Eric S. Levine

BDNF acutely increases tyrosine phosphorylation of the NMDA receptor subunit 2B in cortical and hippocampal postsynaptic densities.

While neurotrophins are critical for neuronal survival and differentiation, recent work suggests that they acutely regulate synaptic transmission as well. Brain-derived neurotrophic factor (BDNF) enhances excitatory postsynaptic currents in cultured dissociated hippocampal neurons within 2-3 min through postsynaptic, phosphorylation-dependent mechanisms. Moreover, BDNF modulates hippocampal long-term potentiation, in which postsynaptic NMDA (N-methyl-D-aspartate) receptors (NRs) play a key role. We now report that BDNF acutely increases tyrosine phosphorylation of the specific NMDA receptor subunit NR2B, which has recently been shown to play a role in long-term potentiation. Incubation of BDNF with cortical or hippocampal postsynaptic densities for 5 min increased tyrosine phosphorylation of the NR2B subunits in a dose-dependent manner. A maximal increase to 165% of control phosphorylation occurred at a BDNF concentration of 2 ng/ml. The BDNF action appeared to be specific, since nerve growth factor, another member of the neurotrophin gene family, had no effect on NR2B phosphorylation. Further, BDNF action was selective, since it did not alter tyrosine phosphorylation of NR2A subunits. Our results suggest that tyrosine phosphorylation of NR2B subunits of the NMDA receptor may contribute to neurotrophin modulation of postsynaptic responsiveness and long-term potentiation.

Induced pluripotent stem cell models of the genomic imprinting disorders Angelman and Prader–Willi syndromes

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.

Kalirin 7 is required for synaptic structure and function

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 (Kal7KO) grew and reproduced normally. Golgi impregnation and electron microscopy revealed decreased hippocampal spine density in Kal7KO mice. Behaviorally, Kal7KO 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. Kal7KO 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 Kal7KO 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 Kal7KO mice, with decreased excitatory synapses apparent only after 21 d in vitro. Expression of exogenous Kal7 in Kal7KO neurons rescued this deficit. Kal7 plays an essential role in synaptic structure and function, affecting a subset of cognitive processes.

Functional trkB neurotrophin receptors are intrinsic components of the adult brain postsynaptic density.

Neurotrophins have long been thought to act as target-derived factors that regulate the survival and differentiation of afferent neurons. Recently, brain-derived neurotrophic factor (BDNF) was shown to elicit rapid increases in synaptic activity of cultured hippocampal neurons by enhancing responsiveness to excitatory input. These findings suggest a postsynaptic localization of neurotrophin receptors. In this study, we examined the expression of trkB, a high-affinity receptor for BDNF, in the postsynaptic density (PSD), a proteinaceous specialization of the postsynaptic membrane. Western blot analyses with antibodies to trkB revealed localization to the PSD in adult rat cerebral cortex and hippocampus. Only the full-length, active form of trkB was detected in PSD samples. BDNF treatment of the adult cortical PSD resulted in a 5-fold increase in trkB autophosphorylation, supporting the contention that the PSD contains functional trkB. Truncated trkB, which does not contain the tyrosine kinase signaling domain, though present in membrane fractions, was undetectable in the PSD. The presence of trkB in the PSD is consistent with a role for neurotrophins in the regulation of synaptic activity via direct postsynaptic mechanisms.

Activity of cat locus coeruleus noradrenergic neurons during the defense reaction

