on selective neurodegeneration

A single nicotine exposure increases dopamine levels in the mesolimbic reward system for hours, but nicotine concentrations experienced by smokers desensitize nAChRs on dopamine neurons in seconds to minutes. Here, we show that persistent modulation of both GABAergic and glutamatergic synaptic transmission by nicotine can contribute to the sustained increase in dopamine neuron excitability. Nicotine enhances GABAergic transmission transiently, which is followed by a persistent depression of these inhibitory inputs due to nAChR desensitization. Simultaneously, nicotine enhances glutamatergic transmission through nAChRs that desensitize less than those on GABA neurons. The net effect is a shift toward excitation of the dopamine reward system. These results suggest that spatial and temporal differences in nicotinic receptor activity on both excitatory and inhibitory neurons in reward areas coordinate to reinforce nicotine self-administration.     

Nicotinic acetylcholine receptors containing α6 subunits contribute to alcohol reward‐related behaviours

Evidence is emerging that neuronal nicotinic acetylcholine receptors (nAChRs) in the mesolimbic dopamine (DA) system are involved in mediating the reinforcing effects of alcohol. Midbrain DA neurons express high levels of α6 subunit‐containing nAChRs that modulate DA transmission, implicating their involvement in reward‐related behaviours. This study assessed the role of α6‐containing nAChRs in modulating alcohol reward using transgenic mice expressing mutant, hypersensitive α6 nAChR subunits (α6L9′S mice). α6L9′S mice and littermate controls were tested in three well‐established models of alcohol reward: 24‐h two‐bottle choice drinking, drinking in the dark (DID), and conditioned place preference (CPP). Confocal microscopy and patch‐clamp electrophysiology were used to show the localization and function of hypersensitive α6 subunit‐containing nAChRs. Results indicate that female α6L9′S mice showed significantly higher alcohol intake at low concentrations of alcohol (3% and 6%) in the two‐bottle choice procedure. Both male and female α6L9′S mice drank significantly more in the DID procedure and displayed an alcohol‐induced place preference using a low dose of alcohol (0.5 g/kg) that was ineffective in littermate controls. Confocal microscopy showed that α6 subunit‐containing nAChRs are selectively expressed on ventral tegmental area (VTA) DAergic, but not GABAergic neurons. Patch‐clamp electrophysiology showed that VTA DA neurons of α6L9′S mice are hypersensitive to ACh. Collectively, these results suggest that α6L9′S mice are more sensitive to the rewarding effects of alcohol, and suggest that VTA α6 subunit‐containing nAChRs modulate alcohol reward. Thus, α6 subunit‐containing nAChRs may be a promising therapeutic target for treatment of alcohol use disorders .     

Endogenous Cholinergic Inputs and Local Circuit Mechanisms Govern the Phasic Mesolimbic Dopamine Response to Nicotine

Nicotine exerts its reinforcing action by stimulating nicotinic acetylcholine receptors (nAChRs) and boosting dopamine (DA) output from the ventral tegmental area (VTA). Recent data have led to a debate about the principal pathway of nicotine action: direct stimulation of the DAergic cells through nAChR activation, or disinhibition mediated through desensitization of nAChRs on GABAergic interneurons. We use a computational model of the VTA circuitry and nAChR function to shed light on this issue. Our model illustrates that the α4β2-containing nAChRs either on DA or GABA cells can mediate the acute effects of nicotine. We account for in vitro as well as in vivo data, and predict the conditions necessary for either direct stimulation or disinhibition to be at the origin of DA activity increases. We propose key experiments to disentangle the contribution of both mechanisms. We show that the rate of endogenous acetylcholine input crucially determines the evoked DA response for both mechanisms. Together our results delineate the mechanisms by which the VTA mediates the acute rewarding properties of nicotine and suggest an acetylcholine dependence hypothesis for nicotine reinforcement.     

Drug-driven AMPA receptor redistribution mimicked by selective dopamine neuron stimulation

Background

Addictive drugs have in common that they cause surges in dopamine (DA) concentration in the mesolimbic reward system and elicit synaptic plasticity in DA neurons of the ventral tegmental area (VTA). Cocaine for example drives insertion of GluA2-lacking AMPA receptors (AMPARs) at glutamatergic synapes in DA neurons. However it remains elusive which molecular target of cocaine drives such AMPAR redistribution and whether other addictive drugs (morphine and nicotine) cause similar changes through their effects on the mesolimbic DA system.

