ER-PM Junctions on GABAergic Interneurons Are Organized by Neuregulin 2/VAP Interactions and Regulated by NMDA Receptors.

Neuregulins (NRGs) signal via ErbB receptors to regulate neural development, excitability, synaptic and network activity, and behaviors relevant to psychiatric disorders. Bidirectional signaling between NRG2/ErbB4 and NMDA receptors is thought to homeostatically regulate GABAergic interneurons in response to increased excitatory neurotransmission or elevated extracellular glutamate levels. Unprocessed proNRG2 forms discrete clusters on cell bodies and proximal dendrites that colocalize with the potassium channel Kv2.1 at specialized endoplasmic reticulum-plasma membrane (ER-PM) junctions, and NMDA receptor activation triggers rapid dissociation from ER-PM junctions and ectodomain shedding by ADAM10. Here, we elucidate the mechanistic basis of proNRG2 clustering at ER-PM junctions and its regulation by NMDA receptors. Importantly, we demonstrate that proNRG2 promotes the formation of ER-PM junctions by directly binding the ER-resident membrane tether VAP, like Kv2.1. The proNRG2 intracellular domain harbors two non-canonical, low-affinity sites that cooperatively mediate VAP binding. One of these is a cryptic and phosphorylation-dependent VAP binding motif that is dephosphorylated following NMDA receptor activation, thus revealing how excitatory neurotransmission promotes the dissociation of proNRG2 from ER-PM junctions. Therefore, proNRG2 and Kv2.1 can independently function as VAP-dependent organizers of neuronal ER-PM junctions. Based on these and prior studies, we propose that proNRG2 and Kv2.1 serve as co-regulated downstream effectors of NMDA receptors to homeostatically regulate GABAergic interneurons.

Developmental, neurochemical, and behavioral analyses of ErbB4 Cyt-1 knockout mice.

Neuregulins (NRGs) and their cognate neuronal receptor ERBB4, which is expressed in GABAergic and dopaminergic neurons, regulate numerous behaviors in rodents and have been identified as schizophrenia at-risk genes. ErbB4 transcripts are alternatively spliced to generate isoforms that either include (Cyt-1) or exclude (Cyt-2) exon 26, which encodes a cytoplasmic domain that imparts ErbB4 receptors the ability to signal via the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway. Although ErbB4 Cyt-1/2 isoforms have been studied in transfected cultured cells, their functions in vivo remain unknown. Here, we generated ErbB4-floxed (ErbB4-Cyt1fl/fl ) mice to investigate the effects of germline (constitutive) and conditional (acute) deletions of the Cyt-1 exon. Overall receptor mRNA levels remain unchanged in germline ErbB4 Cyt-1 knockouts (Cyt-1 KOs), with all transcripts encoding Cyt-2 variants. In contrast to mice lacking all ErbB4 receptor function, GABAergic interneuron migration and number are unaltered in Cyt-1 KOs. However, basal extracellular dopamine (DA) levels in the medial prefrontal cortex are increased in Cyt-1 heterozygotes. Despite these neurochemical changes, Cyt-1 heterozygous and homozygous mice do not manifest behavioral abnormalities previously reported to be altered in ErbB4 null mice. To address the possibility that Cyt-2 variants compensate for the lack of Cyt-1 during development, we microinjected an adeno-associated virus expressing Cre-recombinase (AAV-Cre) into the DA-rich ventral tegmental area of adult ErbB4-Cyt1fl/fl mice to acutely target exon 26. These conditional Cyt-1 KOs were found to exhibit behavioral abnormalities in the elevated plus maze and startle response, consistent with the idea that late exon 26 ablations may circumvent compensation by Cyt-2 variants. Taken together, our observations indicate that ErbB4 Cyt-1 function in vivo is important for DA balance and behaviors in adults.

Structural Similarities between Neuregulin 1-3 Isoforms Determine Their Subcellular Distribution and Signaling Mode in Central Neurons.

