Postsynaptic Sites Research Articles

Dendritic Spine Quantification Using an Automatic Three-Dimensional Neuron Reconstruction Software.

Synaptic connections allow for the exchange and processing of information between neurons. The post-synaptic site of excitatory synapses is often formed on dendritic spines. Dendritic spines are structures of great interest in research centered around synaptic plasticity, neurodevelopment, and neurological and psychiatric disorders. Dendritic spines undergo structural modifications during their lifespan, with properties such as total spine number, dendritic spine size, and morphologically defined subtype altering in response to different processes. Delineating the molecular mechanisms regulating these structural alterations of dendritic spines relies on morphological measurement. This mandates accurate and reproducible dendritic spine analysis to provide experimental evidence. The present study outlines a detailed protocol for dendritic spine quantification and classification using Neurolucida 360 (automatic three-dimensional neuron reconstruction software). This protocol allows for the determination of key dendritic spine properties such as total spine density, spine head volume, and classification into spine subtypes thus enabling effective analysis of dendritic spine structural phenotypes.

Spinal PTP1B Regulated NMDA Receptor-mediated Nociceptive Transmission and Peripheral Inflammation-induced Pain Sensitization.

Protein tyrosine phosphatases (PTPs) catalyze the dephosphorylation of several pain-related substrates in spinal cord dorsal horn and are critically involved in the modification of pain transmission. The current study demonstrated that protein tyrosine phosphatase 1B (PTP1B), a unique endoplasmic reticulum-resident member of PTP family, displayed an activity-dependent increase in its protein expression and synaptic localization in spinal dorsal horn of adult male rats. PTP1B interacted with the Src Homology 3 (SH3) domain of Synapse-Associated Protein 102 (SAP102), one of the postsynaptic scaffolding proteins that anchored PTP1B at postsynaptic sites. The SAP102-tethered PTP1B augmented the synaptic transmission mediated specifically by GluN2B subunit-containing N-methyl-D-aspartate subtype glutamate receptors. Interference with PTP1B activity or disruption of its interaction with SAP102 attenuated GluN2B-mediated nociceptive transmission and ameliorated pain sensitization induced by intraplantar injection of Complete Freund's Adjuvant. These data suggested that the activity-dependent synaptic redistribution of PTP1B served as an important mechanism regulating GluN2B receptor activity and that manipulation of PTP1B synaptic targeting might represent an effective approach for the treatment of chronic inflammatory pain.

A trans-synaptic IgLON adhesion molecular complex directly contacts and clusters a nicotinic receptor.

The localization and clustering of neurotransmitter receptors at appropriate postsynaptic sites is a key step in the control of synaptic transmission. Here, we identify a novel paradigm for the synaptic localization of an ionotropic acetylcholine receptor (AChR) based on the direct interaction of its extracellular domain with a cell adhesion molecule of the IgLON family. Our results show that RIG-5 and ZIG-8, which encode the sole IgLONs in C. elegans, are tethered in the pre- and postsynaptic membranes, respectively, and interact in vivo through their first immunoglobulin-like (Ig) domains. In addition, ZIG-8 traps ACR-16 via a direct cis- interaction between the ZIG-8 Ig2 domain and the base of the large extracellular AChR domain. Such mechanism has never been reported, but all these molecules are conserved during evolution. Similar interactions may directly couple Ig superfamily adhesion molecules and members of the large family of Cys-loop ionotropic receptors, including AChRs, in the mammalian nervous system, and may be relevant in the context of IgLON-associated brain diseases.

SARM1 is essential for NMDA receptor-dependent endocytosis of AMPA receptors in hippocampal neurons

Long-term depression (LTD) is a form of synaptic plasticity thought to be the cellular basis of experience-dependent learning and memory. LTD is caused by an activity-dependent decrease in cell surface α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors (AMPA receptors) at the postsynaptic sites. However, the mechanism through which AMPA receptors are removed from the cell surface via neuronal activity is not fully understood. In this study, we showed that small interfering RNA (siRNA)-mediated knockdown of sterile alpha and toll/interleukin receptor motif containing 1 (SARM1) in cultured hippocampal neurons prevented the N-methyl-d-aspartate (NMDA)-induced reduction in cell surface AMPA receptors. However, the control RNA did not affect NMDA-mediated AMPA receptor trafficking. Overexpression of the siRNA-resistant form of SARM1 in SARM1-knocked-down neurons restored AMPA receptor trafficking. However, overexpression of SARM1, which lacks the mitochondrial transport signal, in the SARM1-knocked-down neurons did not restore NMDA-dependent AMPA receptor endocytosis. Moreover, the inhibition of the NADase activity of SARM1 blocked the NMDA-induced reduction of cell surface AMPA receptors. These results suggest that both the mitochondrial localization and NADase activity of SARM1 are essential for NMDA receptor-dependent AMPA receptor internalization in the hippocampal neurons.

Neural Development and Repair Induced by Femtosecond Laser Stimulation.

