Despite the well-known role of the neurotransmitter dopamine in reinforcement learning, generating the brain’s patterns of dopamine release for conditioned response learning remains unresolved. The Zweifel laboratory discovered that two ion channel subunits, Kv4.3 and BKCa1.1, expressed in ventral tegmental area (VTA) dopamine-producing neurons control the pattern of dopamine neuron firing at the cell body and dopamine release in the nucleus accumbens (NAc) on different time scales to regulate separate phases of reinforced behavior in awake and behaving mice (in vivo). However, whether the impact on dopamine release is dependent on cell body loss of function (LOF) or possible effects of local terminal regulation remains unknown. DLight—a fluorescent, genetically modified dopamine receptor—was injected into the NAc. Chrimson, a photoactivatable ion channel, and CRISPR/Cas9 viruses that knocked out either of the two ion channel subunits were injected into the VTA for selective expression in dopamine neurons. Three conditions were imposed: control (no subunit knockout), Kv4.3 LOF, or BKCa1.1 LOF. After at least six weeks, NAc brain slices were prepared for two-photon imaging of dLight following photoactivation of VTA dopamine terminals. I measured changes in dLight fluorescence, indicating dopamine release, using ImageJ and analyzed them using ImageJ, Excel, and GraphPad Prism. VTA dopamine terminals in the dLight-expressing NAc of control and Kv4.3 LOF mice were photostimulated; the BKCa1.1 LOF mice are in progress. I found that loss of Kv4.3 in VTA dopamine neurons does not impact dopamine release ex vivo. Observed differences in dopamine release in vivo in Kv4.3 LOF mice and lack of differences in ex vivo suggest that the ion channel effects at the cell body impact dopamine release downstream, highlighting that regulators of dopamine neuron cell body activity patterns impact dopamine release and affect dopamine-mediated behaviors like learning and motivation.

The ongoing opioid epidemic has made apparent the consequences of repeat opioid use and the opioid withdrawal that follows. Avoidance of opioid withdrawal and its associated symptoms has led to a need to understand signaling within neural circuits involved. The parabrachial nucleus (PBN) is a region in the brainstem that relays sensory information throughout critical forebrain regions, informing specific behavioral actions and learning mechanisms. A genetically defined cell population with the PBN expressing the calcitonin gene related peptide (CGRP, gene name Calca) has been demonstrated to relay aversive sensory and visceral information when activated. I hypothesized that PBN-CGRP neurons may be a neuron population that is recruited during opioid withdrawal. To investigate whether the aversive experience of opioid withdrawal recruits PBN-CGRP neuron activity, I induced morphine-dependence in mice expressing a yellow fluorescent protein in PBN-CGRP neurons. After establishing morphine dependence, precipitated opioid withdrawal was induced with an injection of naloxone. Ninety minutes after induction of precipitated opioid withdrawal, mice were perfused to fix brain tissue. I then performed immunohistochemistry for cFos, an immediate early gene and a marker of increased neural activity, in brain sections with the PBN. I am quantifying the expression of cFos, a marker of increased neural activity, in PBN neurons that contain the neuropeptide CGRP following naloxone precipitated withdrawal. I expect to see increased cFos expression indicating the recruitment of PBN-CGRP neurons in mice that are suffering from opiod withdrawal compared to the control mice. By understanding whether the PBN is recruited during opioid withdrawal, I can begin to explore options to alter neuronal activity in the PBN to lessen the severity of symptoms felt during opioid withdrawal, a key contributor to the prevalence of relapse in those overusing opiates.