Neuroplasticity in cholinergic neurons of the laterodorsal tegmental nucleus contributes to the development of cocaine addiction

The laterodorsal tegmental nucleus (LDT) is a brainstem nucleus that sends cholinergic, glutamatergic, and gamma‐aminobutyric acid (GABA)‐ergic projections to the ventral tegmental area (VTA), a key brain region associated with reward information processing and reinforcement learning, and thus, with addiction induced by drugs of abuse, including cocaine. Recent studies have revealed that the LDT, in addition to the VTA, plays important roles in the development and expression of cocaine‐induced addiction and stress‐induced enhancement of addictive behaviors. Additionally, neuroplasticity induced in LDT cholinergic neurons by repeated cocaine administration critically contributes to these behaviors. Elucidation of the underlying mechanisms of cocaine‐induced neuroplasticity in the LDT that influences reward circuit activity may lead to the development of therapeutic strategies to treat cocaine addiction and stress‐induced reinstatement of cocaine use. This review summarizes recent progress in the study of the LDT, specifically neuroplasticity in LDT cholinergic neurons induced by cocaine and its functional roles in the development and modulation of addictive behaviors associated with cocaine.     

Cocaine exposure enhances excitatory synaptic drive to cholinergic neurons in the laterodorsal tegmental nucleus

Accumulating evidence indicates that the laterodorsal tegmental nucleus (LDT) is associated with reward processing and addiction. The cholinergic projection from the LDT to the ventral tegmental area is essential for a large dopamine release in the nucleus accumbens, which is critically involved in the reinforcing effects of addictive drugs, including cocaine. In contrast to the large number of studies on plasticity induced after cocaine exposure in the mesocorticolimbic dopaminergic system, it remains unknown whether LDT cholinergic neurons exhibit plastic changes following cocaine administration. To address this issue, we performed ex vivo whole‐cell recordings in LDT cholinergic neurons obtained from rats following cocaine administration. Neurons obtained from 1 day after 5‐day cocaine‐treated rats showed significantly smaller paired‐pulse ratios of evoked EPSCs and higher miniature EPSC frequencies than those from saline‐treated rats, indicating an induction of presynaptic plasticity of increased glutamate release. This plasticity seemed to recover after a 5‐day withdrawal from repeated cocaine exposure, and required NMDA receptor stimulation and nitric oxide production. Additionally, pharmacological suppression of activity of the medial prefrontal cortex inhibited the presynaptic plasticity in the LDT. On the other hand, AMPA/NMDA ratios were not different between saline‐ and cocaine‐treated groups, revealing an absence of postsynaptic plasticity. These findings provide the first direct evidence of cocaine‐induced synaptic plasticity in LDT cholinergic neurons and suggest that the presynaptic plasticity enhances the activity of LDT cholinergic neurons, contributing to the expression of cocaine‐induced addictive behaviors through the dysregulation of the mesocorticolimbic system.     

Chronic cocaine exposure induces noradrenergic modulation of inhibitory synaptic transmission to cholinergic neurons of the laterodorsal tegmental nucleus

The laterodorsal tegmental nucleus (LDT), which sends cholinergic efferent connections to dopaminergic (DA) neurons in the ventral tegmental area (VTA), plays a critical role in the development of addictive behavior and the reinstatement of cocaine‐seeking behavior. Although repeated cocaine exposure elicits plastic changes in excitatory synaptic transmission and intrinsic membrane excitability in LDT cholinergic neurons, it remains unclear whether inhibitory synaptic transmission is modulated by cocaine exposure. The LDT receives fibers containing noradrenaline (NA), a neurotransmitter whose extracellular levels increase with cocaine exposure. Therefore, it is hypothesized that repeated cocaine exposure induces plastic changes in LDT cholinergic neurons via NA. Ex vivo electrophysiological recordings in LDT cholinergic neurons were obtained from rats repeatedly exposed to cocaine. Bath‐application of NA induced similar levels of hyperpolarization in both saline‐ and cocaine‐treated neurons. However, NA attenuated the amplitude of inhibitory postsynaptic currents (IPSCs) in cocaine‐ but not saline‐treated neurons through α2 adrenoceptors. This NA‐induced IPSC attenuation was observed in the presence of strychnine, but not gabazine, indicating that NA modulated GABAergic but not glycinergic neurotransmission. NA increased the paired‐pulse ratios of evoked IPSCs and decreased the frequencies of miniature IPSCs (mIPSCs) without affecting their amplitudes, suggesting a presynaptic mechanism. These findings suggest that repeated cocaine exposure induces neuroplasticity in GABAergic synaptic transmission onto LDT cholinergic neurons by probably modulating presynaptic α2 adrenoceptors. This potentially increases the activity of LDT cholinergic neurons, which might contribute to the development of addictive behavior by enhancing VTA DA neuronal activity.     

