A comparative study of self-administration of morphine into the amygdala and the ventral tegmental area in mice

Genetic analysis of Ntn1 in adult mouse midbrain neurons reveals its function in maintaining excitatory synapses, loss of Ntn1 function in inhibitory neurons is significantly detrimental to mesolimbic system function.

BALB/c mice were bilaterally implanted with two guide cannulae, the tips of which were positioned either 1.5 mm above the amygdala (AMY) and the ventral tegmental area (VTA)(AMY-VTA subjects) or 1.5 mm above the AMY and 2.3 mm above the VTA (D.vta)(AMY-D.vta subjects). On each experimental day, a stainless steel injection cannula was inserted into each brain structure. The experiment was carried out in a Y-maze. During a preliminary phase, which lasted 4 days, animals were allowed to self-inject morphine successively into the AMY and into the VTA, or into the AMY and into the D.vta. From the 5th day, animals of each group were given the possibility of choosing between the two sites. Four subgroups were constituted depending on the dose of morphine used (5 and 50 ng: AMY5ng-VTA5ng, AMY50ng-VTA50ng, AMY5ng-D.vta5ng, and AMY5ng-D.vta50ng). The AMY5ng-VTA5ng group rapidly differentiated between the two injection sites and showed a marked preference for self-injection into the VTA. In the AMY50ng-VTA50ng group, no significant preference was observed, with the animals tending to alternate self-injection into the AMY and VTA. The AMY5ng-D.vta5ng group discriminated between the two sites and self-injected morphine preferentially into the AMY. The discrimination performance of the AMY5ng-D.vta50ng group was not statistically different from that at chance level. These results demonstrate that mice are capable of discriminating, at the intracerebral level, the motivational or rewarding components of morphine when the dose available is low (5 ng). The preference manifested is highly influenced by the location of injection cannulae. The positive effect of a low dose of morphine appeared stronger in the VTA than in the AMY. However, the location of injection cannulae 0.8 mm above the VTA induced a marked preference for self-injection into the AMY. Consequently, the rewarding effects of morphine into the VTA probably results from a local action of the drug and not from a dorsal diffusion.

BALB/c mice were unilaterally implanted with a guide cannula, the tip of which was positioned 1.5 mm above the ventral tegmental area (VTA). On each day of the experimental period, a stainless steel injection cannula was inserted into the VTA in order to study the eventual self-administration of a low dose (1.5 ng/50 nl) of bicuculline, a GABAA-antagonist, using a spatial discrimination task in a Y maze. Mice rapidly discriminated between the arm enabling a micro-injection of bicuculline and the neutral arm of the maze, and robust self-administration of this GABAergic antagonist was observed. Once this self-administration response for bicuculline had been fully acquired, the systemic injection of the dopaminergic D2 antagonist sulpiride (50 mg/kg), 30 min before the test, produced a rapid extinction of the self-administration response. Moreover, if this same sulpiride pretreatment was given during the initial acquisition period mice did not discriminate between the two arms of the Y-maze. These data demonstrate the dopamine D2 dependence of this bicuculline self-administration behavior, and confirm that GABAergic interneurons and/or inputs normally transynaptically inhibit neuronal activity in the mesocorticolimbic dopamine system.

Drug addiction is characterized by compulsive drug use and relapse. Thioredoxin-1 is emerging as an important modulator involved in the cellular protective response against a variety of toxic stressors. Previous study has reported that thioredoxin-1 overexpression prevents the acquisition of methamphetamine-conditioned place preference. Here, we aimed to investigate the effect of thioredoxin-1 on methamphetamine-conditioned place preference extinction and the possible mechanism. (a) An extinction procedure in mice was employed to investigate the effect of thioredoxin-1 on the extinction of methamphetamine-conditioned place preference. After the acquisition of methamphetamine-conditioned place preference, mice underwent the following procedures: the injection of thioredoxin-1 small interfering RNA in the ventral tegmental area followed by the post-conditioned place preference test, four days of extinction training followed by four days of recovery after surgery. (b) The levels of thioredoxin-1, dopamine D1 receptor, tyrosine hydroxylase, phosphorylated extracellular regulated kinase, and phosphorylated cyclic adenosine monophosphate response element binding protein were examined by using Western blot analysis. Thioredoxin-1 downregulation in the ventral tegmental area delayed methamphetamine-conditioned place preference extinction. The expression of thioredoxin-1 was decreased in the ventral tegmental area of mice in control and negative groups after methamphetamine-conditioned place preference extinction, but not in the thioredoxin-1 siRNA group. The levels of dopamine D1 receptor, tyrosine hydroxylase, phosphorylated extracellular regulated kinase, and phosphorylated cyclic adenosine monophosphate response element binding protein were decreased in the ventral tegmental area, nucleus accumbens, and prefrontal cortex of mice in the control and negative groups after methamphetamine-conditioned place preference extinction, but were inversely increased in thioredoxin-1 siRNA group. The results suggest that downregulation of thioredoxin-1 in the ventral tegmental area may delay methamphetamine-conditioned place preference extinction by regulating the mesocorticolimbic dopaminergic signaling pathway.