The Neuregulin (NRG) family of ErbB ligands is comprised of numerous variants originating from the use of different genes, alternative promoters, and splice variants. NRGs have generally been thought to be transported to axons and presynaptic terminals where they signal via ErbB3/4 receptors in paracrine or juxtacrine mode. However, we recently demonstrated that unprocessed pro-NRG2 accumulates on cell bodies and proximal dendrites, and that NMDAR activity is required for shedding of its ectodomain by metalloproteinases. Here we systematically investigated the subcellular distribution and processing of major NRG isoforms in rat hippocampal neurons. We show that NRG1 isotypes I and II, which like NRG2 are single-pass transmembrane proteins with an Ig-like domain, share the same subcellular distribution and ectodomain shedding properties. We furthermore show that NRG3, like CRD-NRG1, is a dual-pass transmembrane protein that harbors a second transmembrane domain near its amino terminus. Both NRG3 and CRD-NRG1 cluster on axons through juxtacrine interactions with ErbB4 present on GABAergic interneurons. Interestingly, although single-pass NRGs accumulate as unprocessed proforms, axonal puncta of CRD-NRG1 and NRG3 are comprised of processed protein. Mutations of CRD-NRG1 and NRG3 that render them resistant to BACE cleavage, as well as BACE inhibition, result in the loss of axonal puncta and in the accumulation of unprocessed proforms in neuronal soma. Together, these results define two groups of NRGs with distinct membrane topologies and fundamentally different targeting and processing properties in central neurons. The implications of this functional diversity for the regulation of neuronal processes by the NRG/ErbB pathway are discussed.SIGNIFICANCE STATEMENT Numerous Neuregulins (NRGs) are generated through the use of different genes, promoters, and alternative splicing, but the functional significance of this evolutionary conserved diversity remains poorly understood. Here we show that NRGs can be categorized by their membrane topologies. Single-pass NRGs, such as NRG1 Types I/II and NRG2, accumulate as unprocessed proforms on cell bodies, and their ectodomains are shed by metalloproteinases in response to NMDA receptor activation. By contrast, dual-pass CRD-NRG1 and NRG3 are constitutively processed by BACE and accumulate on axons where they interact with ErbB4 in juxtacrine mode. These findings reveal a previously unknown functional relationship between membrane topology, protein processing, and subcellular distribution, and suggest that single- and dual-pass NRGs regulate neuronal functions in fundamentally different ways.

ErbB4 reduces synaptic GABAA currents independent of its receptor tyrosine kinase activity.

ErbB4 signaling in the central nervous system is implicated in neuropsychiatric disorders and epilepsy. In cortical tissue, ErbB4 associates with excitatory synapses located on inhibitory interneurons. However, biochemical and histological data described herein demonstrate that the vast majority of ErbB4 is extrasynaptic and detergent-soluble. To explore the function of this receptor population, we used unbiased proteomics, in combination with electrophysiological, biochemical, and cell biological techniques, to identify a clinically relevant ErbB4-interacting protein, the GABAA receptor α1 subunit (GABAR α1). We show that ErbB4 and GABAR α1 are robustly coexpressed in hippocampal interneurons, and that ErbB4-null mice have diminished cortical GABAR α1 expression. Moreover, we characterize a Neuregulin-mediated ErbB4 signaling modality, independent of receptor tyrosine kinase activity, that couples ErbB4 to decreased postsynaptic GABAR currents on inhibitory interneurons. Consistent with an evolving understanding of GABAR trafficking, this pathway requires both clathrin-mediated endocytosis and protein kinase C to reduce GABAR inhibitory currents, surface GABAR α1 expression, and colocalization with the inhibitory postsynaptic protein gephyrin. Our results reveal a 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.

Neuregulin and dopamine modulation of hippocampal gamma oscillations is dependent on dopamine D4 receptors.