Regulation of the ventral tegmental area by the bed nucleus of the stria terminalis is required for expression of cocaine preference

Lateral hypothalamus (LH) orexin neurons are essential for the expression of a cocaine place preference. However, the afferents that regulate the activity of these orexin neurons during reward behaviors are not completely understood. Using tract tracing combined with Fos staining, we examined LH afferents for Fos induction during cocaine preference in rats. We found that the ventral bed nucleus of the stria terminalis (vBNST) was a major input to the LH orexin cell field that was significantly Fos‐activated during cocaine conditioned place preference (CPP). Inactivation of the vBNST with baclofen plus muscimol blocked expression of cocaine CPP. Surprisingly, such inactivation of the vBNST also increased Fos induction in LH orexin neurons; as activity in these cells is normally associated with increased preference, this result indicates that a vBNST–orexin connection is unlikely to be responsible for CPP that is dependent on vBNST activity. Because previous studies have revealed that vBNST regulates dopamine cells in the ventral tegmental area (VTA), which is known to be involved in CPP and other reward functions, we tested whether vBNST afferents to the VTA are necessary for cocaine CPP. We found that disconnection of the vBNST and VTA (using local microinjections of baclofen plus muscimol unilaterally into the vBNST and contralateral VTA) significantly attenuated expression of cocaine preference. However, blocking ionotropic glutamatergic afferents to the VTA from the vBNST did not significantly reduce cocaine preference. These results indicate that a non‐glutamatergic vBNST–VTA projection is involved in expression of cocaine preference.     

Muscarinic receptor blockade in the ventral tegmental area attenuates cocaine enhancement of laterodorsal tegmentum stimulation‐evoked accumbens dopamine efflux in the mouse

The reinforcing properties of cocaine have been related to increased extracellular concentrations of dopamine in the nucleus accumbens (NAc). M5 muscarinic acetylcholine receptors (mAChRs) on dopamine cells in the ventral tegmental area (VTA) facilitate mesoaccumbens dopamine transmission and are critically involved in mediating natural and drug reinforcement. We investigated the effects of pharmacological blockade of mAChRs in the VTA on cocaine's ability to enhance electrically evoked NAc dopamine efflux. Using fixed potential amperometry together with carbon fiber recording microelectrodes positioned in the NAc core, we quantified dopamine oxidation currents (dopamine efflux) evoked by brief stimulation (15 monophasic pulses at 50 Hz every 30 s) of the laterodorsal tegmentum (LDT) in urethane (1.5 g/kg, i.p.) anesthetized mice. Compared to predrug baseline responses, cocaine (5 or 10 mg/kg, i.p.) dose‐dependently enhanced LDT stimulation‐evoked NAc dopamine efflux, whereas the nonsubtype selective mAChR antagonist scopolamine (10 μg/0.5 μl) microinfused into the VTA diminished LDT‐evoked NAc dopamine efflux. Preinfusion of scopolamine into the VTA diminished the facilitatory actions of cocaine on LDT stimulation‐evoked NAc dopamine efflux, and when infused at the peak effect of cocaine attenuated LDT‐evoked dopamine efflux to below predrug baseline levels. These findings suggest that LDT cholinergic inputs to dopamine neurons in the VTA, via activation of mAChRs (probably of the M5 subtype), are involved in modulating the facilitatory effects of cocaine on NAc dopamine neurotransmission. They also suggest that the development of antagonists aimed at selectively disrupting M5 receptor function may be valuable in reducing abuse liability of psychostimulants. Synapse 64:216–223, 2010. © 2009 Wiley‐Liss, Inc.     

