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Neuronal avalanches have become an ubiquitous tool to describe the activity of large neuronal assemblies. The emergence of scale-free statistics with well-defined exponents has led to the belief that the brain might operate near a critical point. Yet not much is known in terms of how the different exponents arise or how robust they are. Using calcium imaging recordings of dissociated neuronal cultures we show that the exponents are not universal, and that significantly different exponents arise with different culture preparations, leading to the existence of different universality classes. Naturally developing cultures show avalanche statistics consistent with those of a mean-field branching process, however, cultures grown in the presence of folic acid metabolites appear to be in a distinct universality class with significantly different critical exponents. Given the increased synaptic density and number of feedback loops in folate reared cultures, our results suggest that network topology plays a leading role in shaping the avalanche dynamics. We also show that for both types of cultures pronounced correlations exist in the sizes of neuronal avalanches indicating size clustering, being much stronger in folate reared cultures.

Axonal transsynaptic signaling between presynaptic neurexin (NX) and postsynaptic neuroligin (NL) is essential for many properties of synaptic connectivity. Here, we demonstrate the existence of a parallel axo-glial signaling pathway between axonal NX and oligodendritic (OL) NL3. We show that this pathway contributes to the regulation of myelinogenesis, the maintenance of established myelination, and the differentiation state of the OL using in vitro models. We first confirm that NL3 mRNA and protein are expressed in OLs and in OL precursors. We then show that OLs in culture form contacts with non-neuronal cells exogenously expressing NL3's binding partners NX1α or NX1β. Conversely, blocking axo-glial NX-NL3 signaling by saturating NX with exogenous soluble NL protein (NL-ECD) disrupts interactions between OLs and axons in both in vitro and ex vivo assays. Myelination by OLs is tied to their differentiation state, and we find that blocking NX-NL signaling with soluble NL protein also caused OL differentiation to stall at an immature stage. Moreover, in vitro knockdown of NL3 in OLs with siRNAs stalls their development and reduces branching complexity. Interestingly, inclusion of an autism related mutation in the NL blocking protein attenuates these effects; OLs differentiate and the dynamics of OL-axon signaling occur normally as this peptide does not disrupt NX-NL3 axo-glial interactions. Our findings provide evidence not only for a new pathway in axo-glial communication, they also potentially explain the correlation between altered white matter and autism. GLIA 2015;63:2023-2039.

PurposeCaV3.2 signaling contributes to nociception, pruritus, gastrointestinal motility, anxiety, and blood pressure homeostasis. This calcium channel, encoded by CACNA1H, overlaps the human tryptase locus, wherein increased TPSAB1 copy number causes hereditary α-tryptasemia. Germ-line CACNA1H variants may contribute to the variable expressivity observed with this genetic trait.MethodsTryptase-encoding sequences at TPSAB1 and TPSB2, and TPSG1 and CACNA1H variants were genotyped in 46 families with hereditary α-tryptasemia syndrome. Electrophysiology was performed on tsA201 HEK cells transfected with wild-type or variant CACNA1H constructs. Effects on clinical phenotypes were interrogated in families with TPSAB1 duplications and in volunteers from the ClinSeq cohort.ResultsThree nonsynonymous variants in CACNA1H (rs3751664, rs58124832, and rs72552056) cosegregated with TPSAB1 duplications in 32/46 families and were confirmed to be in linkage disequilibrium (LD). In vitro, variant CaV3.2 had functional effects: reducing current densities, and altering inactivation and deactivation properties. No clinical differences were observed in association with the CACNA1H haplotype.ConclusionA previously unrecognized haplotype containing three functional CACNA1H variants is relatively common among Caucasians, and is frequently coinherited on the same allele as additional TPSAB1 copies. The variant CACNA1H haplotype, which in vitro imparts partial gain of function, does not result in detectable phenotypic differences in the heterozygous state.