The neuregulin/ErbB signaling network is genetically associated with schizophrenia and modulates hippocampal γ oscillations--a type of neuronal network activity important for higher brain processes and altered in psychiatric disorders. Because neuregulin-1 (NRG-1) dramatically increases extracellular dopamine levels in the hippocampus, we investigated the relationship between NRG/ErbB and dopamine signaling in hippocampal γ oscillations. Using agonists for different D1- and D2-type dopamine receptors, we found that the D4 receptor (D4R) agonist PD168077, but not D1/D5 and D2/D3 agonists, increases γ oscillation power, and its effect is blocked by the highly specific D4R antagonist L-745,870. Using double in situ hybridization and immunofluorescence histochemistry, we show that hippocampal D4R mRNA and protein are more highly expressed in GAD67-positive GABAergic interneurons, many of which express the NRG-1 receptor ErbB4. Importantly, D4 and ErbB4 receptors are coexpressed in parvalbumin-positive basket cells that are critical for γ oscillations. Last, we report that D4R activation is essential for the effects of NRG-1 on network activity because L-745,870 and the atypical antipsychotic clozapine dramatically reduce the NRG-1-induced increase in γ oscillation power. This unique link between D4R and ErbB4 signaling on γ oscillation power, and their coexpression in parvalbumin-expressing interneurons, suggests a cellular mechanism that may be compromised in different psychiatric disorders affecting cognitive control. These findings are important given the association of a DRD4 polymorphism with alterations in attention, working memory, and γ oscillations, and suggest potential benefits of D4R modulators for targeting cognitive deficits.

The importance of the NRG-1/ErbB4 pathway for synaptic plasticity and behaviors associated with psychiatric disorders.

Neuregulin 1 (NRG-1) and its receptor ErbB4 have emerged as biologically plausible schizophrenia risk factors, modulators of GABAergic and dopaminergic neurotransmission, and as potent regulators of glutamatergic synaptic plasticity. NRG-1 acutely depotentiates LTP in hippocampal slices, and blocking ErbB kinase activity inhibits LTP reversal by theta-pulse stimuli (TPS), an activity-dependent reversal paradigm. NRG-1/ErbB4 signaling in parvalbumin (PV) interneurons has been implicated in inhibitory transmission onto pyramidal neurons. However, the role of ErbB4, in particular in PV interneurons, for LTP reversal has not been investigated. Here we show that ErbB4-null (ErbB4(-/-)) and PV interneuron-restricted mutant (PV-Cre;ErbB4) mice, as well as NRG-1 hypomorphic mice, exhibit increased hippocampal LTP. Moreover, both ErbB4(-/-) and PV-Cre;ErbB4 mice lack TPS-mediated LTP reversal. A comparative behavioral analysis of full and conditional ErbB4 mutant mice revealed that both exhibit hyperactivity in a novel environment and deficits in prepulse inhibition of the startle response. Strikingly, however, only ErbB4(-/-) mice exhibit reduced anxiety-like behaviors in the elevated plus maze task and deficits in cued and contextual fear conditioning. These results suggest that aberrant NRG-1/ErbB4 signaling in PV interneurons accounts for some but not all behavioral abnormalities observed in ErbB4(-/-) mice. Consistent with the observation that PV-Cre;ErbB4 mice exhibit normal fear conditioning, we find that ErbB4 is broadly expressed in the amygdala, largely by cells negative for PV. These findings are important to better understand ErbB4's role in complex behaviors and warrant further analysis of ErbB4 mutant mice lacking the receptor in distinct neuron types.

Transcytosis and trans-synaptic retention by postsynaptic ErbB4 underlie axonal accumulation of NRG3.