Repeated exposure to psychostimulants such as methamphetamine (METH) induces neuronal adaptations in the mesocorticolimbic dopamine system, including the ventral tegmental area (VTA). These changes lead to persistently enhanced neuronal activity causing increased dopamine release and addictive phenotypes. A factor contributing to increased dopaminergic activity in this system appears to be reduced GABAB receptor-mediated neuronal inhibition in the VTA. Dephosphorylation of serine 783 (Ser783) of the GABAB2 subunit by protein phosphatase 2A (PP2A) appears to trigger the downregulation GABAB receptors in psychostimulant-addicted rodents. Therefore, preventing the interaction of GABAB receptors with PP2A using an interfering peptide is a promising strategy to restore GABAB receptor-mediated neuronal inhibition. We have previously developed an interfering peptide (PP2A-Pep) that inhibits the GABAB receptors/PP2A interaction and thereby restores receptor expression under pathological conditions. Here, we tested the hypothesis that restoration of GABAB receptor expression in the VTA of METH addicted mice reduce addictive phenotypes. We found that the expression of GABAB receptors was significantly reduced in the VTA and nucleus accumbens but not in the hippocampus and somatosensory cortex of METH-addicted mice. Infusion of PP2A-Pep into the VTA of METH-addicted mice restored GABAB receptor expression in the VTA and inhibited METH-induced locomotor sensitization as assessed in the open field test. Moreover, administration of PP2A-Pep into the VTA also reduced drug seeking behavior in the conditioned place preference test. These observations underscore the importance of VTA GABAB receptors in controlling addictive phenotypes. Furthermore, this study illustrates the value of interfering peptides targeting diseases-related protein-protein interactions as an alternative approach for a potential development of selective therapeutic interventions.

Mechanisms of cannabinoid CB2 receptor-mediated reduction of dopamine neuronal excitability in mouse ventral tegmental area

The ventral tegmental area (VTA), a pivotal hub in the brain's reward circuitry, receives inputs from the lateral hypothalamic area (LHA). However, it remains unclear whether melanin-concentrating hormone (MCH) and orexin-A (OX-A) neurons in the LHA exert individual or cooperative influence on palatable food consumption in the VTA. This study aims to investigate the modulatory role of MCH and OX-A in hedonic feeding within the VTA of high-fat diet (HFD) mice. Male mice were subjected to an 8-week high-fat diet. To visualize the projections from the LHA to VTA, we employed fluorescent gold retrograde tracing combined with immunofluorescence staining. Immunofluorescence staining or enzyme-linked immunosorbent assay was used to detect the activity of the VTA neurons, expression of OX-A or MCH in the LHA, as well as the activity of their receptors (OXR1 and MCHR1) in the VTA following a sucrose preference test. Single-unit extracellular electrical discharge recordings were conducted to assess the effects of OX-A and MCH on VTA neurons in HFD mice. Additionally, chemogenetic inhibition of MCH neurons and immunofluorescence staining were utilized to observe the regulatory roles of MCH in changes of hedonic feeding induced by OX-A in HFD mice. Sucrose intake resulted in lower activation of VTA neurons in the HFD mice compared to CON mice, while OX-Aergic and MCHergic neurons project from the LHA to the VTA. Although sucrose intake increased the expression of OX-A and MCH in HFD mice, it led to diminished activation of OXR1-positive and MCHR1-positive VTA neurons compared to CON mice. Extracellular single-unit recording revealed that MCH significantly suppressed the firing rate of OX-A-responsive neurons in the VTA. MCH attenuated the hedonic feeding response induced by OX-A in HFD mice, and administration of MCHR1 antagonist (SNAP94847) significantly potentiated the effect of OX-A. Chemogenetic inhibition of MCH neurons improved the activity of OXR1-expressing neurons, which could be reversed by pretreatment with an OXR1 antagonist (SB334867). Furthermore, chemogenetic inhibition of MCH enhanced hedonic feeding behavior, which was counteracted by SB334867 treatment in HFD mice. Melanin-concentrating hormone could attenuate the hedonic feeding behavior induced by orexin-A in the VTA of HFD mice.