Loss of the ability to regulate calcium is a central event leading to neuronal cell death during a wide range of pathological conditions including stroke and seizure. Here we present a new dissociated hippocampal cell culture model of acute electrical activity which incorporates the photoconductive stimulation of neuronal networks grown on silicon wafers. This technology allows precise modeling of user defined neuronal activity patterns, and the study of their effect on neuronal physiology. Here, seizure-like conditions were created by continuous stimulation, causing hundreds of neurons to fire synchronously at 50Hz for 4min. This stimulation protocol induced cell death as monitored by propidium iodide staining. The number of dead cells per stimulation region increased from 3.6+/-2.1 preceding stimulation to 81+/-21 30min following stimulation. Excitotoxicity primarily affected excitatory rather than inhibitory neurons, and was preceded by an increase in intracellular calcium as well as changes in the mitochondrial membrane potential, as measured by a tetramethylrhodamine methyl ester (TMRM) assay. Cyclosporin A (CsA), a mitochondrial permeability transition pore (PTP) blocker, was effective in preventing cell death. We propose that photoconductive stimulation is a useful tool for investigating the pathogenesis of excitotoxicity in vitro.

Cognition and behavior primarily arise from the communication that occurs between brain cells. By using photoconductive stimulation to trigger localized regions of neuronal action potentials and astrocyte Ca2+ waves in dissociated rat hippocampal cultures, we can directly study microglia behavior in response to physiological and pathological levels of activity. Connections between neurons can be modified by microglia, which regulate gap junctions and synapses through secretion of proteins such as cytokines, proteases and neurotrophic factors. Activated microglia participate in bidirectional communication with the excitable tissues that they support. Through feedback from the many ion channels and surface receptors they express, microglia are informed of neuronal and astrocytic activity that may indicate disruption in the homeostasis of the CNS. Such disturbances alert microglia to locations of such activity and promote their transformation into a reactive state, in which they perform adaptive functions that can be either neuroprotective, neurotoxic, or neuromodulatory. Under physiological conditions, normal brain activity has the effect of suppressing microglia inflammatory responses. This report summarizes available data about the interaction of microglia and brain activity and presents a new in vitro paradigm to study the mechanisms involved. We propose that photoconductive stimulation is a powerful tool for studying the cellular and molecular mechanisms underlying the dynamic interactions between neurons, astrocytes and microglia.

Synapses established during central nervous system development can be modified through synapse elimination and formation. These processes are, in part, activity dependent and require regulated trafficking of post-synaptic components. Here, we investigate the activity-driven remodeling of cultured rat hippocampal neurons at 14 days in vitro, focusing on the post-synaptic proteins PSD-95, Shank, neuroligin (NL)1 and actin. Using live imaging and photoconductive stimulation, we found that high-frequency activity altered the trajectory, but not velocity, of PSD-95-GFP and Shank-YFP clusters, whereas it reduced the speed and increased the number of NL1 clusters. Actin-CFP reorganized into puncta following activity and approximately 50% of new puncta colocalized with NL1 clusters. Actin reorganization was enhanced by the overexpression of NL1 and decreased by the expression of an NL1 mutant, NL1-R473C. These results demonstrate activity-dependent changes that may result in the formation of new post-synaptic sites and suggest that NL1 modulates actin reorganization. The results also suggest that a common mechanism underlies both the developmental and activity-dependent remodeling of excitatory synapses.

The neuroligin (NL) gene family codes for brain specific cell adhesion molecules that play an important role in synaptic connectivity. Recent studies have identified NL mutations linked to patients with autism spectrum disorders (ASD). Cognitive deficits seen in autistic patients are hypothesized to arise from altered synchronicity both within and between brain regions. Here we show how the expression of autism-associated neuroligin mutation R471C-NL3 affects synchrony in dissociated cultures of rat hippocampal neurons. Spontaneous network activity patterns of cultures expressing wild type and mutant NL3 were measured by optical techniques. Firing events were quantified and compared by cross-correlation analysis. Our results suggest that NL3 overexpression enhances synchrony of spontaneous activity patterns, however, this ability is reduced with the R471C-NL3 mutation. We investigated the structural basis of this phenomenon using fractal dimension analysis to characterize the arrangement of axon trajectories. R471C-NL3 cultures were associated with lower fractal dimensions and higher lacunarity values, indicating a decrease in the complexity of axonal architecture. Transfection of R471C-NL3 into a subpopulation of cells in a network resulted in neuronal degeneration. This degeneration likely affected the inhibitory population of neurons, as there were half as many (P

