Identification of Rat Ventral Tegmental Area GABAergic Neurons

PLOS ONE, Dec 2019

The canonical two neuron model of opioid reward posits that mu opioid receptor (MOR) activation produces reward by disinhibiting midbrain ventral tegmental area (VTA) dopamine neurons through inhibition of local GABAergic interneurons. Although indirect evidence supports the neural circuit postulated by this model, its validity has been called into question by growing evidence for VTA neuronal heterogeneity and the recent demonstration that MOR agonists inhibit GABAergic terminals in the VTA arising from extrinsic neurons. In addition, VTA MOR reward can be dopamine-independent. To directly test the assumption that MOR activation directly inhibits local GABAergic neurons, we investigated the properties of rat VTA GABA neurons directly identified with either immunocytochemistry for GABA or GAD65/67, or in situ hybridization for GAD65/67 mRNA. Utilizing co-labeling with an antibody for the neural marker NeuN and in situ hybridization against GAD65/67, we found that 23±3% of VTA neurons are GAD65/67(+). In contrast to the assumptions of the two neuron model, VTA GABAergic neurons are heterogeneous, both physiologically and pharmacologically. Importantly, only 7/13 confirmed VTA GABA neurons were inhibited by the MOR selective agonist DAMGO. Interestingly, all confirmed VTA GABA neurons were insensitive to the GABAB receptor agonist baclofen (0/6 inhibited), while all confirmed dopamine neurons were inhibited (19/19). The heterogeneity of opioid responses we found in VTA GABAergic neurons, and the fact that GABA terminals arising from neurons outside the VTA are inhibited by MOR agonists, make further studies essential to determine the local circuit mechanisms underlying VTA MOR reward.

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Identification of Rat Ventral Tegmental Area GABAergic Neurons

Citation: Margolis EB, Toy B, Himmels P, Morales M, Fields HL ( Identification of Rat Ventral Tegmental Area GABAergic Neurons Elyssa B. Margolis 0 1 Brian Toy 0 1 Patricia Himmels 0 1 Marisela Morales 0 1 Howard L. Fields 0 1 Laurent Groc, Institute for Interdisciplinary Neuroscience, France 0 1 Ernest Gallo Clinic and Research Center, University of California San Francisco , Emeryville , California, United States of America, 2 Department of Neurology, University of California San Francisco , San Francisco , California, United States of America, 3 Wheeler Center for the Neurobiology of Addiction, University of California San Francisco , San Francisco , California, United States of America, 4 Cellular Neurophysiology, National Institute on Drug Abuse , Baltimore, Maryland , United States of America 1 All animal protocols were conducted under National Institutes Health (NIH). Guidelines using the NIH handbook Animals in Research and were approved by the Institutional Animal Care and Use Committee (Ernest Gallo Clinic and Research Center, University of California at San Francisco , Emeryville, CA), approval ID 10.01.202 The canonical two neuron model of opioid reward posits that mu opioid receptor (MOR) activation produces reward by disinhibiting midbrain ventral tegmental area (VTA) dopamine neurons through inhibition of local GABAergic interneurons. Although indirect evidence supports the neural circuit postulated by this model, its validity has been called into question by growing evidence for VTA neuronal heterogeneity and the recent demonstration that MOR agonists inhibit GABAergic terminals in the VTA arising from extrinsic neurons. In addition, VTA MOR reward can be dopamine-independent. To directly test the assumption that MOR activation directly inhibits local GABAergic neurons, we investigated the properties of rat VTA GABA neurons directly identified with either immunocytochemistry for GABA or GAD65/67, or in situ hybridization for GAD65/67 mRNA. Utilizing co-labeling with an antibody for the neural marker NeuN and in situ hybridization against GAD65/67, we found that 2363% of VTA neurons are GAD65/67(+). In contrast to the assumptions of the two neuron model, VTA GABAergic neurons are heterogeneous, both physiologically and pharmacologically. Importantly, only 7/13 confirmed VTA GABA neurons were inhibited by the MOR selective agonist DAMGO. Interestingly, all confirmed VTA GABA neurons were insensitive to the GABAB receptor agonist baclofen (0/6 inhibited), while all confirmed dopamine neurons were inhibited (19/19). The heterogeneity of opioid responses we found in VTA GABAergic neurons, and the fact that GABA terminals arising from neurons outside the VTA are inhibited by MOR agonists, make further studies essential to determine the local circuit mechanisms underlying VTA MOR reward. - Funding: The project described was supported by Grant Number R01DA030529 from the National Institute On Drug Abuse. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute On Drug Abuse or the National Institutes of Health. This project was also supported by the Irene and Eric Simon Brain Research Foundation, funds from the State of California for medical research on alcohol and substance abuse through the University of California, San Francisco, and funds to the Intramural Research Program of the National Institute on Drug Abuse. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. The essential role of the VTA in the motivational and reinforcing actions of MOR agonists is well established [16], however the local circuit mechanisms are uncertain. Because MOR agonists in the VTA increase both dopamine release in the ventral striatum [79] and the firing of putative VTA dopamine neurons [1012], and because dopamine contributes to the motivational actions of a variety of natural and drug rewards, the idea that activation of VTA dopamine neurons is required for opioid reward has been widely accepted. While local MOR agonists do activate some VTA dopamine neurons, the direct synaptic effects of opioid receptor activation are typically inhibitory. The canonical two neuron model of opioid reward proposes that, as in other brain regions [13], MOR excites midbrain VTA dopamine neurons indirectly by hyperpolarizing local GABAergic interneurons [14,15]. However, in the original studies VTA neurons were identified as GABAergic if they were directly inhibited by a MOR agonist; clearly this is circular reasoning when testing the hypothesis that MOR agonists work by inhibiting GABA release [15,16]. Conversely, VTA neurons were identified as dopaminergic if they were inhibited by dopamine D2 receptor activation but not MOR activation. Subsequent research has demonstrated that the neurons of (...truncated)


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Elyssa B. Margolis, Brian Toy, Patricia Himmels, Marisela Morales, Howard L. Fields. Identification of Rat Ventral Tegmental Area GABAergic Neurons, PLOS ONE, 2012, Volume 7, Issue 7, DOI: 10.1371/journal.pone.0042365