Methodology / Principal Findings

We used in vitro electrophysiological techniques in wild-type and transgenic mice to observe the modulation of excitatory inputs onto DA neurons by addictive drugs. To observe AMPAR redistribution, post-embedding immunohistochemistry for GluA2 AMPAR subunit was combined with electron microscopy. We also used a double-floxed AAV virus expressing channelrhodopsin together with a DAT Cre mouse line to selectively express ChR2 in VTA DA neurons. We find that in mice where the effect of cocaine on the dopamine transporter (DAT) is specifically blocked, AMPAR redistribution was absent following administration of the drug. Furthermore, addictive drugs known to increase dopamine levels cause a similar AMPAR redistribution. Finally, activating DA VTA neurons optogenetically is sufficient to drive insertion of GluA2-lacking AMPARs, mimicking the changes observed after a single injection of morphine, nicotine or cocaine.

Conclusions / Significance

We propose the mesolimbic dopamine system as a point of convergence at which addictive drugs can alter neural circuits. We also show that direct activation of DA neurons is sufficient to drive AMPAR redistribution, which may be a mechanism associated with early steps of non-substance related addictions.     

Role of excitatory amino acids in the ventral tegmental area for central actions of non-competitive NMDA-receptor antagonists and nicotine

Summary The putative role of non-NMDA excitatory amino acid (EAA) receptors in the ventral tegmental area (VTA) for the increase in dopamine (DA) release in the nucleus acumbens (NAC) and the behavioural stimulation induced by systemically administered dizocilpine (MK-801) was investigated. Microdialysis was utilized in rats with probes in the VTA and NAC. The VTA was perfused with the AMPA and kainate receptor antagonist CNQX (0.3 or 1.0 mM) or vehicle and dialysates from the NAC were analyzed with high-performance liquid chromatography for DA. Forty min after onset of CNQX or vehicle perfusion of the VTA MK-801 (0.1 mg/kg) was injected subcutaneously (sc). Subsequently, typical MK-801 induced behaviours were assessed. The MK-801 induced hyperlocomotion was associated with a 50% increase of DA levels in NAC dialysates. Both the MK-801 evoked hyperlocomotion and DA release in the NAC were effectively antagonized by CNQX perfusion of the VTA. However, by itself the CNQX or vehicle perusion of the VTA did not affect DA levels in NAC or the rated behaviours. The results indicate that MK-801 induced hyperlocomotion and increased DA release in the NAC are largely elicited within the VTA via activation of non-NMDA EAA receptors, tentatively caused by locally increased EAA release. In contrast, the enhanced DA output in the NAC induced by systemic nicotine (0.5 mg/kg sc) was not antagonized by intra VTA infusion of CNQX (0.3 or 1.0 mM), but instead by infusion of the NMDA receptor antagonist AP-5 (0.3 or 1.0 mM) into the VTA, which by itself did not alter DA levels in the NAC. Thus, the probably indirect, EAA mediated activation of the mesolimbic DA neurons in the VTA by MK-801 and nicotine, respectively, seems to be mediated via different glutamate receptor subtypes.     

Self-Administration of Ethanol,Cocaine, or Nicotine Does Not Decrease the Soma Size of Ventral Tegmental Area Dopamine Neurons

Our previous observations show that chronic opiate administration, including self-administration, decrease the soma size of dopamine (DA) neurons in the ventral tegmental area (VTA) of rodents and humans, a morphological change correlated with increased firing rate and reward tolerance. Given that a general hallmark of drugs of abuse is to increase activity of the mesolimbic DA circuit, we sought to determine whether additional drug classes produced a similar morphological change. Sections containing VTA were obtained from rats that self-administered cocaine or ethanol and from mice that consumed nicotine. In contrast to opiates, we found no change in VTA DA soma size induced by any of these other drugs. These data suggest that VTA morphological changes are induced in a drug-specific manner and reinforce recent findings that some changes in mesolimbic signaling and neuroplasticity are drug-class dependent.     

Conditional Knockout of NMDA Receptors in Dopamine Neurons Prevents Nicotine-Conditioned Place Preference

Nicotine from smoking tobacco produces one of the most common forms of addictive behavior and has major societal and health consequences. It is known that nicotine triggers tobacco addiction by activating nicotine acetylcholine receptors (nAChRs) in the midbrain dopaminergic reward system, primarily via the ventral tegmental area. Heterogeneity of cell populations in the region has made it difficult for pharmacology-based analyses to precisely assess the functional significance of glutamatergic inputs to dopamine neurons in nicotine addiction. By generating dopamine neuron-specific NR1 knockout mice using cre/loxP-mediated method, we demonstrate that genetic inactivation of the NMDA receptors in ventral tegmental area dopamine neurons selectively prevents nicotine-conditioned place preference. Interestingly, the mutant mice exhibit normal performances in the conditioned place aversion induced by aversive air puffs. Therefore, this selective effect on addictive drug-induced reinforcement behavior suggests that NMDA receptors in the dopamine neurons are critical for the development of nicotine addiction.     