Polyphosphate (polyP) consists of tens to hundreds of phosphates, linked by ATP-like high-energy bonds. Although polyP is present in mammalian mitochondria, its physiological roles there are obscure. Here, we examine the involvement of polyP in mitochondrial energy metabolism and ion transport. We constructed a vector to express a mitochondrially targeted polyphosphatase, along with a GFP fluorescent tag. Specific reduction of mitochondrial polyP, by polyphosphatase expression, significantly modulates mitochondrial bioenergetics, as indicated by the reduction of inner membrane potential and increased NADH levels. Furthermore, reduction of polyP levels increases mitochondrial capacity to accumulate calcium and reduces the likelihood of the calcium-induced mitochondrial permeability transition, a central event in many types of necrotic cell death. This confers protection against cell death, including that induced by β-amyloid peptide, a pathogenic agent in Alzheimer's disease. These results demonstrate a crucial role played by polyP in mitochondrial function of mammalian cells.

Overactivation of neuronal N-methyl-D-aspartate receptors (NMDARs) causes excitotoxicity and is necessary for neuronal death. In the classical view, these ligand-gated Ca(2+)-permeable ionotropic receptors require co-agonists and membrane depolarization for activation. We report that NMDARs signal during ligand binding without activation of their ion conduction pore. Pharmacological pore block with MK-801, physiological pore block with Mg(2+) or a Ca(2+)-impermeable NMDAR variant prevented NMDAR currents, but did not block excitotoxic dendritic blebbing and secondary currents induced by exogenous NMDA. NMDARs, Src kinase and Panx1 form a signaling complex, and activation of Panx1 required phosphorylation at Y308. Disruption of this NMDAR-Src-Panx1 signaling complex in vitro or in vivo by administration of an interfering peptide either before or 2 h after ischemia or stroke was neuroprotective. Our observations provide insights into a new signaling modality of NMDARs that has broad-reaching implications for brain physiology and pathology.

SUMMARY Information processing in the central nervous system is primarily mediated through synaptic connections between neurons. This connectivity in turn defines how large ensembles of neurons may coordinate network output to execute complex sensory and motor functions including learning and memory. The synaptic connectivity between any given pair of neurons is not hard-wired;rather it exhibits a high degree of plasticity, which in turn forms the basis for learning and memory. While there has been extensive research to define the cellular and molecular basis of synaptic plasticity, at the level of either pairs of neurons or smaller networks, analysis of larger neuronal ensembles has proved technically challenging. The ability to monitor the activities of larger neuronal networks simultaneously and non-invasively is a necessary prerequisite to understanding how neuronal networks function at the systems level. Here we describe recent breakthroughs in the area of various bionic hybrids whereby neuronal networks have been successfully interfaced with silicon devices to monitor the output of synaptically connected neurons. These technologies hold tremendous potential for future research not only in the area of synaptic plasticity but also for the development of strategies that will enable implantation of electronic devices in live animals during various memory tasks.

SummaryNonsynaptic clusters of postsynaptic proteins have been documented; however, their role remains elusive. We monitored the trafficking of several candidate proteins implicated in synaptogenesis, when nonsynaptic clusters of scaffold proteins are most abundant. We find a protein complex consisting of two populations that differ in their content, mobility, and involvement in synapse formation. One subpopulation is mobile and relies on actin transport for delivery to nascent and existing synapses. These mobile clusters contain the scaffolding proteins PSD-95, GKAP, and Shank. A proportion of mobile clusters that exhibits slow movement and travels short distances contains neuroligin-1. The second group consists of stationary nonsynaptic scaffold complexes that mainly contain neuroligin-1, can recruit synaptophysin-containing axonal transport vesicles, and are readily transformed to functional presynaptic contacts that recycle the vital dye FM 4-64. These results postulate a mechanism whereby preformed scaffold protein complexes serve as predetermined postsynaptic hotspots for establishment of new functional excitatory synapses.

