UC Berkeley

Neuromodulators are key signaling molecules in the nervous system that regulate neuronal activity by modulating intrinsic excitability, synaptic transmission, and plasticity. The largest known class of neuromodulators are neuropeptides. Traditionally, neurons have been defined primarily by the neurotransmitter they release (i.e. glutamatergic or GABAergic); however, recent single cell RNA sequencing studies have revealed that many neurons also express genes encoding for one or more neuropeptides and/or neuropeptide receptors. Gastrin-releasing peptide (GRP), a bombesin-like neuropeptide, is one such neuropeptide that has mainly been studied in the spinal cord in the context of itch behavior. Our lab previously showed that Grp mRNA is also expressed in the midbrain, in about a third of dopamine (DA) neurons. My research goal was to further explore these Grp-expressing neurons and determine whether a functional GRP/GRPR sub-circuit exists within the basal ganglia.I first used fluorescent in situ hybridization (FISH) to probe for Grpr, which encodes gastrin releasing peptide receptor (GRPR), in the targets of the Grp-expressing DA neurons. I found that Grpr was expressed by specific populations of neurons in the striatum and nucleus accumbens medial shell (NAc MSh). This complementary expression pattern suggests that GRP/GRPR signaling may define novel peptidergic sub-circuits within the basal ganglia. For the purpose of this project, I chose to focus on the Grpr-expressing neurons in the NAc MSh. I performed additional FISH experiments to genetically characterize the Grpr-expressing cells in the context of the basal ganglia. I determined that the Grpr+ cells appeared to be a small subpopulation of GABA-ergic indirect pathway striatal projection neurons (iSPNs). Morphology analyses supported their SPN identity. Next, I performed whole-cell recordings on these cells to determine their electrophysiological properties and intrinsic excitability using a GRPR-eGFP reporter mouse. While genetically and morphologically the GRPR+ cells appeared to be SPNs, their electrophysiological properties were distinct from other types of SPNs. Mainly, a majority of GRPR+ cells were spontaneously active at baseline, they were more depolarized, had higher membrane resistance, and had wider action potentials than 1 SPNs. Their properties were also distinct from neighboring cholinergic interneurons in the NAc MSh. The higher intrinsic excitability and activity of GRPR+ cells was confirmed with FISH where I found that Grpr+ cells were approximately twice as likely to be Fos+ compared to Grpr- cells. I next investigated whether there were other sources of GRP into the NAc mSh, in addition to the previously identified Grp+ DA neurons from the ventral tegmental area. I injected a retrograde tracer into the NAc MSh and probed for expression of Grp mRNA in synaptically connected neurons using FISH. I found that a significant number of the Grp+ inputs into the NAc MSh arise from glutamatergic neurons projecting from the hippocampus as well as the basolateral amygdala. After identifying receptor expression and mapping the GRP inputs to the NAc MSh, I characterized how the GRPR-expressing cells in the NAc MSh respond to GRP both in slice and in vivo. In slice, GRP wash-on led to depolarization and an approximately 140% increase in membrane resistance, effects that could be blocked with a GRPR-specific antagonist. GRP injections in vivo induced Fos expression selectively in Grpr+ cells in the injected hemisphere but not the un-injected hemisphere. Having characterized the GRPR-expressing cells in the NAc MSh, I probed the function of GRP/GRPR signaling in the circuit. To do so, I generated Grpr knock-out (KO) mice and ran them through behavioral tasks known to involve the NAc MSh: progressive ratio lever pressing, accelerating rotarod, and sucrose preference. I also ran the mice through the open field to assess general locomotor activity and spontaneous behaviors. In the open field, mice lacking Grpr travelled significantly less total distance and displayed different behavioral syllable usage identified by motion sequencing (MoSeq), suggesting that disruption of GRPR-signaling globally affects mouse behavior patterns. I saw no consistent changes in the progressive ratio task, indicating that global disruption of GRP- GRPR signaling does not affect motivation. In the accelerating rotarod, however, Grpr KO mice had better performance, but showed no differences in learning, indicating that GRP- GRPR signaling is involved in motor coordination. In the sucrose preference test, I found that Grpr KO mice displayed less of a preference, indicating that GRP signaling may be involved in detecting positive valence and “liking” sensation. Overall, I have identified and characterized a novel subpopulation of iSPNs in the NAc MSh defined by their expression of GRPR. Furthermore, I mapped the inputs to the NAc MSh that express Grp and have shown both in slice and in vivo that GRPR+ cells in the NAc MSh respond to GRP. Finally, I have shown how disruption of GRP/GRPR signaling leads to behavioral changes in NAc MSh-dependent behaviors. Together, my dissertation research has characterized a novel peptidergic circuit in the NAc MSh defined by GRP/GRPR expression that is functionally relevant in NAc MSh-dependent behavior.