The limbic circuitry underlying cocaine seeking encompasses the PPTg/LDT

The direct glutamatergic projection from the medial prefrontal cortex (mPFC) to the nucleus accumbens plays a critical role in mediating the reinstatement of cocaine seeking behavior. The mPFC also sends glutamatergic projections to the pedunculopontine tegmental nucleus (PPTg) and laterodorsal tegmental nucleus (LDT), which in turn send glutamatergic and cholinergic efferents to the ventral tegmental area (VTA) where they synapse on dopaminergic cells that innervate limbic structures including the nucleus accumbens. The goal of these experiments was to examine a potential role for the PPTg/LDT in the reinstatement of cocaine seeking. All rats were trained to self-administer cocaine (0.25 mg, i.v.) on a fixed-ratio 5 schedule of reinforcement. Cocaine self-administration behavior was extinguished and a series of subsequent pharmacological experiments were performed to assess the potential role of the mPFC, PPTg/LDT and VTA in the reinstatement of cocaine seeking. Administration of the D1-like dopamine receptor agonist SKF-81297 (1.0 μg) directly into the mPFC produced a small, but statistically significant, increase in cocaine seeking behavior. Furthermore, microinjection of the ionotropic glutamate receptor antagonist CNQX (0.3 μg) into the PPTg/LDT attenuated the reinstatement of drug seeking induced by a priming injection of cocaine (10 mg/kg, i.p.). Intra-VTA administration of CNQX, the nicotinic receptor antagonist mecamylamine (10.0 μg) or the muscarinic receptor antagonist scopolamine (24.0 μg) also blocked cocaine seeking. Taken together, these results suggest that cocaine priming-induced reinstatement of drug seeking is mediated in part by a serial polysynaptic limbic subcircuit encompassing the mPFC, PPTg/LDT and VTA.     

Acute cocaine exposure alters spine density and long-term potentiation in the ventral tegmental area

Growing evidence indicates that the expression of synaptic plasticity in the central nervous system results in dendritic reorganization and spine remodeling. Although long-term potentiation of glutamatergic synapses after cocaine exposure in the ventral tegmental area (VTA) has been proposed as a cellular mechanism underlying addictive behaviors, the relationship between long-term potentiation and dendritic remodeling induced by cocaine on the dopaminergic neurons of the VTA has not been demonstrated. Here we report that rat VTA cells classified as type I and II showed distinct morphological responses to cocaine, as a single cocaine exposure significantly increased dendritic spine density in type I but not in type II cells. Further, only type I cells had a significant increase in the AMPA receptor:NMDA receptor ratio after a single cocaine exposure. Taken together, our data provide evidence that increased spine density and synaptic plasticity are coexpressed within the same VTA neuronal population and that only type I neurons are structurally and synaptically modified by cocaine.     

Synaptic Adaptations at the Rostromedial Tegmental Nucleus Underlie Individual Differences in Cocaine Avoidance Behavior

Although cocaine is powerfully rewarding, not all individuals are equally prone to abusing this drug. We postulate that these differences arise in part because some individuals exhibit stronger aversive responses to cocaine that protect them from cocaine seeking. Indeed, using conditioned place preference (CPP) and a runway operant cocaine self-administration task, we demonstrate that avoidance responses to cocaine vary greatly between individual high cocaine-avoider and low cocaine-avoider rats. These behavioral differences correlated with cocaine-induced activation of the rostromedial tegmental nucleus (RMTg), measured using both in vivo firing and c-fos, whereas slice electrophysiological recordings from ventral tegmental area (VTA)-projecting RMTg neurons showed that relative to low avoiders, high avoiders exhibited greater intrinsic excitability, greater transmission via calcium-permeable AMPA receptors (CP-AMPARs), and higher presynaptic glutamate release. In behaving animals, blocking CP-AMPARs in the RMTg with NASPM reduced cocaine avoidance. Hence, cocaine addiction vulnerability may be linked to multiple coordinated synaptic differences in VTA-projecting RMTg neurons.SIGNIFICANCE STATEMENT Although cocaine is highly addictive, not all individuals exposed to cocaine progress to chronic use for reasons that remain unclear. We find that cocaine''s aversive effects, although less widely studied than its rewarding effects, show more individual variability, are predictive of subsequent propensity to seek cocaine, and are driven by variations in RMTg in response to cocaine that arise from distinct alterations in intrinsic excitability and glutamate transmission onto VTA-projecting RMTg neurons.     