We have identified a novel interaction between huntingtin (htt) and Ntype calcium channels, a channel key in coupling calcium influx with synaptic vesicle exocytosis. Htt is a widely expressed 350-kDa cytosolic protein bearing an N-terminal polyglutamine tract. Htt is proteolytically cleaved by calpains and caspases and the resultant htt N-terminal fragments have been proposed to be biologically active; however, the cellular function of htt and/or the htt fragments remains enigmatic. We show that N-terminal fragments of htt (consisting of exon1) and fulllength htt associate with the synaptic protein interaction (synprint) region of the N-type calcium channel. Given that synprint has previously been shown to bind syntaxin 1A and that this association elicits inhibition of N-type calcium channels, we tested whether htt exon1 affects the modulation of these channels. Our data indicate that htt exon1 enhances calcium influx by blocking syntaxin 1A inhibition of N-type calcium channels and attributes a key role for htt N-terminal fragments in the fine tuning of neurotransmission. D 2005 Elsevier Inc. All rights reserved.

Axonal transsynaptic signaling between presynaptic neurexin (NX) and postsynaptic neuroligin (NL) is essential for many properties of synaptic connectivity. Here, we demonstrate the existence of a parallel axo-glial signaling pathway between axonal NX and oligodendritic (OL) NL3. We show that this pathway contributes to the regulation of myelinogenesis, the maintenance of established myelination, and the differentiation state of the OL using in vitro models. We first confirm that NL3 mRNA and protein are expressed in OLs and in OL precursors. We then show that OLs in culture form contacts with non-neuronal cells exogenously expressing NL3's binding partners NX1α or NX1β. Conversely, blocking axo-glial NX-NL3 signaling by saturating NX with exogenous soluble NL protein (NL-ECD) disrupts interactions between OLs and axons in both in vitro and ex vivo assays. Myelination by OLs is tied to their differentiation state, and we find that blocking NX-NL signaling with soluble NL protein also caused OL differentiation to stall at an immature stage. Moreover, in vitro knockdown of NL3 in OLs with siRNAs stalls their development and reduces branching complexity. Interestingly, inclusion of an autism related mutation in the NL blocking protein attenuates these effects; OLs differentiate and the dynamics of OL-axon signaling occur normally as this peptide does not disrupt NX-NL3 axo-glial interactions. Our findings provide evidence not only for a new pathway in axo-glial communication, they also potentially explain the correlation between altered white matter and autism. GLIA 2015;63:2023-2039.

The large microtubule-associated/Ca2+-signalling protein p600 (also known as UBR4) is required for hippocampal neuronal survival upon Ca2+ dyshomeostasis induced by glutamate treatment. During this process, p600 prevents aggregation of the Ca2+/calmodulin-dependent kinase IIα (CaMKIIα), a proxy of neuronal death, via direct binding to calmodulin in a microtubuleindependent manner. Using photoconductive stimulation coupled with live imaging of single neurons, we identified a distinct mechanism of prevention of CaMKIIα aggregation by p600. Upon direct depolarization, CaMKIIα translocates to microtubules. In the absence of p600, this translocation is interrupted in favour of a sustained self-aggregation that is prevented by the microtubule-stabilizing drug paclitaxel. Thus, during photoconductive stimulation, p600 prevents the aggregation of CaMKIIα by stabilizing microtubules. The effectiveness of this stabilization for preventing CaMKIIα aggregation during direct depolarization but not during glutamate treatment suggests a model wherein p600 has two modes of action depending on the source of cytosolic Ca2+. Electronic Supplementary Material Supplementary material is available for this article at 10.2478/s11658-014-0201-9 and is accessible for authorized users.

