1. Conservation of ErbB4 expression in GABAergic interneurons in the hippocampus and cortex, from rodents to primates: Knowledge of the cellular and subcellular localization of ErbB4 is important for understanding how Neuregulin (NRG) regulates neuronal network activity and behavior. In collaboration with Dr. McBain's and Dr. Lewis's lab, we unequivocally demonstrated, using a combination of single-cell RT-PCR and electrophysiological profiling in mice, that ErbB4 transcripts are expressed GABAergic interneurons and not in excitatory pyramidal cells. A finding confirmed with our ErbB4 mAb that showed receptor accumulates in the somato-dendritic region of cortical GABAergic interneurons in rodents, rhesus monkey and humans;presynaptic or axonal expression of ErbB4 was rarely detected. This across-species conservation validates the use of rodents to analyze cellular and neural circuit effects of abnormal ErbB4 function as a means to model endophenotypes pertinent to psychiatric disorders. 2. Importance of Neuregulin/ErbB4 signaling in parvalbumin-positive fast-spiking interneurons: We previously showed, using reverse-microdialysis neurochemistry and electrophysiology, that NRG1 signaling via ErbB4 potently triggers dopamine release in the hippocampus and reverses LTP at Schaeffer collateral-to-CA1 (SC-CA1) synapses through activation of dopamine D4 receptors. Importantly, we found that ErbB4 is not detectable in excitatory pyramidal neurons but is abundantly expressed in GABAergic interneurons, in particular fast-spiking parvalbumin-positive basket cells. These observations strongly suggested that effects of NRG/ErbB4 on glutamatergic synaptic plasticity are indirect. To address this question directly, we compared the effects of full vs. parvalbumin (PV) interneuron-specific Erbb4 ablation in genetically engineered mice. As expected, NRG-mediated inhibition of LTP induction and reversal of early-phase LTP were absent in full ErbB4 KO mice. Interestingly, ErbB4 ablation in PV interneurons was sufficient to reproduce the effects of the full ErbB4 knockout. 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 support a possible contribution of this pathway, genetically linked with increased risk for schizophrenia, to the pathophysiology underlying psychiatric disorders. 3. NRG/ErbB4 and dopamine D4 receptor signaling converge in PV neurons and regulate gamma oscillations: We previously reported that 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. In collaboration with Dr.Fisahns lab, we used selective agonists for different dopamine G-coupled receptors that increase (D1 and D5 receptors) or decrease (D2, D3 and D4 receptors) the synthesis of cAMP, and found that only an agonist specific for D4 receptors (PD168077) augmented the power of gamma oscillations. By contrast, agonists for D1/D5 and D2R/D3 receptors were without effect. The increase in gamma oscillation power induced by PD168077 was totally blocked by a highly specific D4 receptor antagonist (L-745,870), further stressing the importance of this receptor for neuronal network activity. Importantly, we found that L-745,870 and clozapine, an antipsychotic that preferentially targets D4 receptors, also blocked increases in gamma oscillation power by NRG1. Using double in situ hybridization (in collaboration with Dr. U. Winzer-Serhan) and immunofluorescence histochemistry, we showed that hippocampal D4 receptor mRNA and protein are highest in a subset of GABAergic interneurons. Importantly, D4 and ErbB4 receptors are coexpressed in PV+ fast-spiking basket cells, a type of somatic-targeting GABAergic interneurons that are critically important for regulating gamma oscillations. This novel cross-talk between D4 and ErbB4 receptor signaling to augment gamma oscillation power, and their coexpression in PV-positive interneurons, suggest a cellular mechanism that may be compromised in different psychiatric disorders affecting cognitive control. 