Although neurotrophins are primarily associated with long-term effects on neuronal survival and differentiation, recent studies have shown that acute changes in synaptic transmission can also be produced. In the hippocampus, an area critically involved in learning and memory, we have found that brain-derived neurotrophic factor (BDNF) rapidly enhanced synaptic efficacy through a previously unreported mechanism-increased postsynaptic responsiveness via a phosphorylation-dependent pathway. Within minutes of BDNF application to cultured hippocampal neurons, spontaneous firing rate was dramatically increased, as were the frequency and amplitude of excitatory postsynaptic currents. The increased frequency of postsynaptic currents resulted from the change in presynaptic firing. However, the increased amplitude was postsynaptic in origin because it was selectively blocked by intracellular injection of the tyrosine kinase receptor (Ntrk2/TrkB) inhibitor K-252a and potentiated by injection of the phosphatase inhibitor okadaic acid. These results suggest a role for BDNF in the modulation of synaptic transmission in the hippocampus.Neurotrophins are important regulators of the survival, development, and differentiation of multiple neuronal populations (1-3). These effects generally occur over the course of hours or even days. Recently, evidence has accumulated that neurotrophins can also modulate transmitter release and synaptic transmission (4-8). Such effects could underly a role for these factors in synaptic plasticity. The potential effects of neurotrophins on synaptic transmission in the hippocampus were studied with primary cultures of embryonic hippocampal neurons. We report that brain-derived neurotrophic factor (BDNF) rapidly enhanced the strength of synaptic connections in these neurons. Both the amplitude and the frequency of excitatory postsynaptic currents (EPSCs) were increased within 2-3 min of neurotrophin application. Moreover, the potentiation of synaptic current amplitude occurred via a previously undescribed postsynaptic mechanism in which BDNF increased the responsiveness of the postsynaptic neuron to the excitatory input. These results indicate a previously unreported role for this neurotrophin in the hippocampus. MATERIALS AND METHODS

Rho GTPases activated by GDP/GTP exchange factors (GEFs) play key roles in the developing and adult nervous system. Kalirin-7 (Kal7), the predominant adult splice form of the multifunctional Kalirin RhoGEF, includes a PDZ [postsynaptic density-95 (PSD-95)/Discs large (Dlg)/zona occludens-1 (ZO-1)] binding domain and localizes to the postsynaptic side of excitatory synapses. In vitro studies demonstrated that overexpression of Kal7 increased dendritic spine density, whereas reduced expression of endogenous Kal7 decreased spine density. To evaluate the role of Kal7 in vivo, mice lacking the terminal exon unique to Kal7 were created. Mice lacking both copies of the Kal7 exon (Kal7 KO ) grew and reproduced normally. Golgi impregnation and electron microscopy revealed decreased hippocampal spine density in Kal7 KO mice. Behaviorally, Kal7 KO mice showed decreased anxiety-like behavior in the elevated zero maze and impaired acquisition of a passive avoidance task, but normal behavior in open field, object recognition, and radial arm maze tasks. Kal7 KO mice were deficient in hippocampal long-term potentiation. Western blot analysis confirmed the absence of Kal7 and revealed compensatory increases in larger Kalirin isoforms. PSDs purified from the cortices of Kal7 KO mice showed a deficit in Cdk5, a kinase known to phosphorylate Kal7 and play an essential role in synaptic function. The early stages of excitatory synaptic development proceeded normally in cortical neurons prepared from Kal7 KO mice, with decreased excitatory synapses apparent only after 21 d in vitro. Expression of exogenous Kal7 in Kal7 KO neurons rescued this deficit. Kal7 plays an essential role in synaptic structure and function, affecting a subset of cognitive processes.

