RESUMO
Ion channel complexes typically consist of both pore-forming subunits and 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 use cell-type-specific viral-mediated CRISPR/Cas9 mutagenesis to target the potassium channel auxiliary subunit Kvß2 (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 toward 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 Kvß2 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.NEW & NOTEWORTHY Here, we utilize CRISPR/Cas9-mediated mutagenesis in dopamine neurons in mice to target the gene encoding Kvß2, an auxiliary subunit that forms a part of Kv1 channel complexes. We find that the absence of Kvß2 alters action potential properties by reducing surface expression of pore-forming subunits and shifting the voltage dependence of channel inactivation. This work establishes a new function for Kvß2 subunits and Kv1 complexes in regulating dopamine neuron activity.
Assuntos
Neurônios Dopaminérgicos , Canais de Potássio , Animais , Neurônios Dopaminérgicos/metabolismo , Camundongos , Canais de Potássio/metabolismo , Superfamília Shaker de Canais de Potássio/genéticaRESUMO
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.