Progesterone has rapid and membrane effects in the facilitation of female mouse sexual behavior

We have recently reported that activation of cannabinoid type 2 receptors (CB2Rs) reduces dopamine (DA) neuron excitability in mouse ventral tegmental area (VTA). Here, we elucidate the underlying mechanisms. Using cell-attached recording in VTA slices, bath-application of CB2R agonists (JWH133 or five other CB2R agonists) significantly reduced VTA DA neuron action potential (AP) firing rate. Under patch-clamp whole-cell recording model, JWH133 (10 µM) mildly reduced the frequency of miniature excitatory postsynaptic currents (mEPSCs) but not miniature inhibitory postsynaptic currents (mIPSCs). JWH133 also did not alter evoked EPSCs or IPSCs. In freshly dissociated VTA DA neurons, JWH133 reduced AP firing rate, delayed AP initiation and enhanced AP after-hyperpolarization. In voltage-clamp recordings, JWH133 enhanced M-type K+ currents and this effect was absent in CB2-/- mice and abolished by co-administration of a selective CB2R antagonist (AM630). CB2R-mediated inhibition in VTA DA neuron firing can be mimicked by M-current opener and blocked by M-current blocker. In addition, enhancement of neuronal cAMP by forskolin reduced M-current and increased DA neuron firing rate. Finally, pharmacological block of synaptic transmission by NBQX, D-APV and picrotoxin in VTA slices failed to prevent CB2R-mediated inhibition, while intracellular infusion of guanosine 5'-O-2-thiodiphosphate (GDP-β-S) through recording electrode to block postsynaptic G-protein function prevented JWH133-induced reduction in AP firing. Collectively, our results suggest that CB2Rs modulate VTA DA neuron excitability mainly through an intrinsic mechanism, including a CB2R-mediated reduction of intracellular cAMP, and in turn enhancement of M-type K+ currents. Funding Statement: This research was supported by the Barrow Neuroscience Foundation, the BNI-BMS Seed Fund, the CNSF (81771437), and the National Institute on Drug Abuse, Intramural Research Program. Declaration of Interests: Dr. Ma, ZG reports no disclosures, Dr. Gao, FF reports no disclosures, Dr. Larsen, B. reports no disclosures, Dr. Gao M reports no disclosures, D. Chen, DJ reports no disclosures, Xiaokuang Ma reports no disclosures, Dr. Qiu, SF reports no disclosures, Dr. Zhou Y reports no disclosures, Dr. Xie, JX reports no disclosures, Dr. Xi, ZX reports no disclosures, Dr. Wu J reports no disclosures. Ethics Approval Statement: All experimental procedures were approved by the Institutional Animal Care and Use Committee at the Barrow Neurological Institute.

In the present report, fast-scan cyclic voltammetry was used to identify the monoamines that were released by electrical stimulation in mouse brain slices containing ventral tegmental area (VTA), substantia nigra (SN) -pars compacta (SNc) and -pars reticulata (SNr). We showed that voltammograms obtained in mouse VTA were consistent with detection of a catecholamine, while those in both subregions of the SN were consistent with detection of an indolamine, based on the reduction peak potentials. We used pharmacological blockade and genetic deletion of monoamine transporters to further confirm the identity of released monoamines in mouse midbrain and to assess the control of monoamines by their transporters in each brain region. Inhibition of dopamine and norepinephrine transporters by nomifensine (1 and 10 microm) decreased uptake rates in the VTA, but did not change uptake rates in either subregion of the SN. Serotonin transporter inhibition by fluoxetine (10 microm) decreased uptake rates in the SNc and SNr, but was without effect in the VTA. Selective inhibition of the norepinephrine transporter by desipramine (10 microm) had no effect in any brain region. Using dopamine transporter- and serotonin transporter-knockout mice, we found decreased uptake rates in VTA and SN subregions, respectively. Peak signals recorded in each midbrain region were pulse number dependent and exhibited limited frequency dependence. Thus, dopamine is predominately detected by voltammetry in mouse VTA, while serotonin is predominately detected in mouse SNc and SNr. Furthermore, active uptake occurs in these areas and can be altered only by specific uptake inhibitors, suggesting a lack of heterologous uptake. In addition, somatodendritic dopamine release in VTA was not mediated by monoamine transporters. This work offers an initial characterization of voltammetric signals in the midbrain of the mouse and provides insight into the regulation of monoamine neurotransmission in these areas.