Netrins are a family of secreted proteins that function as chemotropic axon guidance cues during neural development. Here we demonstrate that netrin-1 continues to be expressed in the adult rat spinal cord at a level similar to that in the embryonic CNS. In contrast, netrin-3, which is also expressed in the embryonic spinal cord, was not detected in the adult.In situhybridization analysis demonstrated that cells in the white matter and the gray matter of the adult spinal cord express netrin-1. Colocalization studies using the neuronal marker NeuN revealed that netrin-1 is expressed by multiple classes of spinal interneurons and motoneurons. Markers identifying glial cell types indicated that netrin-1 is expressed by most, if not all, oligodendrocytes but not by astrocytes. During neural development, netrin-1 has been proposed to function as a diffusible long-range cue for growing axons. We show that in the adult spinal cord the majority of netrin-1 protein is not freely soluble but is associated with membranes or the extracellular matrix. Fractionation of adult spinal cord white matter demonstrated that netrin-1 was absent from fractions enriched for compact myelin but was enriched in fractions containing periaxonal myelin and axolemma, indicating that netrin-1 protein may be localized to the periaxonal space. These findings suggest that in addition to its role as a long-range guidance cue for developing axons, netrin may have a short-range function associated with the cell surface that contributes to the maintenance of appropriate neuronal and axon–oligodendroglial interactions in the mature nervous system.

Depolymerization of actin by latrunculin A transiently promotes neurotransmitter release. The mean rate of mEPSCs increases by a Ca2+-independent process, without a concomitant change in the mean amplitude. The readily releasable vesicle pool size and the rate of refilling of the readily releasable pool remain unaltered by latrunculin treatment. Evoked neurotransmitter release also increases in a manner consistent with an increase in vesicle release probability. The observed enhancement of neurotransmitter release is specific to actin depolymerization mediated by latrunculin A and is not caused by cytochalasin D. Our findings indicate that actin participates in a regulatory mechanism that restrains fusion of synaptic vesicles at the active zone.

Maternal folic acid supplementation is essential to reduce the risk of neural tube defects. We hypothesize that high levels of folic acid throughout gestation may produce neural networks more susceptible to seizure in offspring. We hence administered large doses of folic acid to rats before and during gestation and found their offspring had a 42% decrease in their seizure threshold. In vitro, acute application of folic acid or its metabolite 4Hfolate to neurons induced hyper-excitability and bursting. Cultured neuronal networks which develop in the presence of a low concentration (50 nM) of 4Hfolate had reduced capacity to stabilize their network dynamics after a burst of high-frequency activity, and an increase in the frequency of mEPSCs. Networks reared in the presence of the folic acid metabolite 5M4Hfolate developed a spontaneous, distinctive bursting pattern, and both metabolites produced an increase in synaptic density.

Loss of hippocampal neurons as a result of traumatic brain injury (TBI) is thought to contribute to the observed spatial memory deficits. Using a rodent model of experimental brain injury, we have examined the nature of hippocampal cell death following TBI. Light microscope examination of stained sections showed the presence of a large number of hyperchromatic and dystrophic neurons in the dentate gyrus of the hippocampus. These cells appeared to be undergoing nuclear condensation. Electron microscope examination demonstrated the presence of cell shrinkage, condensed chromatin, nuclear segmentation, and cytoplasmic vacuolization. Detection of a DNA ladder and in situ labeling (TUNEL) were also consistent with the process of apoptosis. However, in some dystrophic neurons these morphologies were also accompanied by the presence of swollen mitochondria and a lack of distinctive rough endoplasmic reticulum which are typically associated with necrosis. These findings show that cortical impact injury produces cell death in the hippocampus which has both apoptotic and necrotic features.