Neuregulins (NRGs) are EGF-like ligands associated with cognitive disorders. Unprocessed proNRG3 is cleaved by BACE1 to generate the mature membrane-bound NRG3 ligand, but the subcellular site of proNRG3 cleavage, mechanisms underlying its transport into axons, and presynaptic accumulation remain unknown. Using an optogenetic proNRG3 cleavage reporter (LA143-NRG3), we investigate the spatial-temporal dynamics of NRG3 processing and sorting in neurons. In dark conditions, unprocessed LA143-NRG3 is retained in the trans-Golgi network but, upon photoactivation, is cleaved by BACE1 and released from the TGN. Mature NRG3 then emerges on the somatodendritic plasma membrane from where it is re-endocytosed and anterogradely transported on Rab4+ vesicles into axons via transcytosis. By contrast, the BACE1 substrate APP is sorted into axons on Rab11+ vesicles. Lastly, by a mechanism we denote "trans-synaptic retention," NRG3 accumulates at presynaptic terminals by stable interaction with its receptor ErbB4 on postsynaptic GABAergic interneurons. We propose that trans-synaptic retention may account for polarized expression of other neuronal transmembrane ligands and receptors.

Neuregulin-1 regulates LTP at CA1 hippocampal synapses through activation of dopamine D4 receptors.

Neuregulin-1 (NRG-1) is genetically linked with schizophrenia, a neurodevelopmental cognitive disorder characterized by imbalances in glutamatergic and dopaminergic function. NRG-1 regulates numerous neurodevelopmental processes and, in the adult, suppresses or reverses long-term potentiation (LTP) at hippocampal glutamatergic synapses. Here we show that NRG-1 stimulates dopamine release in the hippocampus and reverses early-phase LTP via activation of D4 dopamine receptors (D4R). NRG-1 fails to depotentiate LTP in hippocampal slices treated with the antipsychotic clozapine and other more selective D4R antagonists. Moreover, LTP is not depotentiated in D4R null mice by either NRG-1 or theta-pulse stimuli. Conversely, direct D4R activation mimics NRG-1 and reduces AMPA receptor currents and surface expression. These findings demonstrate that NRG-1 mediates its unique role in counteracting LTP via dopamine signaling and opens future directions to study new aspects of NRG function. The novel functional link between NRG-1, dopamine, and glutamate has important implications for understanding how imbalances in Neuregulin-ErbB signaling can impinge on dopaminergic and glutamatergic function, neurotransmitter pathways associated with schizophrenia.

Selective expression of ErbB4 in interneurons, but not pyramidal cells, of the rodent hippocampus.

NRG1 and ERBB4 have emerged as some of the most reproducible schizophrenia risk genes. Moreover, the Neuregulin (NRG)/ErbB4 signaling pathway has been implicated in dendritic spine morphogenesis, glutamatergic synaptic plasticity, and neural network control. However, despite much attention this pathway and its effects on pyramidal cells have received recently, the presence of ErbB4 in these cells is still controversial. As knowledge of the precise locus of receptor expression is crucial to delineating the mechanisms by which NRG signaling elicits its diverse physiological effects, we have undertaken a thorough analysis of ErbB4 distribution in the CA1 area of the rodent hippocampus using newly generated rabbit monoclonal antibodies and ErbB4-mutant mice as negative controls. We detected ErbB4 immunoreactivity in GABAergic interneurons but not in pyramidal neurons, a finding that was further corroborated by the lack of ErbB4 mRNA in electrophysiologically identified pyramidal neurons as determined by single-cell reverse transcription-PCR. Contrary to some previous reports, we also did not detect processed ErbB4 fragments or nuclear ErbB4 immunoreactivity. Ultrastructural analysis in CA1 interneurons using immunoelectron microscopy revealed abundant ErbB4 expression in the somatodendritic compartment in which it accumulates at, and adjacent to, glutamatergic postsynaptic sites. In contrast, we found no evidence for presynaptic expression in cultured GAD67-positive hippocampal interneurons and in CA1 basket cell terminals. Our findings identify ErbB4-expressing interneurons, but not pyramidal neurons, as a primary target of NRG signaling in the hippocampus and, furthermore, implicate ErbB4 as a selective marker for glutamatergic synapses on inhibitory interneurons.

Correction to: NMDA Receptors Regulate Neuregulin 2 Binding to ER-PM Junctions and Ectodomain Release by ADAM10.