In vivo activation of midbrain dopamine neurons via sensitized, high-affinity alpha 6 nicotinic acetylcholine receptors

Alpha6-containing (alpha6*) nicotinic ACh receptors (nAChRs) are selectively expressed in dopamine (DA) neurons and participate in cholinergic transmission. We generated and studied mice with gain-of-function alpha6* nAChRs, which isolate and amplify cholinergic control of DA transmission. In contrast to gene knockouts or pharmacological blockers, which show necessity, we show that activating alpha6* nAChRs and DA neurons is sufficient to cause locomotor hyperactivity. alpha6(L9'S) mice are hyperactive in their home cage and fail to habituate to a novel environment. Selective activation of alpha6* nAChRs with low doses of nicotine, by stimulating DA but not GABA neurons, exaggerates these phenotypes and produces a hyperdopaminergic state in vivo. Experiments with additional nicotinic drugs show that altering agonist efficacy at alpha6* provides fine tuning of DA release and locomotor responses. alpha6*-specific agonists or antagonists may, by targeting endogenous cholinergic mechanisms in midbrain or striatum, provide a method for manipulating DA transmission in neural disorders.     

Hierarchical control of dopamine neuron-firing patterns by nicotinic receptors

Nicotine elicits dopamine release by stimulating nicotinic acetylcholine receptors (nAChRs) on dopaminergic neurons. However, a modulation of these neurons by endogenous acetylcholine has not been described. We recorded, in vivo, the spontaneous activity of dopaminergic neurons in the VTA of anaesthetized wt and nAChR knockout mice and their response to nicotine injections. Deleting alpha7 or beta2 subunits modified the spontaneous firing patterns, demonstrating their direct stimulation by endogenous acetylcholine. Quantitative analysis further revealed four principal modes of firing, each depending on the expression of particular nAChR subunits and presenting unique responses to nicotine. The prominent role of the beta2 subunit was further confirmed by its selective lentiviral reexpression in the VTA. These data suggest a hierarchical control of dopaminergic neuron firing patterns by nAChRs: activation of beta2*-nAChR switches cells from a resting to an excited state, whereas activation of alpha7*-nAChRs finely tunes the latter state but only once beta2*-nAChRs have been activated.     

Nicotinic activation of mesolimbic neurons assessed by rubidium efflux in rat accumbens and ventral tegmentum

Dopaminergic mesolimbic neurons, with cell bodies in the ventral tegmental area (VTA) projecting to the nucleus accumbens (NAc), have been shown to be involved in the development of drug dependence. The application of nicotine to either the VTA or NAc produces an increase in dopamine release; however, the positive reinforcement produced by the systemic injection of nicotine is primarily due to stimulation of nicotinic acetylcholine receptors (nAChRs) in the VTA. Because the brain levels of nicotine would likely be the same in both brain areas, the nAChRs in the NAc may be less sensitive than those in the VTA. This study was undertaken to make a direct comparison of the native nAChRs in intact slices of NAc and VTA by measuring nicotine-stimulated efflux of (86)Rb(+) in a superfusion assay. The potency of nicotine and several other agonists was similar in both brain areas, but nicotine was somewhat more efficacious in the NAc. The effects of treatment duration, calcium and nicotinic antagonists were also determined. The results suggest that the predominant effect of nicotine in the VTA following systemic administration is due to differences in neuronal circuitry or firing patterns rather than inherent differences in the two nAChR populations.     

Distributed network actions by nicotine increase the threshold for spike-timing-dependent plasticity in prefrontal cortex

Nicotine enhances attention and working memory by activating nicotinic acetylcholine receptors (nAChRs). The prefrontal cortex (PFC) is critical for these cognitive functions and is also rich in nAChR expression. Specific cellular and synaptic mechanisms underlying nicotine's effects on cognition remain elusive. Here we show that nicotine exposure increases the threshold for synaptic spike-timing-dependent potentiation (STDP) in layer V pyramidal neurons of the mouse PFC. During coincident presynaptic and postsynaptic activity, nicotine reduces dendritic calcium signals associated with action potential propagation by enhancing GABAergic transmission. This results from a series of presynaptic actions involving different PFC interneurons and multiple nAChR subtypes. Pharmacological block of nAChRs or GABA(A) receptors prevented nicotine's actions and restored STDP, as did increasing dendritic calcium signals with stronger postsynaptic activity. Thus, by activating nAChRs distributed throughout the PFC neuronal network, nicotine affects PFC information processing and storage by increasing the amount of postsynaptic activity necessary to induce STDP.     