Role of MEK‐ERK pathway in morphine‐induced conditioned place preference in ventral tegmental area of rats

A major goal of research on drug addiction is to develop the effective treatments to deal with the long‐term behavioral disorders especially reinstatement induced by the addictive drugs such as opiates, cocaine, and cannabinoid. The molecular mechanisms underlying these substance‐related disorders remain unclear so far. Here we used the model of morphine‐induced conditioned place preference (CPP) in rats to mimic the progress of drug‐taking, withdrawal and relapse in human. The tissue of ventral tegmental area (VTA), one of the most important brain structures associated with abused drug‐related disorders, was taken and two‐dimensional electrophoresis (2‐DE) was performed to analyze and compare the changes of protein expression patterns during the different stages of morphine‐induced CPP. First, we found that there were 80 proteins identified to be changed in the process of morphine‐induced CPP. Furthermore, as the mitogen‐activated protein kinase kinase 1 (MAPKK1) was increased significantly in the stages of establishment and reinstatement, we confirmed the change of activated extracellular signal‐regulated kinase (ERK) by Western blotting in VTA tissue and cultured cell. The results demonstrated that the activated MEK‐ERK pathway by chronic morphine treatment in VTA was involved in morphine‐induced reinstatement. Moreover, inhibition of MEK‐ERK pathway by infusion the MEK inhibitor U0126 in VTA blocked the establishment of morphine‐induced CPP. The present study found significant changes in a group of protein expressions in VTA during morphine‐induced CPP and further confirmed the role of MEK‐ERK cell signaling pathway of VTA in morphine addiction. © 2010 Wiley‐Liss, Inc.     

Pontine cholinergic neurons simultaneously innervate two thalamic targets

Cholinergic neurons located in the lateral dorsal tegmental (LDT) and pedunculopontine tegmental (PPT) nuclei have been shown to principally innervate the thalamus. In order to determine whether some of these neurons might simultaneously project to two thalamic targets we made microinjections of rhodamine-conjugated microbeads into the central-lateral nucleus of the thalamus and fluorescein isothiocyanate (FITC)-conjugated microbeads into the dorso-lateral geniculate nucleus. We then determined whether both tracers were found in immunohistochemically identified cholinergic somata in the LDT and PPT nuclei. Results showed that some cholinergic and non-cholinergic neurons in the LDT and PPT nuclei projected to both thalamic sites. This finding extends our understanding of the projections of the LDT-PPT cholinergic neurons and further supports the role of these neurons in complex behaviors.     

Opioid-induced rewards,locomotion, and dopamine activation: A proposed model for control by mesopontine and rostromedial tegmental neurons

Opioids, such as morphine or heroin, increase forebrain dopamine (DA) release and locomotion, and support the acquisition of conditioned place preference (CPP) or self-administration. The most sensitive sites for these opioid effects in rodents are in the ventral tegmental area (VTA) and rostromedial tegmental nucleus (RMTg). Opioid inhibition of GABA neurons in these sites is hypothesized to lead to arousing and rewarding effects through disinhibition of VTA DA neurons. We review findings that the laterodorsal tegmental (LDTg) and pedunculopontine tegmental (PPTg) nuclei, which each contain cholinergic, GABAergic, and glutamatergic cells, are important for these effects. LDTg and/or PPTg cholinergic inputs to VTA mediate opioid-induced locomotion and DA activation via VTA M5 muscarinic receptors. LDTg and/or PPTg cholinergic inputs to RMTg also modulate opioid-induced locomotion. Lesions or inhibition of LDTg or PPTg neurons reduce morphine-induced increases in forebrain DA release, acquisition of morphine CPP or self-administration. We propose a circuit model that links VTA and RMTg GABA with LDTg and PPTg neurons critical for DA-dependent opioid effects in drug-naïve rodents.     