THE requirement of protein and messenger RNA synthesis for long-term memory1,2 suggests that neural activity induced by learning initiates a cascade of gene expression3. Here we use differential screening to identify five immediate–early genes induced by neuronal activity. One of these is tissue-plasminogen activator (tPA), an extracellular serine protease, which is induced with different spatial patterns in the brain by three activity-dependent events: (1) convulsive seizure increases expression of tPA in the whole brain; (2) stimulation of the perforant path produces an epileptiform after-discharge that ultimately leads to kindling increases the levels of tPA throughout the hippocampus bilaterally; and (3) brief high-frequency stimulation of the perforant path that produces long-term potentiation (LTP) causes an NMDA (N -methyl-D-aspartate) receptor-mediated increase in the levels of tPA mRNA which is restricted to the granule cells of the ipsilateral dentate gyrus. As release of tPA is correlated with morphological differentiation4–6, the increased expression of tPA may play a role in the structural changes that accompany activity-dependent plasticity7–10.

The denV gene from bacteriophage T4 encodes a pyrimidine dimer-specific endonuclease that has the capacity to initiate excision repair of DNA. Cells from excision repair-deficient xeroderma pigmentosum (XP) patients are able to carry out excision repair initiated by the denV gene product and introduction of the denV gene into XP cells results in the partial restoration of colony-forming ability after irradiation with UV light. In this work we have constructed a helper-independent recombinant human adenovirus, Ad5denV, which contains the denV gene. A 1.9 kb cartridge consisting of the denV gene flanked by the long terminal repeat (LTR) promoter from Rous sarcoma virus (RSV) and the simian virus 40 (SV40) polyadenylation (poly A) splice signals, was inserted into the E3 region of an E3 deletion mutant (Ad5d1E3) of adenovirus type 5. Infection of human fibroblasts and other permissive human cells with Ad5denV resulted in lytic infection and expression of the denV gene was confirmed by primer extension of infected cell RNA. The ability of the denV gene to restore the DNA repair deficiency in XP fibroblasts was examined using host cell reactivation of viral structural antigen formation for UV-irradiated adenovirus. The control virus, Ad5VSV, was also a recombinant which contained the gene for vesicular stomatitis virus glycoprotein G inserted into the E3 region of Ad5d1E3. UV survival of Ad5denV was similar to that of Ad5VSV following infection of two normal fibroblast strains and a Cockayne syndrome fibroblast strain, CS7SE, from complementation group B. In contrast, UV survival of Ad5denV was significantly greater than that for Ad5VSV after infection of three unrelated XP fibroblast strains from complementation groups A, C and E. However, UV survival of Ad5denV in the XP fibroblasts did not reach levels obtained in normal fibroblasts, indicating that restoration of the XP defect was partial.

Photoconductive stimulation allows the noninvasive depolarization of neurons cultured on a silicon wafer. This technique relies on a beam of light to target a cell of interest while applying a voltage bias across the silicon wafer. The targeted cell is excited with minimal physiological manipulation, and, therefore, long-term modulation of activity patterns and investigations of biochemical mechanisms sensitive to physiological perturbations are possible. Ideologically similar to transistor-based neuronal interfaces, the photoconductive-stimulation method has the advantage of being able to extracellularly excite any neuron in a network regardless of its spatial position on the silicon substrate. This protocol can be easily implemented on a conventional reflected-light fluorescence microscope using materials and resources that are readily available. Time requirements are comparable to standard cell-culture and electrophysiology techniques. When combined with fluorescence imaging of various molecular probes, activity-dependent cellular processes can be dynamically monitored.

Recent evidence demonstrates that low-frequency oscillations of intracellular calcium on timescales of seconds to minutes drive distinct aspects of neuronal development, but the mechanisms by which these calcium transients are coupled to signaling cascades are not well understood. Here we test the hypothesis that spontaneous electrical activity activates protein kinase A (PKA). We use live-cell indicators to observe spontaneous and evoked changes in cAMP levels and PKA activity in developing retinal neurons. Expression of cAMP and PKA indicators in neonatal rat retinal explants reveals spontaneous oscillations in PKA activity that are temporally correlated with spontaneous depolarizations associated with retinal waves. In response to short applications of forskolin, dopamine, or high-potassium concentration, we image an increase in cAMP levels and PKA activity, indicating that this second-messenger pathway can be activated quickly by neural activity. Depolarization-evoked increases in PKA activity were blocked by the removal of extracellular calcium, indicating that they are mediated by a calcium-dependent mechanism. These findings demonstrate for the first time that spontaneous activity in developing circuits is correlated with activation of the cAMP/PKA pathway and that PKA activity is turned on and off on the timescale of tens of seconds. These results show a link between neural activity and an intracellular biochemical cascade associated with plasticity, axon guidance, and neural differentiation.