The single-unit activity of locus coeruleus noradrenergic (LC-NE) neurons was recorded in freely moving cats during naturally induced defense reactions. Defense reactions, consisting of arched back, piloerection, flattened ears and mydriasis, were elicited by exposing the cat either to a dog, or to a cat displaying aggressive behavior induced by electrical stimulation of the hypothalamus. LC-NE neurons were identified using previously established criteria, including suppression of firing during rapid eye movement (REM) sleep and in response to clonidine administration. Exposure to a dog evoked defense reactions and increased the tonic firing rate of LC-NE neurons (n = 8) from a baseline of approximately 0.9 spikes/s to approximately 2.5 spikes/s. Exposure to an aggressive cat evoked defense reactions that were qualitatively very similar to those produced by dog exposure, and elevated the tonic firing rate of LC-NE neurons (n = 8) from a baseline of approximately 1.0 spikes/s to approximately 2.5 spikes/s. In addition to these tonic elevations of activity, LC-NE neurons discharged in phasic bursts (as high as 10 spikes in a 500 ms period) in close association with specific threatening acts made by the dog or hypothalamically stimulated cat. The mere presence of a dog was sufficient to evoke tonic activation of LC-NE neurons, even in the absence of threatening advances by the dog, whereas exposure to a hypothalamically stimulated cat produced LC-NE neuronal activation only when the stimulated cat showed aggressive behavior. These results extend our previous work, which examined the response of LC-NE neurons to environmental and physiological stressors, into a more ethologically relevant domain, and suggest that LC-NE neuronal activation may play a role in the response to threatening or challenging situations.

Brain‐derived neurotrophic factor increases activity of NR2B‐containing N‐methyl‐D‐aspartate receptors in excised patches from hippocampal neurons

Growth factors, including members of the neurotrophin gene family, play a central role in the regulation of neuronal survival and differentiation during development. In addition to these relatively long‐term actions of neurotrophins, recent studies have shown that these factors also rapidly modulate synaptic transmission. Brain‐derived neurotrophic factor (BDNF), in particular, regulates both pre‐ and postsynaptic aspects of hippocampal synaptic transmission. The postsynaptic effects include an increase in glutamate responsiveness, mediated by the N‐methyl‐D‐aspartate (NMDA) glutamate receptor subtype. It is not clear, however, where BDNF‐trkB signal transduction is initiated, because trkB receptors are located in both pre‐ and postsynaptic membranes. In the present study, we used excised membrane patches from cultured hippocampal neurons to determine whether BDNF directly modulates postsynaptic NMDA receptor activity. The results indicate that acute exposure to BDNF increases NMDA single channel open probability via postsynaptic trkB receptors and that this effect is dependent on the presence of the NR2B subunit of the NMDA receptor. J. Neurosci. Res. 62:357–362, 2000.

NMDA receptor subunits in the postsynaptic density of rat brain : expression and phosphorylation by endogenous protein kinases

N-methyl-D-aspartate (NMDA) receptors (NRs) play critical roles in diverse synaptic processes in the brain. However, subcellular distribution, spatiotemporal expression and regulation of NR subunits in brain synapses are unknown. We report that NR1 and NR2A-2C subunits are all enriched in the postsynaptic density (PSD), which plays critical roles in trophin-mediated synaptic plasticity. Significant expression of NRs was observed the first two weeks after birth, during synaptogenesis, and in adulthood. Functional diversity of NRs, resulting from heterogeneous composition, was supported by the finding that different NR2 subunits were associated in a region-specific manner with NR1. Phosphorylation of NR1, a key subunit of the NMDA receptor-channel complex, was significantly enhanced by activators of calmodulin (CaM) kinases (CKs) or protein kinase C (PKC), but not by those of PKA. Co-immunoprecipitation studies revealed that NR1 was physically associated with functionally active PKCgamma and the major PSD protein (mPSDp) through noncovalent interactions. Our results suggest that NMDA receptors play roles in postsynaptic mechanisms in a subunit-, composition-, brain region- and developmental-specific manner. Our findings also indicate that the PSD is a coherent functional unit containing protein kinases that potentially regulate NMDA receptor function via phosphorylation.