Acetylcholine from the mesopontine tegmental nuclei differentially affects methamphetamine induced locomotor activity and neurotransmitter levels in the mesolimbic pathway

Methamphetamine (MA) increases dopamine (DA) levels within the mesolimbic pathway and acetylcholine (ACh), a neurotransmitter known to increase DA cell firing and release and mediate reinforcement, within the ventral tegmental area (VTA). The laterodorsal tegmental (LDT) and pedunculopontine tegmental (PPT) nuclei provide cholinergic input to the VTA; however, the contribution of LDT- and PPT-derived ACh to MA-induced DA and ACh levels and locomotor activation remains unknown. The first experiment examined the role of LDT-derived ACh in MA locomotor activation by reversibly inhibiting these neurons with bilateral intra-LDT microinjections of the M2 receptor agonist oxotremorine (OXO). Male C57BL/6 J mice were given a bilateral 0.1 μl OXO (0, 1, or 10 nM/side) microinjection immediately prior to IP saline or MA (2 mg/kg). The highest OXO concentration significantly inhibited both saline- and MA-primed locomotor activity. In a second set of experiments we characterized the individual contributions of ACh originating in the LDT or pedunculopontine tegmental nucleus (PPT) to MA-induced levels of ACh and DA by administering intra-LDT or PPT OXO and performing in vivo microdialysis in the VTA and NAc. Intra-LDT OXO dose-dependently attenuated the MA-induced increase in ACh within the VTA but had no effect on DA in NAc. Intra-PPT OXO had no effect on ACh or DA levels within the VTA or NAc, respectively. We conclude that LDT, but not PPT, ACh is important in locomotor behavior and the cholinergic, but not dopaminergic, response to systemic MA.     

Sound‐specific plasticity in the primary auditory cortex as induced by the cholinergic pedunculopontine tegmental nucleus

Brain cholinergic modulation is essential for learning‐induced plasticity of the auditory cortex. The pedunculopontine tegmental nucleus (PPTg) is an important cholinergic nucleus in the brainstem, and appears to be involved in learning and subcortical plasticity. This study confirms the involvement of the PPTg in the plasticity of the auditory cortex in mice. We show here that electrical stimulation of the PPTg paired with a tone induced drastic changes in the frequency tunings of auditory cortical neurons. Importantly, the changes in frequency tuning were highly specific to the frequency of the paired tone; the best frequency of auditory cortical neurons shifted towards the frequency of the paired tone. We further demonstrated that such frequency‐specific plasticity was largely eliminated by either thalamic or cortical application of the muscarinic acetylcholine receptor antagonist atropine. Our finding suggests that the PPTg significantly contributes to auditory cortical plasticity via the auditory thalamus and cholinergic basal forebrain.     

Activity of cholinergic neurons in the laterodorsal tegmental nucleus during emission of 22kHz vocalization in rats

It has been postulated that the ascending cholinergic tegmental system is responsible for the initiation of the aversive emotional state with a concomitant alarm vocalization in the rat. It is assumed that the activity of cholinergic neurons of the laterodorsal tegmental nucleus (LDT) will cause release of acetylcholine in the target areas and will initiate the emission of 22 kHz vocalizations. The goal of the present study was to test the hypothesis that the cholinergic neurons of the LDT increase their activity during emission of 22 kHz alarm calls. Vocalizations were induced by an air puff or by intrahypothalamic-preoptic injection of carbachol. The activity of the LDT cholinergic neurons was studied by a double histochemical labelling for choline acetyltransferase, as a marker of cholinergic somata, and for c-Fos protein, as a marker of cells with heighten metabolic activity. Both air puff stimulation and intracerebral carbachol induced comparable 22 kHz alarm vocalizations. The activity of neurons in the LDT was significantly higher during prolonged emission of 22 kHz alarm calls induced by air puff or injection of carbachol than in the non-vocalizing or low-vocalizing controls. There were approximately two times more of all c-Fos-labelled cells in the LDT of vocalizing animals and 2.5 times more active cholinergic neurons during prolonged 22 kHz vocalization than in the control conditions without vocalization. However, the active cholinergic neurons constituted only a small proportion of all active LDT cells (2.3%). At the same time, there were no significant increases in the number of c-Fos-labelled cells in the neighbouring pedunculopontine nucleus (PPT). These findings lead to the conclusion that the neurons of the LDT, including cholinergic neurons, but not those of the PPT, significantly increased their activity during prolonged emission of alarm vocalizations, as evidenced by the c-Fos immunoreactivity.     