The original version of this article unfortunately contained an error in Title as the authors inadvertently deleted during revisions the last two words of the paper's title ("by ADAM10"), which is very important for explaining the article's content and impact.

NMDA Receptors Regulate Neuregulin 2 Binding to ER-PM Junctions and Ectodomain Release by ADAM10 [corrected].

Unprocessed pro-neuregulin 2 (pro-NRG2) accumulates on neuronal cell bodies at junctions between the endoplasmic reticulum and plasma membrane (ER-PM junctions). NMDA receptors (NMDARs) trigger NRG2 ectodomain shedding from these sites followed by activation of ErbB4 receptor tyrosine kinases, and ErbB4 signaling cell-autonomously downregulates intrinsic excitability of GABAergic interneurons by reducing voltage-gated sodium channel currents. NMDARs also promote dispersal of Kv2.1 clusters from ER-PM junctions and cause a hyperpolarizing shift in its voltage-dependent channel activation, suggesting that NRG2/ErbB4 and Kv2.1 work together to regulate intrinsic interneuron excitability in an activity-dependent manner. Here we explored the cellular processes underlying NMDAR-dependent NRG2 shedding in cultured rat hippocampal neurons. We report that NMDARs control shedding by two separate but converging mechanisms. First, NMDA treatment disrupts binding of pro-NRG2 to ER-PM junctions by post-translationally modifying conserved Ser/Thr residues in its intracellular domain. Second, using a mutant NRG2 protein that cannot be modified at these residues and that fails to accumulate at ER-PM junctions, we demonstrate that NMDARs also directly promote NRG2 shedding by ADAM-type metalloproteinases. Using pharmacological and shRNA-mediated knockdown, and metalloproteinase overexpression, we unexpectedly find that ADAM10, but not ADAM17/TACE, is the major NRG2 sheddase acting downstream of NMDAR activation. Together, these findings reveal how NMDARs exert tight control over the NRG2/ErbB4 signaling pathway, and suggest that NRG2 and Kv2.1 are co-regulated components of a shared pathway that responds to elevated extracellular glutamate levels.

Neuregulins and neuronal plasticity: possible relevance in schizophrenia.

Polymorphisms in the Neuregulin 1 (NRG1) and ErbB4 receptor genes have been associated with schizophrenia in numerous cohort and family studies, and biochemical measurements from postmortem prefrontal cortex homogenates suggest that NRG/ErbB signalling is altered in schizophrenia. Moreover, recent work from our group, and from others, indicates that NRG/ErbB signalling has a role in regulating glutamatergic transmission--an intriguing finding given that glutamatergic hypofunction has been proposed to be involved in the pathogenesis underlying schizophrenia. Here we will provide a brief background of the complexity of the NRG/ErbB signalling system. We will then focus on how NRG1 reverses (depotentiates) long-term potentiation (LTP) at hippocampal Schaeffer collateral--CA1 glutamatergic synapses in the adult brain. Specifically, we found that NRG1 depotentiates LTP in an activity- and time-dependent manner. A role of endogenous NRG for regulating plasticity at hippocampal synapses is supported by experiments demonstrating that ErbB receptor antagonists completely block LTP depotentiation by brief theta-pulse stimuli, a subthreshold stimulus paradigm that reverses LTP in live animals. Preliminary results indicate that NRG1-mediated LTP depotentiation is NMDA receptor independent, and manifests as an internalization of GluR1-containing AMPA receptors. The importance of the NRG/ ErbB signalling pathway in regulating homeostasis at glutamatergic synapses, and its possible implications for schizophrenia, will be discussed.

Neuregulin-1 reverses long-term potentiation at CA1 hippocampal synapses.