Endocannabinoid signalling selectively targets perisomatic inhibitory inputs to pyramidal neurones in juvenile mouse neocortex

Retrograde synaptic signalling has long been recognized as a fundamental feature of neural systems. However, the cellular specificity and functional consequences of fast retrograde communication are not well understood. We have focused our efforts on understanding the role that endocannabinoids play in regulating synaptic inhibition in sensory neocortex. Recent studies have implicated endocannabinoids as the retrograde signalling molecules that underlie depolarization‐induced suppression of inhibition, or DSI. This short‐term form of presynaptic depression is triggered by postsynaptic depolarization and is likely to play an important role in information processing. In the present study we investigated the cellular and synaptic specificity of endocannabinoid signalling in sensory cortex using whole‐cell recordings from layer 2/3 pyramidal neurones (PNs) in acute brain slices. We report that GABAergic interneurones that are depolarized by muscarinic receptor stimulation provided the majority of DSI‐susceptible inputs to neocortical PNs. This subclass of interneurones generated large, fast postsynaptic currents in PNs which were transiently suppressed by either postsynaptic depolarization or a brief train of action potentials. Neocortical DSI required activation of the type 1 cannabinoid receptor (CB1R) but not metabotropic glutamate or GABA receptors. Using focal drug application, we found that the DSI‐susceptible afferents preferentially synapse on the perisomatic membrane of PNs, and not on the apical dendrites. Together, these results suggest that endocannabinoid‐mediated DSI in the cortex can transiently and selectively depress a subclass of PN inputs. Although the physiological implications remain to be explored, this suppression of somatic inhibition may alter the excitability of principal neurones and thereby modulate cortical output.

Presynaptic and Postsynaptic NMDA Receptors Mediate Distinct Effects of Brain-Derived Neurotrophic Factor on Synaptic Transmission

In addition to its effects on neuronal survival and differentiation, brain-derived neurotrophic factor (BDNF) plays an important role in modulating synaptic transmission and plasticity in many brain areas, most notably the neocortex and hippocampus. These effects may underlie a role for BDNF in learning and memory as well as developmental plasticity. Consistent with localization of the tropomyosin-related kinase B receptor to both sides of the synapse, BDNF appears to have pre- and postsynaptic effects, but the underlying cellular mechanisms are unclear and it is not known whether pre- and postsynaptic modulations by BDNF occur simultaneously. To address these issues, we recorded dual-component (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid [AMPA] and N-methyl-D-aspartate [NMDA]) miniature excitatory postsynaptic currents (mEPSCs) from cortical and hippocampal pyramidal neurons and dentate gyrus granule cells from acute brain slices. BDNF had no effect on the fast component of mEPSC decay or on the peak amplitude, suggesting that BDNF did not modulate postsynaptic AMPA receptors, although BDNF rapidly modulated NMDA receptors, as seen by an enhancement of the slow component of mEPSC decay that was prevented by blocking postsynaptic NMDA receptors. At the same time, BDNF acted presynaptically to enhance mEPSC frequency. Surprisingly, the effect on frequency was also NMDA receptor dependent, but required activation of presynaptic, not postsynaptic, NMDA receptors. BDNF also enhanced action potential-dependent glutamate release via presynaptic NMDA receptors, an effect that was unmasked when voltage-gated calcium channels were partially inhibited. Our results indicate that BDNF acutely modulates presynaptic release and postsynaptic responsiveness through simultaneous effects on pre- and postsynaptic NMDA receptors.

Selective role for trkB neurotrophin receptors in rapid modulation of hippocampal synaptic transmission

Neurotrophins regulate neuronal survival and phenotypic differentiation. Recent evidence also suggests a role in the modulation of synaptic activity. Using neuronal cell cultures from embryonic hippocampus, we previously found that application of brain-derived neurotrophic factor rapidly enhanced synaptic transmission. We now report that application of neurotrophin-4, another ligand for the trkB neurotrophin receptor, was equally effective in enhancing synaptic currents. In contrast, nerve growth factor, neurotrophin-3, basic fibroblast growth factor and epidermal growth factor did not share this action. Our results suggest that activation of trkB receptors plays a selective role in the regulation of synaptic efficacy in the hippocampus.