4. Neuregulin directly decreases voltage-gated sodium currents in hippocampal ErbB4-expresssing interneurons: One of our primary goals has been to investigate if and how NRG1 may directly affect 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 initially analyzed the effects of NRG1 on the excitability of ErbB4+ and ErbB4- neurons, identified in live-labeling by an ectodomain-targeting antibody, using whole-cell current-clamp recordings. We found that NRG1 decreased firing of ErbB4+ but not ErbB4- neurons by shifting the action potential threshold. Using voltage-clamp recordings, we determined that 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 can directly impact GABAergic interneuron activity and potentially affect excitatory/inhibitory balance. 5. NRG1 signaling directly influences AMPA receptor (AMPAR) activity in glutamatergic ErbB4-expressing cerebellar granule cells (CGCs): Previous studies demonstrated how NRG1 indirectly affects plasticity at glutamatergic synapses in principal glutamatergic neurons in the hippocampus and frontal cortex. We analyzed the effects of NRG1 on developing cultured CGCs in collaboration with Dr. C. Fenster. These cultures consist predominantly of granule neurons that express ErbB4. We found that 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-synaptic currents, in baseline conditions of CGCs grown for 10-12 days in vitro. However, we found that high-glycine induces a form of chemical potentiation (chemLTP) in CGCs characterized by an increase in AMPAR-mEPSC frequency that is decreased by NRG1 treatment. Our data suggest that the NRG1 effect is mediated via GluA4 subunits in CGCs. This study for the first time shows that high-glycine can induce plasticity at glutamatergic synapses in CGCs, and that acute NRG1/ErbB signaling can regulate glutamatergic plasticity in CGCs in a fashion similar to SC-CA1 synapses. 6. Proteomics approach to understanding NRG/ErbB4 proximal signaling targets: To discover potentially novel and direct biological targets of NRG/ErbB4, in collaboration with Dr. S. Markeys group (NIMH), we are employing an unbiased liquid chromatography-tandem mass spectrometry (LC/MS/MS) proteomics approach to determine the composition of the ErbB4 receptor proteome. We recently reported validation of using our receptor mAb for affinity purification ErbB4 complexes and their analysis by LC/MS/MS. We are using a similar approach to isolate endogenous ErbB4 complexes from metabolically active synaptosomes following NRG-1 stimulation. Using this approach, we discovered that NRG/ErbB4 signaling alters the ion channel composition of cellular microdomains associated with ErbB4. Our goal is to determine the functional consequences of these alterations.

Project Start
Project End
Budget Start
Budget End
Support Year
23
Fiscal Year
2012
Total Cost
$1,384,153
Indirect Cost
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State
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Yan, L; Shamir, A; Skirzewski, M et al. (2017) Neuregulin-2 ablation results in dopamine dysregulation and severe behavioral phenotypes relevant to psychiatric disorders. Mol Psychiatry :
Skirzewski, M; Karavanova, I; Shamir, A et al. (2017) ErbB4 signaling in dopaminergic axonal projections increases extracellular dopamine levels and regulates spatial/working memory behaviors. Mol Psychiatry :
Vullhorst, Detlef; Ahmad, Tanveer; Karavanova, Irina et al. (2017) Structural Similarities between Neuregulin 1-3 Isoforms Determine Their Subcellular Distribution and Signaling Mode in Central Neurons. J Neurosci 37:5232-5249
Canfield, Kaleigh; Li, Jiaqi; Wilkins, Owen M et al. (2015) Receptor tyrosine kinase ERBB4 mediates acquired resistance to ERBB2 inhibitors in breast cancer cells. Cell Cycle 14:648-55
Vullhorst, Detlef; Mitchell, Robert M; Keating, Carolyn et al. (2015) A negative feedback loop controls NMDA receptor function in cortical interneurons via neuregulin 2/ErbB4 signalling. Nat Commun 6:7222
Padmakumar, Vc; Masiuk, Katelyn E; Luger, Dror et al. (2014) Detection of differential fetal and adult expression of chloride intracellular channel 4 (CLIC4) protein by analysis of a green fluorescent protein knock-in mouse line. BMC Dev Biol 14:24
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Mitchell, Robert M; Janssen, Megan J; Karavanova, Irina et al. (2013) ErbB4 reduces synaptic GABAA currents independent of its receptor tyrosine kinase activity. Proc Natl Acad Sci U S A 110:19603-8
Furth, Katrina E; Mastwal, Surjeet; Wang, Kuan H et al. (2013) Dopamine, cognitive function, and gamma oscillations: role of D4 receptors. Front Cell Neurosci 7:102

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