Neuregulin-1 (NRG-1) has been identified genetically as a schizophrenia susceptibility gene, but its function in the adult brain is unknown. Here, we show that NRG-1beta does not affect basal synaptic transmission but reverses long-term potentiation (LTP) at hippocampal Schaffer collateral-->CA1 synapses in an activity- and time-dependent manner. Depotentiation by NRG-1beta is blocked by two structurally distinct and selective ErbB receptor tyrosine kinase inhibitors. Moreover, ErbB receptor inhibition increases LTP at potentiated synapses and blocks LTP reversal by theta-pulse stimuli. NRG-1beta selectively reduces AMPA, not NMDA, receptor EPSCs and has no effect on paired-pulse facilitation ratios. Live imaging of hippocampal neurons transfected with receptors fused to superecliptic green fluorescent protein, as well as quantitative analysis of native receptors, show that NRG-1beta stimulates the internalization of surface glutamate receptor 1-containing AMPA receptors. This novel regulation of LTP by NRG-1 has important implications for the modulation of synaptic homeostasis and schizophrenia.

A negative feedback loop controls NMDA receptor function in cortical interneurons via neuregulin 2/ErbB4 signalling.

The neuregulin receptor ErbB4 is an important modulator of GABAergic interneurons and neural network synchronization. However, little is known about the endogenous ligands that engage ErbB4, the neural processes that activate them or their direct downstream targets. Here we demonstrate, in cultured neurons and in acute slices, that the NMDA receptor is both effector and target of neuregulin 2 (NRG2)/ErbB4 signalling in cortical interneurons. Interneurons co-express ErbB4 and NRG2, and pro-NRG2 accumulates on cell bodies atop subsurface cisternae. NMDA receptor activation rapidly triggers shedding of the signalling-competent NRG2 extracellular domain. In turn, NRG2 promotes ErbB4 association with GluN2B-containing NMDA receptors, followed by rapid internalization of surface receptors and potent downregulation of NMDA but not AMPA receptor currents. These effects occur selectively in ErbB4-positive interneurons and not in ErbB4-negative pyramidal neurons. Our findings reveal an intimate reciprocal relationship between ErbB4 and NMDA receptors with possible implications for the modulation of cortical microcircuits associated with cognitive deficits in psychiatric disorders.

Receptor tyrosine kinase ERBB4 mediates acquired resistance to ERBB2 inhibitors in breast cancer cells.

Approximately 25% of breast cancers overexpress and depend on the receptor tyrosine kinase ERBB2, one of 4 ERBB family members. Targeted therapies directed against ERBB2 have been developed and used clinically, but many patients continue to develop resistance to such therapies. Although much effort has been focused on elucidating the mechanisms of acquired resistance to ERBB2-targeted therapies, the involvement of ERBB4 remains elusive and controversial. We demonstrate that genetic ablation of ERBB4, but not ERBB1-3, led to apoptosis in lapatinib-resistant cells, suggesting that the efficacy of pan-ERBB inhibitors was, at least in part, mediated by the inhibition of ERBB4. Moreover, ERBB4 was upregulated at the protein level in ERBB2+ breast cancer cell lines selected for acquired lapatinib resistance in vitro and in MMTV-Neu mice following prolonged lapatinib treatment. Knockdown of ERBB4 caused a decrease in AKT phosphorylation in resistant cells but not in sensitive cells, suggesting that ERBB4 activated the PI3K/AKT pathway in lapatinib-resistant cells. Importantly, ERBB4 knockdown triggered apoptosis not only in lapatinib-resistant cells but also in trastuzumab-resistant cells. Our results suggest that although ERBB4 is dispensable for naïve ERBB2+ breast cancer cells, it may play a key role in the survival of ERBB2+ cancer cells after they develop resistance to ERBB2 inhibitors, lapatinib and trastuzumab.

Dopamine, cognitive function, and gamma oscillations: role of D4 receptors.

