Project description:Post-mitotic neurons exhibit DNA methylation changes, contrary to the longstanding belief that the epigenetic pattern in terminally differentiated cells is essentially unchanging. While the mechanism and physiological significance of DNA demethylation in neurons have been extensively elucidated, occurrence of de novo DNA methylation and its impacts have been much less investigated. Here we show that neuronal activation induces global de novo DNA methylation at enhancer regions that can repress target genes in primary cultured hippocampal neurons. The functional significance of this de novo DNA methylation was underpinned by the demonstration that inhibition of DNA methyltransferase (DNMT) activity decreased neuronal activity-induced excitatory synaptogenesis. Overexpression of WW and C2 Domain containing 1 (Wwc1), a representative target gene of de novo DNA methylation, could phenocopy this DNMT inhibition-induced decrease in the synaptogenesis. We found that both DNMT1 and DNMT3a were required for the neuronal activity-induced de novo DNA methylation of Wwc1 enhancer. Taking these findings altogether, we concluded that activity-induced de novo DNA methylation affecting gene expression has impacts on neuronal physiology comparable to those of DNA demethylation. Overall design: Examination of DNA methylation and mRNA profiles of 10 DIV hippocampal neurons treated with TTX, Bic/4AP 3 hrs and H3K27ac of 10 DIV hippocampal neurons before and after Bic/4AP treatment
Project description:Synapses are asymmetric cellular adhesions that are critical for nervous system development and function, but the mechanisms that induce their formation are not well understood. We have previously identified thrombospondin as an astrocyte-secreted protein that promotes central nervous system (CNS) synaptogenesis. Here, we identify the neuronal thrombospondin receptor involved in CNS synapse formation as alpha2delta-1, the receptor for the anti-epileptic and analgesic drug gabapentin. We show that the VWF-A domain of alpha2delta-1 interacts with the epidermal growth factor-like repeats common to all thrombospondins. alpha2delta-1 overexpression increases synaptogenesis in vitro and in vivo and is required postsynaptically for thrombospondin- and astrocyte-induced synapse formation in vitro. Gabapentin antagonizes thrombospondin binding to alpha2delta-1 and powerfully inhibits excitatory synapse formation in vitro and in vivo. These findings identify alpha2delta-1 as a receptor involved in excitatory synapse formation and suggest that gabapentin may function therapeutically by blocking new synapse formation.
Project description:VGCCs are multisubunit complexes that play a crucial role in neuronal signaling. Auxiliary ?2? subunits of VGCCs modulate trafficking and biophysical properties of the pore-forming ?1 subunit and trigger excitatory synaptogenesis. Alterations in the expression level of ?2? subunits were implicated in several syndromes and diseases, including chronic neuropathic pain, autism, and epilepsy. However, the contribution of distinct ?2? subunits to excitatory/inhibitory imbalance and aberrant network connectivity characteristic for these pathologic conditions remains unclear. Here, we show that ?2?1 overexpression enhances spontaneous neuronal network activity in developing and mature cultures of hippocampal neurons. In contrast, overexpression, but not downregulation, of ?2?3 enhances neuronal firing in immature cultures, whereas later in development it suppresses neuronal activity. We found that ?2?1 overexpression increases excitatory synaptic density and selectively enhances presynaptic glutamate release, which is impaired on ?2?1 knockdown. Overexpression of ?2?3 increases the excitatory synaptic density as well but also facilitates spontaneous GABA release and triggers an increase in the density of inhibitory synapses, which is accompanied by enhanced axonaloutgrowth in immature interneurons. Together, our findings demonstrate that ?2?1 and ?2?3 subunits play distinct but complementary roles in driving formation of structural and functional network connectivity during early development. An alteration in ?2? surface expression during critical developmental windows can therefore play a causal role and have a profound impact on the excitatory-to-inhibitory balance and network connectivity.SIGNIFICANCE STATEMENT The computational capacity of neuronal networks is determined by their connectivity. Chemical synapses are the main interface for transfer of information between individual neurons. The initial formation of network connectivity requires spontaneous electrical activity and the calcium channel-mediated signaling. We found that, in early development, auxiliary ?2?3 subunits of calcium channels foster presynaptic release of GABA, trigger formation of inhibitory synapses, and promote axonal outgrowth in inhibitory interneurons. In contrast, later in development, ?2?1 subunits promote the glutamatergic neurotransmission and synaptogenesis, as well as strongly enhance neuronal network activity. We propose that formation of connectivity in neuronal networks is associated with a concerted interplay of ?2?1 and ?2?3 subunits of calcium channels.
