Neuropeptides play an important role in modulating mesolimbic system function. However, while synaptic inputs to the ventral tegmental area (VTA) have been extensively mapped, the sources of many neuropeptides are not well resolved. Here, we mapped the anatomical locations of three neuropeptide inputs to the VTA: neurotensin (NTS), corticotrophin releasing factor (CRF), and neurokinin B (NkB). Among numerous labeled inputs we identified the bed nucleus of the stria terminalis (BNST) as a major source of all three peptides, containing similar numbers of NTS, CRF, and NkB VTA projection neurons. Approximately 50% of BNST to VTA inputs co-expressed two or more of the peptides examined. Consistent with this expression pattern, analysis of calcium dynamics in the terminals of these inputs in the VTA revealed both common and distinct patterns of activation during appetitive and aversive conditioning. These data demonstrate additional diversification of the mesolimbic dopamine system through partially overlapping neuropeptidergic inputs.
Ion channel complexes typically consist of both pore-forming subunits as well as auxiliary subunits that do not directly conduct current but can regulate trafficking or alter channel properties. Isolating the role of these auxiliary subunits in neurons has proved difficult due to a lack of specific pharmacological agents and the potential for developmental compensation in constitutive knockout models. Here we employ cell-type specific viral-mediated CRISPR/Cas9 mutagenesis to target the potassium channel auxiliary subunit Kvβ2 (Kcnab2) in dopamine neurons in the adult mouse brain. We find that mutagenesis of Kcnab2 reduces surface expression of Kv1.2, the primary Kv1 pore forming subunit expressed in dopamine neurons, and shifts the voltage dependence of inactivation of potassium channel currents towards more hyperpolarized potentials. Loss of Kcnab2 broadens the action potential waveform in spontaneously firing dopamine neurons recorded in slice, reduces the afterhyperpolarization amplitude, and increases spike timing irregularity and excitability, all of which is consistent with a reduction in potassium channel current. Similar effects were observed with mutagenesis of the pore-forming subunit Kv1.2 (Kcna2). These results identify Kv1 currents as important contributors to dopamine neuron firing and demonstrate a role for Kvβ2 subunits in regulating the trafficking and gating properties of these ion channels. Furthermore, they demonstrate the utility of CRISPR-mediated mutagenesis in the study of previously difficult to isolate ion channel subunits.
Fast-acting neurotransmitters and slow, modulatory neuropeptides are commonly co-released from neurons in the central nervous system (CNS), albeit from distinct synaptic vesicles 1 . The mechanisms of how co-released neurotransmitters and neuropeptides that have opposing actions, e.g., stimulatory versus inhibitory, work together to exert control of neural circuit output remain unclear. This question has been difficult to resolve due to the inability to selectively isolate these signaling pathways in a cell-and circuit-specific manner. To overcome these barriers, we developed a genetic-based anatomical disconnect procedure that utilizes distinct DNA recombinases to independently facilitate conditional in vivo CRISPR/Cas9 mutagenesis 2 of neurotransmitter-and neuropeptide-related genes in distinct cell types in two different brain regions simultaneously. With this approach we demonstrate that the stimulatory neuropeptide neurotensin (Nts) and the inhibitory neurotransmitter γ-aminobutyric acid (GABA), which are co-released from neurons in the lateral hypothalamus (LH), work coordinately to activate dopamine neurons of the ventral tegmental area (VTA-DA). We show that GABA release from LH-Nts neurons acts on GABA neurons within the VTA to rapidly disinhibit VTA-DA neurons, while Nts signals through the Nts receptor 1 (Ntsr1) on VTA-DA neurons to promote a slow depolarization of these cells. Thus, these two signals act on distinct time scales through different cell types to enhance mesolimbic dopamine neuron activation, which optimizes behavioral reinforcement. These data demonstrate a circuitbased mechanism for the coordinated action of a neurotransmitter and neuropeptide with opposing effects on cell physiology.Numerous cell types within the CNS co-release combinations of neuropeptides and neurotransmitters with opposing actions on cellular physiology [3][4][5] . A major unresolved question is how these opposing signals work to coordinate circuit function. An example of this paradoxical co-release is neurons in the LH that release stimulatory Nts and inhibitory GABA.The majority of Nts-producing neurons in the LH, like most LH afferents to the VTA, are reported to be GABAergic [6][7][8] , and LH-Nts neurons are proposed to activate dopamine-producing neurons within the VTA to regulate behavioral reinforcement [8][9][10][11][12][13][14][15] , but it remains unclear what the roles of Nts and GABA are in this process. We propose a circuit mechanism whereby GABA release from LH-Nts neurons acts on local VTA-GABA neurons to rapidly disinhibit VTA-DA cells, while Nts acts through Ntsr1 on VTA-DA neurons to provide a slow stimulatory depolarization.Consistent with previous reports 6,7 , we found that the majority of Nts neurons in the LH are GABAergic, as evidenced by co-expression of Nts and Slc32a1 (Vgat, the vesicular GABA transporter) (Fig. 1a-c, Supplementary Table 1). To establish the connectivity of LH-Nts neurons
SummaryFast-acting neurotransmitters and slow, modulatory neuropeptides are commonly co-released from neurons in the central nervous system (CNS), albeit from distinct synaptic vesicles1. The mechanisms of how co-released neurotransmitters and neuropeptides that have opposing actions, e.g., stimulatory versus inhibitory, work together to exert control of neural circuit output remain unclear. This question has been difficult to resolve due to the inability to selectively isolate these signaling pathways in a cell- and circuit-specific manner. To overcome these barriers, we developed a genetic-based anatomical disconnect procedure that utilizes distinct DNA recombinases to independently facilitate conditional in vivo CRISPR/Cas9 mutagenesis2 of neurotransmitter- and neuropeptide-related genes in distinct cell types in two different brain regions simultaneously. With this approach we demonstrate that the stimulatory neuropeptide neurotensin (Nts) and the inhibitory neurotransmitter γ-aminobutyric acid (GABA), which are co-released from neurons in the lateral hypothalamus (LH), work coordinately to activate dopamine neurons of the ventral tegmental area (VTA-DA). We show that GABA release from LH-Nts neurons acts on GABA neurons within the VTA to rapidly disinhibit VTA-DA neurons, while Nts signals through the Nts receptor 1 (Ntsr1) on VTA-DA neurons to promote a slow depolarization of these cells. Thus, these two signals act on distinct time scales through different cell types to enhance mesolimbic dopamine neuron activation, which optimizes behavioral reinforcement. These data demonstrate a circuit-based mechanism for the coordinated action of a neurotransmitter and neuropeptide with opposing effects on cell physiology.
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