The developing retina is characterized by 'retinal waves', spontaneous depolarizations that propagate through a developing network of interneurons and retinal ganglion cells. Although the circuitry underlying retinal waves is well characterized, the secreted factors that are critical for its normal development are not defined. Dissociated cell culture provides an ideal system for defining these factors; however, it is difficult to recapitulate retinal circuitry in culture. Here we demonstrate that by culturing dissociated retinal neurons in the presence of cells from the superior colliculus (SC), retinal neurons form networks that are similar to those described in the intact retina. Whole-cell voltage clamp recordings reveal the presence of a spontaneously active network of interneurons. In addition, we observed spontaneous, propagating activity reminiscent of that observed in the intact retina. We propose that the presence of factors secreted from the SC results in the development of networks that reproduce critical features of the intact retina. Copyright (C) 2004 S. Karger AG, Basel.

Use-dependent synapse remodeling is thought to provide a cellular mechanism for encoding durable memories, yet whether activity triggers an actual structural change has remained controversial. We use photoconductive stimulation to demonstrate activity-dependent morphological synaptic plasticity by video imaging of GFP-actin at individual synapses. A single tetanus transiently moves presynaptic actin toward and postsynaptic actin away from the synaptic junction. Repetitive spaced tetani induce glutamate receptor-dependent stable restructuring of synapses. Presynaptic actin redistributes and forms new puncta that label for an active synapse marker FM5-95 within 2 hr. Postsynaptic actin sprouts projections toward the new presynaptic actin puncta, resembling the axon-dendrite interaction during synaptogenesis. Our results indicate that activity-dependent presynaptic structural plasticity facilitates the formation of new active presynaptic terminals.

Seizure causes neuronal cell loss in both animal models and human epilepsy. To determine the contribution of apoptotic mechanisms to seizure-induced neuronal cell death, rat brains were examined for the occurrence of terminal deoxynucleotidyl transferase-mediated UTP nick end labeling (TUNEL)-positive nuclei after pilocarpine-induced seizure. Numerous TUNEL-positive cells were observed throughout the postseizure hippocampus, piriform cortex, and entorhinal cortex. Combined TUNEL/NeuN immunocytochemistry demonstrated that the vast majority of TUNEL-positive cells were neurons. To identify components of the signal transduction cascade promoting postseizure apoptosis, the expression of the p75 neurotrophin receptor (p75NTR) was examined. Seizure-induced increases in p75NTR protein and mRNA were detected in hippocampus, piriform cortex, and entorhinal cortex. Immunohistochemical double labeling revealed almost complete correspondence between TUNEL-positive and p75NTR-expressing cells, suggesting that seizure-induced neuronal loss within the CNS occurs through apoptotic signaling cascades involving p75NTR.

Clinical and experimental studies show that loss of neurons in the hippocampus and/or the entorhinal cortex can impede formation and storage of spatial memory. Using a controlled cortical impact model of traumatic brain injury (TBI) in rats, we have examined the temporal and spatial pattern of neuronal death using silver impregnation and cresyl violet staining. Dystrophic neurons can be detected in the dentate gyrus, and the CA1 and CA3 subfields of the hippocampus for up to 2 weeks following injury. These dystrophic cells appeared shrunken and possessed features of apoptosis. Areas containing the dystrophic cells suffer substantial cell loss as demonstrated by thinning of the neuronal layers. Dystrophic cells are also found in the amygdala, entorhinal and piriform cortices, thalamic and hypothalamic regions, and surrounding the contusion site. The loss of these cells may contribute to the memory deficits observed following TBI.

