1. Importance of Neuregulin/ErbB4 signaling in parvalbumin-positive fast-spiking interneurons: Using neurochemistry and electrophysiology, we showed that NRG-ErbB4 activation inhibits and reverses LTP by triggering dopamine (DA) release and activating D4 receptors (D4Rs). Because ErbB4 is not expressed in pyramidal neurons but is highly expressed in GABAergic interneurons, especially fast-spiking parvalbumin (PV)-positive basket cells, we compared the effects of full vs. PV interneuron-specific ErbB4 ablation using knockout (KO) mice. Interestingly, NRG-mediated reversal of LTP was absent in both ErbB4 full and PV-specific KO mice. We also compared these two ErbB4 mutant mouse strains in a battery of behavioral tests relevant for psychiatric disorders and found that PV-restricted mutants replicated many, albeit not all, abnormalities observed in full ErbB4 mutant mice. These findings highlight the role of PV interneurons as a critical nexus of NRG/ErbB4 signaling, and provide further evidence in support of a possible contribution of this pathway, genetically linked with increased risk for schizophrenia, to the pathophysiology underlying psychiatric disorders. 2. NRG/ErbB4 and dopamine D4 receptor signaling converge in PV neurons and regulate gamma oscillations: NRG/ErbB4 signaling modulates the power of kainate-induced hippocampal gamma oscillations. Because NRG1 dramatically increases extracellular dopamine levels in the hippocampus, we investigated the relationship between NRG/ErbB and dopamine signaling in hippocampal gamma oscillations. We used selective agonists for different dopamine G-coupled receptors that increase (D1Rs and D5Rs) or decrease (D2R, D3R and D4Rs) the synthesis of cAMP, and found that only an agonist specific for D4Rs (PD168077) augmented the power of gamma oscillations. By contrast, agonists for D1/D5Rs and D2R/D3Rs were without effect. The effects of PD168077 on increasing gamma power were blocked the D4R antagonist L-745,870, further stressing the importance of this receptor for neuronal network activity. Importantly, L-745,870 and clozapine, an antipsychotic that preferentially targets D4Rs, also blocked increases in gamma oscillation power by NRG1. Importantly, we found that D4Rs and ErbB4 are coexpressed in PV+ fast-spiking basket cells, an interneuron subtype critically important for regulating gamma oscillations. This novel cross-talk between D4R and ErbB4 signaling to augment gamma oscillation power in PV+ interneurons suggest a cellular mechanism possibly compromised in psychiatric disorders exhibiting cognitive deficits. 3. Neuregulin directly decreases voltage-gated sodium currents in hippocampal ErbB4-expresssing interneurons: A major goal is to investigate if and how NRG1 directly affects the intrinsic properties of ErbB4-positive (ErbB4+) interneurons. To this end, we resorted to using dissociated hippocampal cultures, which are devoid of all hippocampal-projecting afferent connections, and to blocking all synaptic activity pharmacologically. We found that NRG1 decreases firing of ErbB4+ but not ErbB4- neurons by shifting the action potential threshold. These effects are primarily attributable to decreased voltage-gated sodium channel activity, as current density was attenuated by 60% after 20 minutes of NRG1 treatment. This was the first study to identify direct actions of NRG1 on voltage-gated sodium channel function in ErbB4-expressing interneurons, thereby offering novel insights into how NRG1/ErbB4 signaling modulates GABAergic interneuron activity and excitatory/inhibitory balance. 4. NRG1 signaling regulates AMPA receptor (AMPAR) activity in glutamatergic ErbB4-expressing cerebellar granule cells (CGCs): With Dr. Fenster, we found that in baseline conditions NRG1 does not affect whole-cell AMPAR or NMDAR mediated currents, nor the frequency or amplitude of spontaneous NMDAR- or AMPAR-mediated miniature excitatory post-synapttic currents, CGCs grown for 10-12 days in vitro. However, opening of NMDARs by exogenous glycine induces a """"""""chemical potentiation"""""""" (chemLTP) in CGCs characterized by an increase in AMPAR-mEPSC frequency that is decreased by NRG1 treatment. Because in our culture conditions CGCs express very low levels of the AMPAR GluA1 subunit but high levels of GluA4, our data suggest that the NRG1 effect could be mediated via GluA4 subunits. This study shows for the first time that high-glycine can induce plasticity at glutamatergic synapses in CGCs that is regulated by NRG1. Our results suggest that, similar to SC-CA1 synapses, NRG1 effects on CGCs are activity dependent and mediated via modulation of synaptic AMPARs. 5. Functional analysis of the extrasynaptic ErbB4 receptor proteome using proteomics approach: Our laboratory has used, in collaboration with S. Markey's group, biochemical and immunological approaches to identify novel proteins that interact with extrasynaptic ErbB4, a pool constituting 80-to 90% of total cortical and hippocampal receptor that has gone mostly unstudied. Using a novel rabbit monoclonal ErbB4 antibody (mAb10) to immunoprecipitate (IP) receptor from metabolically active cortical synaptosome preparations, we employed liquid chromatographytandem mass spectrometry (LC/MS/MS)-based proteomics to determine the proteome composition of ErbB4 complexes. The specificity of this approach was confirmed by comparing the proteins identified in IPs performed from wild-type vs. ErbB4 knockout mice. Among the receptors, signaling proteins and cytoskeletal components IPed selectively by mAb10 and identified by LC/MS/MS (including ErbB4), we identified the the GABAA receptor alpha1 subunit (GABARa1). 6. ErbB4 reduces synaptic GABAA currents independent of its receptor tyrosine kinase activity: Using the aforementioned unbiased approach of ErbB4 IP combined with LC/MS/MS, we identified a novel and clinically relevant ErbB4-interacting protein - the GABARa1. Co-IP and double-immunofluorescence experiments, using GABARa1- and ErbB4-specific antibodies, indicate that both proteins interact in GABAergic interneurons in vivo and in dissociated cultures. Using a combination of electrophysiological, biochemical and cell biological techniques, we identified a novel NRG-mediated ErbB4 signaling pathway that couples ErbB4 to decreased postsynaptic GABAR currents onto inhibitory interneurons. Remarkably, we found that this novel ErbB4 signaling pathway, which decreases postsynaptic GABAR currents on GABAergic interneurons, acts independently of ErbB4s canonical receptor tyrosine kinase activity. We demonstrate inhibition of ErbB4's tyrosine kinase activity by the selective antagonist PD158780, which we previously demonstrated totally blocks the effects of NRG on plasticity and sodium channel currents, in this case fails to block the effects of NRG on reducing GABARa1-mediated currents. Cultured neurons prepared from ErbB4 knockout mice do not down-regulate GABARa1 receptor-mediated currents in response to NRG, but can do so if rescued by infection with AAV viruses expressing ErbB4 harboring a point mutation that inactivates its canonical tyrosine kinase activity. While the effects of NRG on GABARa1 internalization do not require ErbB4 receptor tyrosine kinase activity, both clathrin-mediated endocytosis and protein kinase C are necessary to reduce GABAR inhibitory currents and synaptic alpha 1. Our results reveal a new function of ErbB4, independent of its tyrosine kinase activity, that modulates postsynaptic inhibitory control of hippocampal interneurons and may provide a novel pharmacological target in the treatment of neuropsychiatric disorders and epilepsy.
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