Cholinergic control of morphine‐induced locomotion in rostromedial tegmental nucleus versus ventral tegmental area sites

M5 muscarinic acetylcholine receptors expressed on ventral tegmental dopamine (DA) neurons are needed for opioid activation of DA outputs. Here, the M5 receptor gene was bilaterally transfected into neurons in the ventral tegmental area (VTA) or the adjacent rostromedial tegmental nucleus (RMTg) in mice by means of a Herpes simplex viral vector (HSV) to increase the effect of endogenous acetylcholine. Three days after HSV‐M5 gene infusion in VTA sites, morphine‐induced locomotion more than doubled at two doses, while saline‐induced locomotion was unaffected. When the HSV‐M5 gene was infused into the adjacent RMTg, morphine‐induced locomotion was strongly inhibited. The sharp boundary between these opposing effects was found where tyrosine hydroxylase (TH) and cholinesterase labelling decreases (?4.00 mm posterior to bregma). The same HSV‐M5 gene transfections in M5 knockout mice induced even stronger inhibitory behavioural effects in RMTg but more variability in VTA sites due to stereotypy. The VTA sites where HSV‐M5 increased morphine‐induced locomotion receive direct inputs from many RMTg GAD‐positive neurons, and from pontine ChAT‐positive neurons, as shown by cholera‐toxin B retrograde tracing. Therefore, morphine‐induced locomotion was decreased by M5 receptor gene expression in RMTg GABA neurons that directly inhibit VTA DA neurons. Conversely, enhancing M5 receptor gene expression on VTA DA neurons increased morphine‐induced locomotion via cholinergic inputs.     

Insulin in the ventral tegmental area reduces cocaine‐evoked dopamine in the nucleus accumbens in vivo

Mesolimbic dopamine circuits, implicated in incentive motivation, are sensitive to changes in metabolic state such as weight loss and diet‐induced obesity. These neurons are important targets for metabolic hormones such as leptin, glucagon‐like peptide‐1, ghrelin and insulin. Insulin receptors are located on dopamine neurons in the ventral tegmental area (VTA) and we have previously demonstrated that insulin induces long‐term depression of excitatory synapses onto VTA dopamine neurons. While insulin can decrease dopamine concentration in somatodendritic regions, it can increase dopamine in striatal slices. Whether insulin directly targets the VTA to alter dopamine release in projection areas, such as the nucleus accumbens (NAc), remains unknown. The main goal of the present experiments was to examine NAc dopamine concentration following VTA administration of insulin. Using in vivo FSCV to detect rapid fluctuations in dopamine concentration, we showed that intra‐VTA insulin via action at insulin receptors reduced pedunculopontine nucleus‐evoked dopamine release in the NAc. Furthermore, intra‐VTA insulin reduced cocaine‐potentiated NAc dopamine. Finally, intra‐VTA or intranasal insulin decreased locomotor responses to cocaine, an effect blocked by an intra‐VTA administered insulin receptor antagonist. Together, these data demonstrate that mesolimbic dopaminergic projections are important targets of the metabolic hormone, insulin.     

Corticotropin‐releasing factor type‐2 receptor and corticotropin‐releasing factor‐binding protein coexist in rat ventral tegmental area nerve terminals originated in the lateral hypothalamic area