Cognitive deficits in individuals with schizophrenia (SCZ) are considered core symptoms of this disorder, and can manifest at the prodromal stage. Antipsychotics ameliorate positive symptoms but only modestly improve cognitive symptoms. The lack of treatments that improve cognitive abilities currently represents a major obstacle in developing more effective therapeutic strategies for this debilitating disorder. While D4 receptor (D4R)-specific antagonists are ineffective in the treatment of positive symptoms, animal studies suggest that D4R drugs can improve cognitive deficits. Moreover, recent work from our group suggests that D4Rs synergize with the neuregulin/ErbB4 signaling pathway, genetically identified as risk factors for SCZ, in parvalbumin (PV)-expressing interneurons to modulate gamma oscillations. These high-frequency network oscillations correlate with attention and increase during cognitive tasks in healthy subjects, and this correlation is attenuated in affected individuals. This finding, along with other observations indicating impaired GABAergic function, has led to the idea that abnormal neural activity in the prefrontal cortex (PFC) in individuals with SCZ reflects a perturbation in the balance of excitation and inhibition. Here we review the current state of knowledge of D4R functions in the PFC and hippocampus, two major brain areas implicated in SCZ. Special emphasis is given to studies focusing on the potential role of D4Rs in modulating GABAergic transmission and to an emerging concept of a close synergistic relationship between dopamine/D4R and neuregulin/ErbB4 signaling pathways that tunes the activity of PV interneurons to regulate gamma frequency network oscillations and potentially cognitive processes.

Acute neuregulin-1 signaling influences AMPA receptor mediated responses in cultured cerebellar granule neurons.

Neuregulin-1 (NRG1) is a trophic and differentiation factor that signals through ErbB receptor tyrosine kinases to regulate nervous system development. Previous studies have demonstrated that NRG1 affects plasticity at glutamatergic synapses in principal glutamatergic neurons of the hippocampus and frontal cortex; however, immunohistochemical and genetic analyses strongly suggest these effects are indirect and mediated via ErbB4 receptors on GABAergic interneurons. Here, we used cultured cerebellar granule cells (CGCs) that express ErbB4 to analyze the cell-autonomous effects of NRG1 stimulation on glutamatergic function. These cultures have the advantage that they are relatively homogenous and consist primarily of granule neurons that express ErbB4. We show that acute NRG1 treatment does not affect whole-cell AMPA or NMDA receptor (NMDAR) mediated currents in CGCs at 10-12 days in vitro. NRG1 also does not affect the frequency or amplitude of spontaneous AMPAR or NMDAR mediated miniature excitatory post-synaptic currents (mEPSCs). To further investigate the effects of NRG1 on activity-dependent plasticity of glutamatergic synapses in CGCs, we characterized the effects of high-glyine/0 Mg(2+) (which activates synaptic NMDARs) on AMPAR-mEPSC frequency and amplitude. We show that high-glycine induces a form of chemical long-term potentiation (chemLTP) in CGCs characterized by an increase in AMPAR-mEPSC frequency but not amplitude. Moreover, NRG1 induces a decrease in AMPAR-mEPSC frequency following chemLTP, but does not affect AMPAR-mEPSC amplitude. CGCs in our cultures conditions express low levels of GluR1, in contrast to dissociated hippocampal cultures, but do express the long isoform of GluR4. This study provides first evidence that (1) high-glycine can induce plasticity at glutamatergic synapses in CGCs, and (2) that acute NRG1/ErbB-signaling can regulate glutamatergic plasticity in CGCs. Taken together with previous reports, our results suggest that, similar to Schaeffer collateral to CA1 synapses, NRG1 effects are activity dependent and mediated via modulation of synaptic AMPARs.

Conserved interneuron-specific ErbB4 expression in frontal cortex of rodents, monkeys, and humans: implications for schizophrenia.