Project description:Facet joint injury induces persistent pain that may be maintained by structural plasticity in the spinal cord. Astrocyte-derived thrombospondins, especially thrombospondin-4 (TSP4), have been implicated in synaptogenesis and spinal sensitization in neuropathic pain, but the TSP4 response and its relationship to synaptic changes in the spinal cord have not been investigated for painful joint injury. This study investigates the role of TSP4 in the development and maintenance of persistent pain following injurious facet joint distraction in rats and tests the hypothesis that excitatory synaptogenesis contributes to such pain. Painful facet joint loading induces dorsal horn excitatory synaptogenesis along with decreased TSP4 in the DRG and increased astrocytic release of TSP4 in the spinal cord, all of which parallel the time course of sustained tactile allodynia. Blocking injury-induced spinal TSP4 expression with antisense oligonucleotides or reducing TSP4 activity at its neuronal receptor in the spinal cord with gabapentin treatment both attenuate the allodynia and dorsal horn synaptogenesis that develop after painful facet joint loading. Increased spinal TSP4 also facilitates the development of allodynia and spinal hyperexcitability, even after non-painful physiological loading of the facet joint. These results suggest that spinal TSP4 plays an important role in the development and maintenance of persistent joint-mediated pain by inducing excitatory synaptogenesis and facilitating the transduction of mechanical loading of the facet joint that leads to spinal hyperexcitability.
Project description:Vagal Nerve Stimulation (VNS) Therapy® is a United States Food and Drug Administration approved neurotherapeutic for medically refractory partial epilepsy and treatment-resistant depression. The molecular mechanisms underlying its beneficial effects are unclear. We hypothesized that one mechanism involves neuronal activity-dependent modifications of central nervous system excitatory synapses. To begin to test this hypothesis, we asked whether VNS modifies the activity of neurons in amygdala and hippocampus. Neuronal recordings from adult, freely moving rats revealed that activity in both amygdala and hippocampus was modified by VNS immediately after its application, and changes were detected following 1 week of stimulation. To investigate whether VNS modifies the proteome of excitatory synapses, we established a label-free, quantitative liquid chromatography-tandem mass spectrometry workflow that enables global analysis of the constituents of the postsynaptic density (PSD) proteome. PSD proteins were biochemically purified from amygdala/piriform cortex of VNS- or dummy-treated rats following 1-week stimulation, and individual PSD protein levels were quantified by liquid chromatography-tandem mass spectrometry analysis. We identified 1899 unique peptides corresponding to 425 proteins in PSD fractions, of which expression levels of 22 proteins were differentially regulated by VNS with changes greater than 150%. Changes in a subset of these proteins, including significantly increased expression of neurexin-1?, cadherin 13 and voltage-dependent calcium channel ?2?1, the primary target of the antiepileptic drug gabapentin, and decreased expression of voltage-dependent calcium channel ?3, were confirmed by western blot analysis of PSD samples. These results demonstrate that VNS modulates excitatory synapses through regulating a subset of the PSD proteome. Our study reveals molecular targets of VNS and point to possible mechanisms underlying its beneficial effects, including activity-dependent formation of excitatory synapses.
Project description:Highly coordinated and coincidental patterns of activity-dependent mechanisms ("fire together wire together") are thought to serve as inductive signals during synaptogenesis, enabling neuronal pairing between specific sub-sets of excitatory partners. However, neither the nature of activity triggers, nor the "activity signature" of long-term neuronal firing in developing/regenerating neurons have yet been fully defined. Using a highly tractable model system comprising of identified cholinergic neurons from Lymnaea, we have discovered that intrinsic trophic factors present in the Lymnaea brain-conditioned medium (CM) act as a natural trigger for activity patterns in post- but not the presynaptic neuron. Using microelectrode array recordings, we demonstrate that trophic factors trigger stereotypical activity patterns that include changes in frequency, activity and variance. These parameters were reliable indicators of whether a neuron expressed functional excitatory or inhibitory nAChRs and synapse formation. Surprisingly, we found that the post- but not the presynaptic cell exhibits these changes in activity patterns, and that the functional expression of excitatory nAChRs required neuronal somata, de novo protein synthesis and voltage gated calcium channels. In summary, our data provides novel insights into trophic factor mediated actions on neuronal activity and its specific regulation of nAChR expression.