All addictive drugs target the mesolimbic dopamine system that originates in the ventral tegmental area (VTA). VTA dopamine neurons project mainly to the nucleus accumbens (NAc), from where inhibitory medium spiny neurons (MSNs) project back to the VTA. Cocaine-evoked synaptic plasticity has been observed in excitatory transmission of the NAc and in the VTA but it remains unknown whether the GABAergic projections from the NAc to the VTA also undergo synaptic plasticity in response to cocaine treatment. Here, we used optogenetic projection targeting to selectively stimulate axons of NAc MSNs to record inhibitory postsynaptic currents (IPSCs) in acute VTA slices. We observed a preferential connectivity of MSNs onto VTA GABA neurons, which was confirmed in vivo. Extracellular single unit recordings in the VTA of anaesthetised mice revealed a strong inhibition of GABA neurons in response to the optogenetic stimulation of MSN terminals. In contrast, dopamine neurons showed increased firing rates in response to light stimulation, confirming a disinhibitory action of MSNs onto dopamine cells. Combining retrograde labeling with attenuated cholera toxine and immunohistochemistry we further show that the MSNs projecting directly to the VTA express D1Rs. We tested whether the synapses between MSNs and VTA GABA neurons undergo synaptic plasticity. A high frequency light stimulation protocol successfully potentiatiated IPSCs in VTA GABA cells by 81 ± 20%. Synaptic potentiation was insensitive to the Ca2+ buffer BAPTA in the postsynaptic cell but was prevented by blocking L-type voltage gated Ca2+ channels, indicating that the potentiation is induced presynaptically. The potentiation was mimicked by the adenylat cyclase activator forskolin, indicating that the cAMP-PKA cascade is involved in the mechanism. Further, using 2-photon imaging of activity induced uptake of the styryl dye FM4-64 at MSN terminals, we show that HFS induced potentiation of MSN-VTA GABA neuron synapses is expressed by an increased number of active release sites. We then treated mice with five daily cocaine injections and observed that HFS and forskolin were no longer able to induce inhibitory potentiation ex vivo 24 hours after the last injection. In accordance with an occlusion scenario, we observed paired pulse depression of light evoked IPSCs after cocaine treatment, indicative of increased GABA release at MSN-VTA GABA neuron synapses. Our data suggest that cocaine potentiates inhibitory transmission of D1-MSNs onto VTA GABA neurons via a PKA dependent mechanism. Taken together, these results suggest that cocaine may disinhibit VTA dopamine neurons by increasing GABA release onto VTA GABA neurons.

Background Mounting evidence has shown that sirtuin 1 (SIRT1), a class III histone deacetylase, alleviated several types of neuropathic pain in the spinal cord and dorsal root ganglion and regulated some aberrant behaviors in the ventral tegmental area (VTA) and the nucleus accumbens (NAc). Methods In this context, the effect of SIRT1 on neuropathic pain in the VTA-NAc pathway was investigated in the model of chronic constrictive injury (CCI). Results SIRT1 was localized in the VTA neurons in naive mice. The expression of SIRT1 was decreased in the contralateral VTA of CCI mice. After microinjection of SRT1720 (an activator of SIRT1) in the contralateral VTA of CCI mice, the established thermal hyperalgesia was attenuated. However, it was further exacerbated by EX-527 (an inhibitor of SIRT1). The elevated level of acetyl-histone 3 was reduced by SRT1720 but further elevated by EX-527 in the contralateral VTA of CCI mice. The increased expression of Fos in both VTA and NAc was downregulated by SRT1720 but further upregulated by EX-527 in CCI mice. Conclusions The discovery of the effect of SIRT1 on neuropathic pain in the VTA represents an important step forward in understanding the analgesic mechanisms of the VTA-NAc pathway.

