Cannabinoid-induced Vta Da Neurons, Ltd,

Cannabis is the most commonly abused illegal drug in the world and its main psychoactive ingredient, delta-9-tetrahydrocannabinol (THC), produces rewarding effects in humans and non-human primates. However, there is no effective treatment for cannabis addiction in humans, largely due to our poor understanding of its underlying mechanisms. Recently, considerable evidence suggests that neuroadaptations leading to addiction involve the same glutamate-dependent cellular mechanisms that enable learning and memory. Long-term potentiation (LTP) and long-term depression (LTD) have therefore become an important focus of addiction research. In addition, most psychoactive drugs that have reinforcing or rewarding effects in experimental animals and are abused by humans increase dopamine levels in the nucleus accumbens shell, indicating VTA DA neurons are activated during drug exposure. LTP induction in VTA dopamine neurons following exposure to most drug (nicotine, cocaine, amphetamine, morphine and ethanol), indicated an essential contribution of LTP induction in the VTA dopamine circuitry to the development of drug addiction. It is entirely unknown, however, whether cannabinoids are able to induce LTP or LTD in the VTA.More importantly, whether such alterations in VTA synaptic plasticity causatively contribute to drug addictive behavior has not previously been addressed.In present study, we employed a combination of field potential recordings from both young and adult rats, whole cell recordings of the EPSCs from both dopamine and GABA neurons in the young rat VTA, and molecular biological, biochemical, immunohistochemica to exhibit the changes of VTA synaptic plasticity following chronic cannabinoid exposure. The purposes of this study are to describe the change of synaptic plasticity in VTA following cannobinoid exposure and underlying molecular and cellular mechanisms, and to provide candidate therapeutic strategies for treating cannabis addiction. We first observed that chronic cannabinoid (THC and HU210) exposure in vivo facilitated LTD induction in the VTA. Then, we provide the first evidence suggesting that cannabinoids induce in vivo LTD in VTA dopamine neurons via cannabinoid CB1 receptors (CB1R) probably on VTA astrocytes and AMPAR on VTA dopamine neurons. The results are as followings:1 Cannabinoids act on VTA CB1R to facilitate LTD induction in the DA neuronWithout blocking inhibitory GABA neurons, neither HFS nor LFS of excitatory afferents induced LTP or LTD in the field EPSPs recording in the VTA of both naive rats and vehicle-treated rats. In contrast, LFS induced LTD, but HFS still failed to induce LTP, from in midbrain slices prepared at 24 hours after 5 days chronic cannabinoid exposure. The selective CB1R antagonist AM281 prevented cannabinoid-facilitated LTD induction. Whole cell recording identified that this LTD was expressed in VTA dopamine neurons but not GABA neurons.2 Cannabinoid facilitates LTD induction in the VTA via post-synaptic NMDAR and requiring AMPAR endocytosisThe paired pulse ratio (PPR) recording shows that the in vivo HU210 exposure did not cause a change in probability of glutamate transmitter release, indicating a postsynaptic mechanism for the induction of cannabinoid-facilitated LTD in the VTA. This postsynaptic LTD was blocked by the NMDAR antagonist, but not mGluR antagonists, suggesting that cannabinoid facilitates LTD induction in the VTA via post-synaptic NMDAR.TAT-GluR2 peptide, the blocker of GluR2 endocytosis procedure, abolished LTD induction in HU210-treated rats. GluR2 endocytosis likely occurs in the post-synaptic membrane of VTA dopamine neurons, but not VTA GABA neurons, because GluR2-positive neurons were doubly stained with the dopamine-synthesizing enzyme tyrosine hydroxylase (TH), but not with the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD) 65/67. These results further strongly support the idea that the expression of cannabinoid-facilitated LTD in the VTA occurs in VTA dopamine neurons but not GABA neurons.3 Cannabinoid induces in vivo LTD in VTA dopamine neuronsCompared with the slices prepared at 24 hours after chronic HU210 injection, LFS failed to induce LTD at the slices prepared at 30 minutes after the last HU210 injection, but not at 24 hours, by a preceding HU210-induced LTD in vivo, indicating the occlusion of LFS-induced LTD in vitro at 30 minutes. Cell surface levels of GluR2 in the VTA significantly reduced at 30 minutes, but not at 24 hours, after the fifth HU210 injection. The AMPAR/NMDAR current ratio of glutamatergic synaptic currents in VTA dopamine neurons was significantly smaller at 30 minutes than both that at 24 hours after the fifth HU210 injection and that in naive rats. NR2B/NR2A EPSC ratio of VTA dopamine neurons was significantly larger in HU210-treated rats than that in naive rats. All these evidences support that cannabinoid likely induces in vivo LTD in VTA dopamine neurons.4 Role of VTA astrocytes in cannabinoid-induced in vivo LTD in VTA dopamine neuronsIt were reported that astrocytes express CB1R. So we if selective knocking down of CB1R expression in the VTA using CB1R shRNA expressed by adenoviral vectors could disturb cannabinoid-induced in vivo LTD. Through immunoblotting method, we firstly confirmed that bilateral injection of CB1R shRNA expressed by adenoviral vectors into the VTA significantly suppressed CB1R expression; then we show that VTA astrocytes are the major targets of intra-VTA injection of the vectors by double staining technology; finally, we found that LFS-induced LTD in HU210-treated rats was inhibited after vectors intra-VTA microinjection. Furthermore, we observed that riluzole, a drug can inhibit pre-synaptic glutamate release and enhance glutamate uptake into astrocytes, daily intra-VTA infusion before systemic HU210 injection prevented LFS-induced LTD in the VTA.It is generally agreed that NR2B-containing NMDAR induces GluR2 endocytosis and subsequent LTD induction. Here we show that an increase in NR2B/NR2A EPSC ratio of VTA dopamine neurons at 24 hours after the fifth HU210 injection. Immunofluorescent staining results show that the majority of NR2B-and NR2A-containing NMDARs in the VTA are located in extra-synaptic and synaptic zones, respectively, indicating that the extra-synaptic NR2B activation by glutamate released from nearby astrocytes is response to cannabinoid-induced LTD.5 Counteractive effects of cannabinoid and nicotineWhile we show evidence here strongly suggesting that cannabinoid exposure in vivo induces LTD in vivo in VTA dopamine circuitry, previous studies show that many other drugs of abuse including nicotine, cocaine, amphetamine, morphine and ethanol may induce LTP in vivo in these neurons(10-12).It would be of interest to examine co-administration of cannabinoid and one of these drugs of abuse may neutralize their opposite effects on VTA synaptic plasticity.We obtained two lines of evidence to support this hypothesis. First, HFS, but not LFS, induced VTA LTP from rats receiving chronic nicotine injection. Second, neither HFS nor LFS induced VTA LTP or LTD from rats receiving chronic co-administration of HU210 and nicotine.ConclusionCannabinoid exposure in vivo induced in vivo LTD in the VTA that lasts for less than 24 hr, and facilitated LTD induction in the DA neuron 24 hours later. The signaling process of this LTD induction in the VTA is that cannabinoid via CB1R on astrocytes activate post-extrasynaptic NR2B subunits of NMDAR and then lead GluR2 subunits of AMPAR endocytosis. Furthermore, LTD induced by cannabinoid and TLP induced by nicotine could be neutralized each other.