There is significant functional evidence showing that corticotropin‐releasing factor type‐2 receptor (CRF₂R) and corticotropin‐releasing factor‐binding protein (CRF‐BP) regulate glutamatergic synapses onto ventral tegmental area (VTA) dopaminergic neurons. It has been shown that CRF requires CRF‐BP to potentiate N‐methyl‐d ‐aspartate receptors in dopaminergic neurons through CRF₂R, and that increases glutamate release in cocaine‐treated rats through the activation of CRF₂R only by agonists with high affinity to CRF‐BP. Furthermore, this CRF‐mediated increase in VTA glutamate is responsible for stress‐induced relapse to cocaine‐seeking behaviour. However, there is a lack of anatomical evidence to explain the mechanisms of CRF actions in VTA. Thus, it was studied whether CRF₂R and CRF‐BP are expressed in VTA nerve terminals, using a synaptosomal preparation devoid of postsynaptic elements. The current results show that both proteins are co‐expressed in glutamatergic and γ‐aminobutyric acid (GABA)ergic VTA synaptosomes. A main glutamatergic input to the VTA that has been associated to addictive behaviour is originated in the lateral hypothalamic area (LHA). Thus, this study was focused in the LHA–VTA input using orexin as a marker of this input. The results show that CRF₂R and CRF‐BP mRNA and protein are expressed in the LHA, and that both proteins are present in orexin‐positive VTA synaptosomes. The results showing that CRF₂R and CRF‐BP are expressed in the LHA–VTA input give anatomical support to suggest that this input plays a role in stress‐induced relapse to cocaine‐seeking behaviour.     

Phosphodiesterase 4 inhibition impairs cocaine-induced inhibitory synaptic plasticity and conditioned place preference

Endocannabinoid-mediated long-term depression of inhibitory synaptic transmission (I-LTD) in the ventral tegmental area (VTA) is implicated in cocaine-induced inhibitory synaptic plasticity and behavioral effects. It remains poorly understood, however, how this I-LTD is regulated and whether this regulation affects cocaine-seeking behavior. I-LTD requires cyclic adenosine 3', 5'-monophosphate (cAMP)-dependent protein kinase A (PKA) signaling, raising the possibility that modulators of cAMP/PKA signaling may regulate I-LTD and the reinforcement behavior. Phosphodiesterase (PDE) 4 hydrolyses cAMP and terminates cAMP/PKA signaling. Here, we report that selective PDE4 inhibitors rolipram and Ro 20-1724 blocked I-LTD and acute depression of inhibitory postsynaptic currents (IPSCs) induced by D(2) dopamine receptor and cannabinoid CB(1) receptor agonists in VTA dopamine neurons. We also show that intra-VTA microinjections of PDE4 inhibitor rolipram impaired the acquisition, but not the expression, of conditioned place preference (CPP) to cocaine. Systemic administration of rolipram also increased cAMP response element-binding protein (CREB) phosphorylation and activation in the VTA. Together, our results suggest that blockade of cocaine-induced inhibitory synaptic plasticity (I-LTD) and enhancement of CREB activation are two putative cellular mechanisms by which PDE4 inhibition impairs the acquisition of cocaine CPP.     

Effect of optogenetic manipulation of accumbal medium spiny neurons expressing dopamine D2 receptors in cocaine‐induced behavioral sensitization

Repetitive exposure to addictive drugs causes synaptic modification in the mesocorticolimbic dopamine (DA) system. Dopamine D1 receptors (D1R) or D2 receptors (D2R) expressed in the medium spiny neurons (MSNs) of the nucleus accumbens (NAc) play critical roles in the control of addictive behaviors. Optogenetic activation of D2R‐expressing MSNs (D2R‐MSNs) in the NAc previously demonstrated that these neurons play a key role in withdrawal‐induced plasticity. Here, we examined the effect of optogenetic inhibition of D2R‐MSNs in the NAc on cocaine‐induced behavioral sensitization. Adeno‐associated viral vectors encoding archaerhodopsin (ArchT) were delivered into the NAc of D2‐Cre transgenic mice. Activation of ArchT produced photoinhibition of D2R‐MSNs and caused disinhibition of neighboring MSNs in the NAc. However, such optogenetic silencing of D2R‐MSNs in the NAc in vivo affected neither the initiation nor the expression of cocaine‐induced behavioral sensitization. Similarly, photoinhibition of NAc D2R‐MSNs in the NAc during the drug withdrawal period did not affect the expression of cocaine‐induced behavioral sensitization. More detailed analysis of the effects of optogenetic activation of D2R‐MSNs suggests that D2R‐MSNs in the NAc exert important modulatory effects on neighboring MSN neurons, which may control the balanced output of NAc MSNs to control addictive behaviors.