BACKGROUND: Neuregulin-1 and ErbB4 are genetically associated with schizophrenia, and detailed knowledge of the cellular and subcellular localization of ErbB4 is important for understanding how neuregulin-1 regulates neuronal network activity and behavior. Expression of ErbB4 is restricted to interneurons in the rodent hippocampus and cortex. However, controversy remains about the cellular expression pattern in primate brain and its subcellular distribution in postsynaptic somatodendritic locations versus presynaptic terminals. METHODS: ErbB4 expression was analyzed in pyramidal cells and interneurons in the frontal cortex of five species: C57BL6 mice (n = 3), ErbB4⁻/⁻ mice (n = 2), Sprague-Dawley rats (n = 3), two macaque species (n = 3 + 2), and humans (normal control subjects, n = 2). We investigated 1) messenger RNA in mice, macaques, and humans; 2) protein expression in all species using highly specific monoclonal antibodies; and 3) specificity tests of several ErbB4 antibodies on brain samples (mouse, macaque, human). RESULTS: ErbB4 RNA is restricted to interneurons in the frontal cortex of mice. ErbB4 protein is undetectable in pyramidal cells of rodents, macaques, and human frontal cortex, whereas most interneurons positive for parvalbumin, calretinin, or cholecystokinin, but only a minority of calbindin-positive cells, co-express ErbB4 in macaques. Importantly, no presynaptic ErbB4 expression was detected in any species. CONCLUSIONS: The interneuron-selective somatodendritic expression of ErbB4 is consistent with a primary role of neuregulin-ErbB4 signaling in the postsynaptic modulation of gamma-aminobutyric acidergic function in rodents and primates. Our data validate the use of rodents to analyze effects of abnormal ErbB4 function as a means to model endophenotypes of psychiatric disorders.

Neuregulin links dopaminergic and glutamatergic neurotransmission to control hippocampal synaptic plasticity.

Neuregulin-1 (NRG-1) and its receptor ErbB4 are genetically associated with schizophrenia, a complex developmental disorder of high heritability but unknown etiology that has been proposed to result from deficits in functional connectivity and synaptic plasticity. Based on pharmacological evidence, imbalances in dopaminergic and glutamatergic transmission systems are believed to contribute to its pathophysiology, but genetic data supporting a causative role for either are sparse. Stimulation of NRG-1/ErbB4 signaling inhibits or reverts hippocampal long-term potentiation (LTP) at glutamatergic synapses between Schaeffer collateral afferents and CA1 pyramidal neurons (SC-->CA1). We have recently demonstrated that NRG-1 regulates glutamatergic plasticity by rapidly increasing extracellular hippocampal dopamine levels and activation of D4 dopamine receptors.7 These new findings position the NRG-1/ErbB4 signaling pathway at the crossroads between dopaminergic and glutamatergic neurotransmission and offer novel ways to consolidate genetic, functional and pharmacological data toward a better understanding of the etiological processes underlying schizophrenia, and the role of NRG-1 for normal synaptic function and plasticity. The currently available data suggest that hippocampal interneurons might play a crucial role in mediating NRG-1 induced depotentiation. This interpretation is in line with other evidence pointing towards an involvement of GABAergic cells in the etiology of schizophrenia.

Transcription factor TEAD4 specifies the trophectoderm lineage at the beginning of mammalian development.

Specification of cell lineages in mammals begins shortly after fertilization with formation of a blastocyst consisting of trophectoderm, which contributes exclusively to the placenta, and inner cell mass (ICM), from which the embryo develops. Here we report that ablation of the mouse Tead4 gene results in a preimplantation lethal phenotype, and TEAD4 is one of two highly homologous TEAD transcription factors that are expressed during zygotic gene activation in mouse 2-cell embryos. Tead4(-/-) embryos do not express trophectoderm-specific genes, such as Cdx2, but do express ICM-specific genes, such as Oct4 (also known as Pou5f1). Consequently, Tead4(-/-) morulae do not produce trophoblast stem cells, trophectoderm or blastocoel cavities, and therefore do not implant into the uterine endometrium. However, Tead4(-/-) embryos can produce embryonic stem cells, a derivative of ICM, and if the Tead4 allele is not disrupted until after implantation, then Tead4(-/-) embryos complete development. Thus, Tead4 is the earliest gene shown to be uniquely required for specification of the trophectoderm lineage.