Cellular and synaptic mechanisms of nicotine addiction

The tragic health effects of nicotine addiction highlight the importance of investigating the cellular mechanisms of this complex behavioral phenomenon. The chain of cause and effect of nicotine addiction starts with the interaction of this tobacco alkaloid with nicotinic acetylcholine receptors (nAChRs). This interaction leads to activation of reward centers in the CNS, including the mesoaccumbens DA system, which ultimately leads to behavioral reinforcement and addiction. Recent findings from a number of laboratories have provided new insights into the biologic processes that contribute to nicotine self-administration. Examination of the nAChR subtypes expressed within the reward centers has identified potential roles for these receptors in normal physiology, as well as the effects of nicotine exposure. The high nicotine sensitivity of some nAChR subtypes leads to rapid activation followed in many cases by rapid desensitization. Assessing the relative importance of these molecular phenomena in the behavioral effects of nicotine presents an exciting challenge for future research efforts.     

Conjunctive processing of locomotor signals by the ventral tegmental area neuronal population

The ventral tegmental area (VTA) plays an essential role in reward and motivation. How the dopamine (DA) and non-DA neurons in the VTA engage in motivation-based locomotor behaviors is not well understood. We recorded activity of putative DA and non-DA neurons simultaneously in the VTA of awake mice engaged in motivated voluntary movements such as wheel running. Our results revealed that VTA non-DA neurons exhibited significant rhythmic activity that was correlated with the animal's running rhythms. Activity of putative DA neurons also correlated with the movement behavior, but to a lesser degree. More importantly, putative DA neurons exhibited significant burst activation at both onset and offset of voluntary movements. These findings suggest that VTA DA and non-DA neurons conjunctively process locomotor-related motivational signals that are associated with movement initiation, maintenance and termination.     

Pre-synaptic nicotinic and D2 receptors functionally interact on dopaminergic nerve endings of rat and mouse nucleus accumbens

The existence of pre-synaptic auto- and hetero receptors which modulate neurotransmitter release is well documented. Emerging evidence show that in some cases these pre-synaptic receptors may also cross-talk with each other. The aim of the present work was to investigate whether acetylcholine receptors (nAChRs) and dopamine (DA) autoreceptors, which are both able to modulate DA release, functionally interact on the same nerve endings. We used rat and mouse nucleus accumbens synaptosomes pre-labeled with [³H]DA and exposed to nicotinic and dopaminergic receptor ligands. Both nicotinic agonists and 4-aminopyridine (4-AP) provoked [³H]DA release which was inhibited by quinpirole and blocked by sulpiride and raclopride. Both the inhibitory effect of quinpirole and the stimulatory effect of (−)nicotine did not change when the nAChRs or the DA receptors were desensitized. (−)Nicotine and 4-AP were able to stimulate [³H]DA overflow also in mouse synaptosomes and this overflow was partially inhibited by quinpirole. In the β₂ knockout mice quinpirole was still able to inhibit the [³H]DA overflow elicited by 4-AP. To conclude: in rat and mouse the (−)nicotine evoked-release can be modulated by D₂/D₃ autoreceptors present on the DA terminals and nAChRs function is independent from D₂/D₃ autoreceptors which themselves may function independently from the activation of nAChRs.     

Modulation of dopamine release by the nicotinic agonist epibatidine in the frontal cortex and the nucleus accumbens of naive and chronic nicotine treated rats

The effect of the nicotinic acetylcholine receptors (nAChRs) agonist (+/-)epibatidine on the modulation of dopamine (DA) release was investigated by microdialysis in vivo in the frontal cortex and the nucleus accumbens of naive and chronic nicotine-treated awake rats. (+/-)Epibatidine (2.5 microg/kg, s.c.), contrary to (-)nicotine (0.5 mg/kg, s.c.), decreased the extracellular concentrations of DA in the brain of naive rats. Subchronic nicotine treatment (0.45 mg/kg, s.c., twice daily for 7 days) attenuated the (+/-)epibatidine induced decrease in the DA level. The extracellular concentrations of the DA metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were elevated by (+/-)epibatidine administration in both na?ve and subchronic treated rats. The findings suggest that the decrease in DA extracellular concentrations induced by the high affinity nAChRs agonist (+/-)epibatidine might be due to inactivation of nAChRs, which can be overcome by subchronic treatment with nicotine. Different mechanisms in modulation of DA release appears to be involved in the rat brain by (+/-)epibatidine compare to (-)nicotine.