Most psychiatric disorders are characterized by deficits in the ability to interact socially with others. Ghrelin, a hormone normally associated with the regulation of glucose utilization and appetite, is also implicated in the modulation of motivated behaviors including those associated with food and sex rewards. Here we hypothesized that deficits in ghrelin receptor (growth hormone secretagogue receptor; GHSR) signaling are also associated with deficits in social motivation in male mice. To test this hypothesis, we compared social motivation in male mice lacking GHSR or mice treated with the GHSR antagonist JMV2959 with that of WT or vehicle-treated mice. GHSR signaling in dopamine cells of the ventral tegmental area (VTA) has been implicated in the control of sexual behavior, thus we further hypothesized that GHSR signaling in the VTA is important for social motivation. Thus, we conducted studies where we delivered JMV2959 to block GHSR in the VTA of mice, and studies where we rescued the expression of GHSR in the VTA of GHSR knockout (KO) mice. Mice lacking GHSR or injected with JMV2959 peripherally for 3 consecutive days displayed lower social motivation as reflected by a longer latency to approach a novel conspecific and shorter interaction time compared to WT or vehicle-treated controls. Furthermore, intra-VTA infusion of JMV2959 resulted in longer latencies to approach a novel conspecific, whereas GHSR KO mice with partial rescue of the GHSR showed decreased latencies to begin a novel social interaction. Together, these data suggest that GHSR in the VTA facilitate social approach in male mice, and GHSR-signaling deficits within the VTA result in reduced motivation to interact socially.

BACKGROUND AND PURPOSEThe majority of studies examining desensitization of the μ-opioid receptor (MOR) have examined those located at cell bodies. However, MORs are extensively expressed at nerve terminals throughout the mammalian nervous system. This study is designed to investigate agonist-induced MOR desensitization at nerve terminals in the mouse ventral tegmental area (VTA).EXPERIMENTAL APPROACHMOR function was measured in mature mouse brain slices containing the VTA using whole-cell patch-clamp electrophysiology. Presynaptic MOR function was isolated from postsynaptic function and the functional selectivity, time-dependence and mechanisms of agonist-induced MOR desensitization were examined.KEY RESULTSMORs located at GABAergic nerve terminals in the VTA were completely resistant to rapid desensitization induced by the high-efficacy agonists DAMGO and Met-enkephalin. MORs located postsynaptically on GABAergic cell bodies readily underwent rapid desensitization in response to DAMGO. However, after prolonged (>7 h) treatment with Met-enkephalin, profound homologous MOR desensitization was observed. Morphine could induce rapid MOR desensitization at nerve terminals when PKC was activated.CONCLUSIONS AND IMPLICATIONSAgonist-induced MOR desensitization in GABAergic neurons in the VTA is compartment-selective as well as agonist-selective. When MORs are located at cell bodies, higher-efficacy agonists induce greater levels of rapid desensitization than lower-efficacy agonists. However, the converse is true at nerve terminals where agonists that induce MOR desensitization via PKC are capable of rapid agonist-induced desensitization while higher-efficacy agonists are not. MOR desensitization induced by higher-efficacy agonists at nerve terminals only takes place after prolonged receptor activation.LINKED ARTICLESThis article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2

Serotonin and its signaling pathways are involved in the central regulation of feeding behavior and energy metabolism. The most important role in this regulation is played by dopaminergic neurons of the dorsal raphe nucleus (DRN) and ventral tegmental area (VTA) which express tryptophan hydroxylase 2 (TPH2) catalyzing the rate-limiting step in serotonin synthesis. Changes in activity of the serotonin-synthesizing system in the DRN and VTA may significantly contribute to the development of metabolic disorders, including obesity. However, data on TPH2 expression in these brain regions in obesity are unavailable. The aim of this work was to compare immunohistochemically TPH2 expression in the DRN and VTA of C57Bl/6J (a/a) female mice with a diet-induced obesity (DIO) (caused by 8- and 16-week intake of high-calorie food) and C57Bl/6J (Ay/a) agouti mice with genetically predetermined melanocortin obesity. Double immunolabeling demonstrated that in DIO mice with a 16-week high-calorie diet TPH2 expression in the DRN (the main cerebral source of serotonin) decreases while in the VTA increases. In agouti mice, TPH2 expression in the DRN did not change while in the VTA increased. In DIO mice with a 8-week high-calorie diet and in C57Bl/6J (a/a) mice resistant to this diet, TPH2 expression did not change both in the DRN and VTA. Thus, DIO mice kept on a long-term (16 weeks) diet and agouti mice with melanocortin obesity exhibit specific changes in the serotonin synthesis that are lacking in diet-resistant mice. This finding indicates a crucial role of serotonin signaling in the organismal response to excessive caloric intake.