Project description:Secreted Wnts play crucial roles in synaptogenesis and synapse maintenance, but endogenous factors promoting synapse elimination in central neurons remain unknown. Here we show that proline-rich 7 (PRR7) induces specific removal of excitatory synapses and acts as a Wnt inhibitor. Remarkably, transmembrane protein PRR7 is activity-dependently released by neurons via exosomes. Exosomal PRR7 is uptaken by neurons through membrane fusion and eliminates excitatory synapses in neighboring neurons. Conversely, PRR7 knockdown in sparse neurons greatly increases excitatory synapse numbers in all surrounding neurons. These non-cell autonomous effects of PRR7 are effectively negated by augmentation or blockade of Wnt signaling. PRR7 exerts its effect by blocking the exosomal secretion of Wnts, activation of GSK3?, and promoting proteasomal degradation of PSD proteins. These data uncover a proximity-dependent, reciprocal mechanism for the regulation of excitatory synapse numbers in local neurons and demonstrate the significance of exosomes in inter-neuronal signaling in the vertebrate brain.
Project description:Neuronal activity-inducible gene transcription correlates with rapid and transient increases in histone acetylation at promoters and enhancers of activity-regulated genes. Exactly how histone acetylation modulates transcription of these genes has remained unknown. We used single-cell in situ transcriptional analysis to show that Fos and Npas4 are transcribed in stochastic bursts in mouse neurons and that membrane depolarization increases mRNA expression by increasing burst frequency. We then expressed dCas9-p300 or dCas9-HDAC8 fusion proteins to mimic or block activity-induced histone acetylation locally at enhancers. Adding histone acetylation increased Fos transcription by prolonging burst duration and resulted in higher Fos protein levels and an elevation of resting membrane potential. Inhibiting histone acetylation reduced Fos transcription by reducing burst frequency and impaired experience-dependent Fos protein induction in the hippocampus in vivo. Thus, activity-inducible histone acetylation tunes the transcriptional dynamics of experience-regulated genes to affect selective changes in neuronal gene expression and cellular function.
Project description:During development of the central nervous system, precise synaptic connections between presynaptic and postsynaptic neurons are formed. While significant progress has been made in our understanding of AMPA receptor trafficking during synaptic plasticity, less is known about the molecules that recruit AMPA receptors to nascent synapses during synaptogenesis. Here we identify a type II transmembrane protein (SynDIG1) that regulates AMPA receptor content at developing synapses in dissociated rat hippocampal neurons. SynDIG1 colocalizes with AMPA receptors at synapses and at extrasynaptic sites and associates with AMPA receptors in heterologous cells and brain. Altered levels of SynDIG1 in cultured neurons result in striking changes in excitatory synapse number and function. SynDIG1-mediated synapse development is dependent on association with AMPA receptors via its extracellular C terminus. Intriguingly, SynDIG1 content in dendritic spines is regulated by neuronal activity. Altogether, we define SynDIG1 as an activity-regulated transmembrane protein that regulates excitatory synapse development.
Project description:A systematic assessment of global neural network connectivity through direct electrophysiological assays has remained technically infeasible, even in simpler systems like dissociated neuronal cultures. We introduce an improved algorithmic approach based on Transfer Entropy to reconstruct structural connectivity from network activity monitored through calcium imaging. We focus in this study on the inference of excitatory synaptic links. Based on information theory, our method requires no prior assumptions on the statistics of neuronal firing and neuronal connections. The performance of our algorithm is benchmarked on surrogate time series of calcium fluorescence generated by the simulated dynamics of a network with known ground-truth topology. We find that the functional network topology revealed by Transfer Entropy depends qualitatively on the time-dependent dynamic state of the network (bursting or non-bursting). Thus by conditioning with respect to the global mean activity, we improve the performance of our method. This allows us to focus the analysis to specific dynamical regimes of the network in which the inferred functional connectivity is shaped by monosynaptic excitatory connections, rather than by collective synchrony. Our method can discriminate between actual causal influences between neurons and spurious non-causal correlations due to light scattering artifacts, which inherently affect the quality of fluorescence imaging. Compared to other reconstruction strategies such as cross-correlation or Granger Causality methods, our method based on improved Transfer Entropy is remarkably more accurate. In particular, it provides a good estimation of the excitatory network clustering coefficient, allowing for discrimination between weakly and strongly clustered topologies. Finally, we demonstrate the applicability of our method to analyses of real recordings of in vitro disinhibited cortical cultures where we suggest that excitatory connections are characterized by an elevated level of clustering compared to a random graph (although not extreme) and can be markedly non-local.