Angelman syndrome (AS) and Prader-Willi syndrome (PWS) are neurodevelopmental disorders of genomic imprinting. AS results from loss of function of the ubiquitin protein ligase E3A (UBE3A) gene, whereas the genetic defect in PWS is unknown. Although induced pluripotent stem cells (iPSCs) provide invaluable models of human disease, nuclear reprogramming could limit the usefulness of iPSCs from patients who have AS and PWS should the genomic imprint marks be disturbed by the epigenetic reprogramming process. Our iPSCs derived from patients with AS and PWS show no evidence of DNA methylation imprint erasure at the cis-acting PSW imprinting center. Importantly, we find that, as in normal brain, imprinting of UBE3A is established during neuronal differentiation of AS iPSCs, with the paternal UBE3A allele repressed concomitant with up-regulation of the UBE3A antisense transcript. These iPSC models of genomic imprinting disorders will facilitate investigation of the AS and PWS disease processes and allow study of the developmental timing and mechanism of UBE3A repression in human neurons.antisense transcript | epigenetic | neuronal differentiation A ngelman syndrome (AS) is a neurogenetic disorder characterized by profound intellectual disability, absent speech, frequent seizures, motor dysfunction, and a characteristic happy demeanor (1, 2). Prader-Willi syndrome (PWS) is characterized hyperphagia/obesity; small stature, hands, and feet; and behavioral problems that are likened to obsessive compulsive disorder (3). AS is caused by loss of function of the maternally inherited allele of the E3 ubiquitin ligase UBE3A. UBE3A is subject to tissuespecific genomic imprinting; although both alleles are expressed in most tissues, the paternally inherited allele is repressed in the brain (4-6). Imprinted expression of UBE3A is thought to occur as a result of reciprocal expression of a long noncoding antisense transcript, UBE3A-ATS, which is part of a >600-kb transcript initiating at the differentially methylated PWS imprinting center (IC) located in exon 1 of the SNURF-SNRPN gene (7-9). PWS is associated with the loss of several species of small nucleolar RNAs (snoRNAs) (10); however, its genetic basis is currently unknown, and there is no mouse model that recapitulates all features of PWS.Mouse models of AS have proved significant in studying important aspects of the AS disease mechanism. There are, however, differences in the tissue specificity of the transcript that harbors UBE3A-ATS between humans and mice (11), indicating that the timing and mechanism of UBE3A repression may diverge between these species. The ability to study the developmental timing and mechanism of brain-specific repression of the paternal UBE3A allele in a model of human development is critical for better understanding the AS disease process and for using live neurons from patients with AS to discover previously undescribed therapeutic interventions. Here, we have developed such a model via human induced pluripotent stem cell (iPSC)-technology. ResultsHu...

Neurotrophins (NTs) have recently been found to regulate synaptic transmission in the hippocampus. Whole-cell and single-channel recordings from cultured hippocampal neurons revealed a mechanism responsible for enhanced synaptic strength. Specifically, brain-derived neurotrophic factor augmented glutamate-evoked, but not acetylcholine-evoked, currents 3-fold and increased N-methyl-Daspartic acid (NMDA) receptor open probability. Activation of trkB NT receptors was critical, as glutamate currents were not affected by nerve growth factor or NT-3, and increased open probability was prevented by the tyrosine kinase inhibitor K-252a. In addition, the NMDA receptor antagonist MK-801 blocked brain-derived neurotrophic factor enhancement of synaptic transmission, further suggesting that NTs modulate synaptic efficacy via changes in NMDA receptor function.Members of the neurotrophin (NT) gene family play important roles in a wide range of developmental events (1, 2). In addition to promoting neuronal survival and differentiation, we and others have shown that these factors also acutely modulate the efficacy of synaptic transmission (3-9) and play a role in long-term potentiation (LTP;. The specific targets of NT modulation in the synapse have not been identified. In the hippocampus, activation of the trkB neurotrophin receptor by brain-derived neurotrophic factor (BDNF) or NT-4 rapidly increases the amplitude of postsynaptic currents (4, 15). This effect can be blocked by postsynaptic injection of protein kinase inhibitors, suggesting that the specific synaptic proteins targeted by neurotrophin signaling include postsynaptic neurotransmitter receptors, whose function is modulated by phosphorylation (16)(17)(18). In recent studies, we have found that functional trkB receptors are localized to the postsynaptic density (PSD), a synaptic specialization that contains neurotransmitter receptors and second messenger signaling molecules (19). Moreover, we have found that BDNF rapidly and selectively enhances phosphorylation of N-methyl-D-aspartic acid (NMDA) receptor subunits 1 and 2B in isolated hippocampal PSDs (20,21). In the present report, we investigate neurotrophin modulation of neurotransmitter receptors and identify a mechanism by which BDNF acutely modulates hippocampal synaptic function. MATERIALS AND METHODSCell Culture. Hippocampal cultures were grown as described (22). Briefly, hippocampi were obtained from embryonic day 18 Sprague-Dawley rats and cells were plated on poly-D-lysinecoated Petri dishes at a final density of 10 6 cells͞35 mm dish.Cultures were maintained in serum-free medium at 37°C in a 95% air͞5% CO 2 humidified incubator. These cultures contained virtually pure neurons, as judged by neuron-specific enolase immunocytochemistry.Electrophysiological Recordings. Voltage clamp recordings were obtained from pyramidal-type cells after 12-16 days in vitro using standard techniques (23). Cells were recorded in voltage clamp mode and held at a resting potential of Ϫ40 mV to reduce Mg 2ϩ blockade of N...

