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Item The Behavioral Role of Mu Opioid Receptors in Glutamatergic Neurons(2021-10) Reeves, Kaitlin C.; Sheets, Patrick; Baucum, Anthony II; Yamamoto, Bryan; McKinzie, David; Yoder, KarmenMu opioid receptors (MORs) mediate the analgesic and rewarding effects of opioids. Most research has focused on MORs in GABAergic neurons; however, MORs are also in glutamatergic neurons and their role in opioid-related behaviors was unclear. Our lab previously showed that MORs inhibit glutamate transmission from vesicular glutamate transporter 2 (vGluT2)-expressing thalamostriatal synapses. The behavioral relevance of MORs in vGluT2-expressing neurons was unknown; therefore, I utilized a conditional MOR knockout mouse with MORs deleted in vGluT2-expressing neurons (MORflox-vGluT2cre). MORflox-vGluT2cre mice have disrupted opioid reward, locomotor stimulation, and withdrawal, compared to cre-recombinase negative littermate controls. However, other MOR-mediated behaviors, including opioid-induced antinociception, alcohol reward, and palatable substance consumption are intact. MORs are expressed in vGluT2 neurons in several reward-related brain regions, including the thalamus and lateral habenula (LHb). To determine whether MORs in these brain regions modulate opioid-related behaviors, an adeno-associated viral (AAV) vector encoding cre-recombinase was stereotaxically injected into the thalamus or LHb of MORflox mice to specifically delete MORs in these brain regions. Opioid reward and locomotor stimulation remained intact in both thalamic and LHb MOR knockout mice; however, basal locomotor activity was increased in LHb MOR knockout mice. Sucrose consumption was also intact in LHb MOR knockout mice. Interestingly, in LHb MOR KO mice opioid withdrawal-induced paw shakes were increased, while withdrawal-induced jumping was completely ablated. Our lab previously showed that MORs inhibit glutamate transmission from the anterior insular cortex (AIC), which is disrupted by in vivo alcohol exposure. To determine the role of AIC MORs, AIC MORs were deleted with AAV vectors. AIC MOR knockout mice had intact opioid, sucrose, and alcohol reward, but had increased basal locomotor activity. MORs in glutamatergic neurons are critical mediators of opioid reward; however, the specific glutamatergic neurons mediating the rewarding effects of opioids remains to be determined.Item In situ three-dimensional reconstruction of mouse heart sympathetic innervation by two-photon excitation fluorescence imaging(Elsevier, 2014-01-15) Freeman, Kim; Tao, Wen; Sun, Hongli; Soonpaa, Mark H.; Rubart, Michael; Department of Medicine, IU School of MedicineBackground Sympathetic nerve wiring in the mammalian heart has remained largely unexplored. Resolving the wiring diagram of the cardiac sympathetic network would help establish the structural underpinnings of neurocardiac coupling. New Method We used two-photon excitation fluorescence microscopy, combined with a computer-assisted 3-D tracking algorithm, to map the local sympathetic circuits in living hearts from adult transgenic mice expressing enhanced green fluorescent protein (EGFP) in peripheral adrenergic neurons. Results Quantitative co-localization analyses confirmed that the intramyocardial EGFP distribution recapitulated the anatomy of the sympathetic arbor. In the left ventricular subepicardium of the uninjured heart, the sympathetic network was composed of multiple subarbors, exhibiting variable branching and looping topology. Axonal branches did not overlap with each other within their respective parental subarbor nor with neurites of annexed subarbors. The sympathetic network in the border zone of a 2-week-old myocardial infarction was characterized by substantive rewiring, which included spatially heterogeneous loss and gain of sympathetic fibers and formation of multiple, predominately nested, axon loops of widely variable circumference and geometry. Comparison with Existing Methods In contrast to mechanical tissue sectioning methods that may involve deformation of tissue and uncertainty in registration across sections, our approach preserves continuity of structure, which allows tracing of neurites over distances, and thus enables derivation of the three-dimensional and topological morphology of cardiac sympathetic nerves. Conclusions Our assay should be of general utility to unravel the mechanisms governing sympathetic axon spacing during development and disease.