Behavioral, cellular and molecular changes were examined following axonal injury in the marine mollusc Aplysia californica. Unilateral nerve injury was performed by crushing the pleural-pedal connective and the peripheral pedal nerves innervating one side of the posterior body wall and the tail. The injury procedure severs the axons of the pleural sensory neurons resulting in the blockade of the tail-elicited siphon- withdrawal reflex. Partial reflex recovery is observed within 3 d and reaches 50% of the pretest value by six weeks postinjury. Retrograde staining of injured nerves combined with electrophysiological recordings from siphon motor neurons show that axons can regenerate through the crushed site and reconnect with the tail by three weeks postinjury. Moreover, the behavioral and electrophysiological measurements suggest that the contralateral sensory neurons may contribute to the early recovery of the siphon-withdrawal reflex. The levels of mRNAs coding for actin and calreticulin are elevated while the mRNAs coding for intermediate filament protein, sensorin A, FMRFamide are reduced in the ipsilateral pleural ganglia as detected by Northern blots. In the contralateral pleural ganglia, the levels of mRNAs coding for actin, sensorin A and FMRFamide are elevated. These molecular changes in both the ipsi- and contralateral sides are consistent with the hypothesis that both sides are participating in the behavioral recovery following unilateral axonal injury.

Summary At synapses, cell adhesion molecules (CAMs) provide the molecular framework for coordinating signaling events across the synaptic cleft. Among synaptic CAMs, the integrins, receptors for extracellular matrix proteins and counterreceptors on adjacent cells, are implicated in synapse maturation and plasticity and memory formation. However, little is known about the molecular mechanisms of integrin action at central synapses. Here, we report that postsynaptic β3 integrins control synaptic strength by regulating AMPA receptors (AMPARs) in a subunit-specific manner. Pharmacological perturbation targeting β3 integrins promotes endocytosis of GluR2-containing AMPARs via Rap1 signaling, and expression of β3 integrins produces robust changes in the abundance and composition of synaptic AMPARs without affecting dendritic spine structure. Importantly, homeostatic synaptic scaling induced by activity deprivation elevates surface expression of β3 integrins, and in turn, β3 integrins are required for synaptic scaling. Our findings demonstrate a key role for integrins in the feedback regulation of excitatory synaptic strength.

Synapses mediate communication between neurons, thus playing a fundamental role in information processing in the CNS. Neuroligins form a family of heterophilic synaptic cell adhesion molecules, and neuroligin 1 (NL1) has been shown to be involved in the formation of excitatory synapses and have been suggested to associate indirectly with NMDA receptors by common binding to PSD95. A mutation in neuroligin 3 (Arg451Cys-NL3, human sequence numbering) identified in autistic patients is associated with altered spine density and has reduced binding capacity for its presynaptic partner β-neurexin. Here, we investigated the role of NL1 and the homologous NL1 mutation Arg473Cys-NL1 (R473C-NL1) in excitatory synaptic transmission and NMDA receptor distribution. We demonstrate that R473C-NL1, when expressed in cultured hippocampal neurons, can induce a dramatic increase in NMDA current amplitude and that this change is accompanied by NMDA receptor clustering in the postsynaptic cell.

Rem2 is a small GTP-binding protein of the RGK family. It is targeted to the cell membrane where it interacts with the beta subunit of calcium channels and abolishes or reduces endogenous or exogenous calcium currents, and also has known interactions with calmodulin and 14-3-3. Rem2 is unique in the RGK family, being found predominantly in the brain and upregulated in response to stimulation. Knockdown of Rem2 in neuronal cultures results in fewer glutamatergic synapses. We have found that fluorescent-labeled Rem2 changes its subcellular localization in neurons from a diffuse to a punctuate distribution after neuronal stimulation or after activation of NMDA receptors. This rearrangement is calcium dependent and involves the C-terminal 30 residues, suggesting the presence of a self-association domain as well as an autoinhibitory domain that keeps Rem2 diffusely distributed until stimulation. A calmodulin-binding deficient mutant shows very little rearrangement upon stimulation, supporting a role for calcium in this phenomenon.