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Although neurotrophins have traditionally been regarded as neuronal survival factors, recent work has suggested a role for these factors in synaptic plasticity. In particular, brain-derived neurotrophic factor (BDNF) rapidly enhances synaptic transmission in hippocampal neurons through trkB receptor stimulation and postsynaptic phosphorylation mechanisms. Activation of trkB also modulates hippocampal long-term potentiation, in which postsynaptic N-methyl-D-aspartate glutamate receptors play a key role. However, the final common pathway through which BDNF increases postsynaptic responsiveness is unknown. We now report that BDNF, within 5 min of exposure, elicits a dosedependent increase in phosphorylation of the N-methyl-Daspartate receptor subunit 1. This acute effect occurred in hippocampal synaptoneurosomes, which contain pre-and postsynaptic elements, and in isolated hippocampal postsynaptic densities. Nerve growth factor, in contrast, caused no enhancement of phosphorylation. These results suggest a potential mechanism for trophin-induced potentiation of synaptic transmission.Neurotrophins play important roles in the survival and differentiation of specific neuronal populations during development and adulthood (1-3). Four members of the mammalian neurotrophin gene family have been identified: nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and NT-4͞5 (4, 5). The neurotrophins bind to receptor tyrosine kinases of the trk protooncogene family with high affinity. trkA is a high-affinity receptor for NGF (6, 7), trkB for BDNF and NT-4͞5 (8, 9) as well as NT-3 (10), and trkC for .In addition to classical trophic activities, we and others have shown that neurotrophins acutely modulate synaptic transmission (12-19) and long-term potentiation (20). Experiments with dissociated cultures of hippocampal neurons indicate that BDNF and NT-4͞5 rapidly and selectively potentiate excitatory synaptic currents (17-19), and this trkB-mediated synaptic enhancement involves phosphorylation-dependent modulation of postsynaptic responsiveness (18). The exact mechanisms by which trkB activation modulates synaptic transmission remain to be established.We recently found that trkB is a functionally active, intrinsic component of the adult rat postsynaptic density (PSD) isolated from cerebral cortex and hippocampus (21). The PSD is a proteinaceous disc-shaped subcellular organelle apposed to the inner surface of the postsynaptic membrane of chemical synapses (22). Neurotransmitter receptors, protein kinases, and ion channel proteins are anchored to the PSD (22), suggesting that the PSD participates in signal transduction and even receptor regulation. In fact, our recent studies revealed that N-methyl-D-aspartate (NMDA) receptors can be phosphorylated by intrinsic PSD kinases (23,24). Phosphorylation of NMDA receptors is known to enhance receptor binding, channel conductance, and synaptic transmission (25-27). Because trkB activation enhances synaptic activity via postsynaptic phosphor...

The paired oocyte cell-cell channel assay was used to investigate the mechanisms involved in the process of formation of gap junction channels. Single oocytes, injected with connexin-specific mRNAs, accumulate a pool of precursors from which cell-cell channels can form rapidly upon pairing. Several lines of evidence, including immunohistochemistry and surface labeling, indicate that part of this precursor pool is located in the cell membrane, probably in the form of closed hemichannels. The homophilic binding of hemichannels to each other can be mimicked by synthetic peptides representing the extracellular loop sequences of connexin32. The peptides specifically suppress channel formation. A crucial role is established for the six cysteines in the extracellular domains that are conserved in all vertebrate gap junction proteins. Change of any of these cysteines into serines results in absolute loss of function of the mutant connexin. The effects of thiol-specific reagents on channel formation suggest that docking and/or opening of channels involves disulfide exchange. Several of the variable amino acids in the extracellular loop sequences were found to determine specificity of connexin-connexin interactions.

The oocyte cell-cell channel assay was used to demonstrate that connexin-43 is a cell-cell channel-forming protein as previously shown for connexin-32. Expression of connexin-32 in one and connexin43 in the other oocyte ofa pair results in the formation of junctional conductances at rates similar to those observed when only one or the other connexin is expressed in both oocytes of a pair. This suggests that hybrid cell-cell channels form in the oocyte system. Hybrid channels also form when a connexin43 mRNA-injected oocyte is pailed with a noninjected oocyte expressing endogenous connexin. The latter hybrids have properties apparently contributed by both types of hemichannels. Pure connexin43 channels are not voltage gated, whereas pure oocyte channels are voltage dependent; hybrids of these channels rectify.