Dendritic spines function as postsynaptic sites, receiving excitatory signals from presynaptic axons. The synaptic plasticity of spines underlies the refinement of neuronal circuits. Neural cognitive disorders are commonly associated with the impairment and elimination of dendritic spines. In this study, we report an all-optical method to activate dendritic spine growth and regeneration by a single short flash of femtosecond laser stimulation. The inhibited development and loss of spines can be rescued by a transient illumination of the laser inside a micrometer region of the soma by activating the extracellular signal-regulated kinase (ERK) signaling pathway. The rescued neurons exhibit function. Hence we provide a potential noninvasive method for the regeneration of dendritic spines.

Tumor necrosis factor alpha induces NOX2-dependent reactive oxygen species production in hypothalamic paraventricular nucleus neurons following angiotensin II infusion

There is evidence that tumor necrosis factor alpha (TNFα) influences autonomic processes coordinated within the hypothalamic paraventricular nucleus (PVN), however, the signaling mechanisms subserving TNFα′s actions in this brain area are unclear. In non-neuronal cell types, TNFα has been shown to play an important role in canonical NADPH oxidase (NOX2)-mediated production of reactive oxygen species (ROS), molecules also known to be critically involved in hypertension. However, little is known about the role of TNFα in NOX2-dependent ROS production in the PVN within the context of hypertension. Using dual labeling immunoelectron microscopy and dihydroethidium (DHE) microfluorography, we provide structural and functional evidence for interactions between TNFα and NOX2 in the PVN. The TNFα type 1 receptor (TNFR1), the major mediator of TNFα signaling in the PVN, was commonly co-localized with the catalytic gp91phox subunit of NOX2 in postsynaptic sites of PVN neurons. Additionally, there was an increase in dual labeled dendritic profiles following fourteen-day slow-pressor angiotensin II (AngII) infusion. Using DHE microfluorography, it was also shown that TNFα application resulted in a NOX2-dependent increase in ROS in isolated PVN neurons projecting to the spinal cord. Further, TNFα-mediated ROS production was heightened after AngII infusion. The finding that TNFR1 and gp91phox are positioned for rapid interactions, particularly in PVN-spinal cord projection neurons, provides a molecular substrate by which inflammatory signaling and oxidative stress may jointly contribute to AngII hypertension.

Presynaptic Rac1 in the hippocampus selectively regulates working memory

One of the most extensively studied members of the Ras superfamily of small GTPases, Rac1 is an intracellular signal transducer that remodels actin and phosphorylation signaling networks. Previous studies have shown that Rac1-mediated signaling is associated with hippocampal-dependent working memory and longer-term forms of learning and memory and that Rac1 can modulate forms of both pre- and postsynaptic plasticity. How these different cognitive functions and forms of plasticity mediated by Rac1 are linked, however, is unclear. Here, we show that spatial working memory in mice is selectively impaired following the expression of a genetically encoded Rac1 inhibitor at presynaptic terminals, while longer-term cognitive processes are affected by Rac1 inhibition at postsynaptic sites. To investigate the regulatory mechanisms of this presynaptic process, we leveraged new advances in mass spectrometry to identify the proteomic and post-translational landscape of presynaptic Rac1 signaling. We identified serine/threonine kinases and phosphorylated cytoskeletal signaling and synaptic vesicle proteins enriched with active Rac1. The phosphorylated sites in these proteins are at positions likely to have regulatory effects on synaptic vesicles. Consistent with this, we also report changes in the distribution and morphology of synaptic vesicles and in postsynaptic ultrastructure following presynaptic Rac1 inhibition. Overall, this study reveals a previously unrecognized presynaptic role of Rac1 signaling in cognitive processes and provides insights into its potential regulatory mechanisms.

Presynaptic Rac1 in the hippocampus selectively regulates working memory.

One of the most extensively studied members of the Ras superfamily of small GTPases, Rac1 is an intracellular signal transducer that remodels actin and phosphorylation signaling networks. Previous studies have shown that Rac1-mediated signaling is associated with hippocampal-dependent working memory and longer-term forms of learning and memory and that Rac1 can modulate forms of both pre- and postsynaptic plasticity. How these different cognitive functions and forms of plasticity mediated by Rac1 are linked, however, is unclear. Here, we show that spatial working memory in mice is selectively impaired following the expression of a genetically encoded Rac1 inhibitor at presynaptic terminals, while longer-term cognitive processes are affected by Rac1 inhibition at postsynaptic sites. To investigate the regulatory mechanisms of this presynaptic process, we leveraged new advances in mass spectrometry to identify the proteomic and post-translational landscape of presynaptic Rac1 signaling. We identified serine/threonine kinases and phosphorylated cytoskeletal signaling and synaptic vesicle proteins enriched with active Rac1. The phosphorylated sites in these proteins are at positions likely to have regulatory effects on synaptic vesicles. Consistent with this, we also report changes in the distribution and morphology of synaptic vesicles and in postsynaptic ultrastructure following presynaptic Rac1 inhibition. Overall, this study reveals a previously unrecognized presynaptic role of Rac1 signaling in cognitive processes and provides insights into its potential regulatory mechanisms.

MC4R Localizes at Excitatory Postsynaptic and Peri-Postsynaptic Sites of Hypothalamic Neurons in Primary Culture.

The melanocortin-4 receptor (MC4R) is a G protein-coupled receptor (GPCR) that is expressed in several brain locations encompassing the hypothalamus and the brainstem, where the receptor controls several body functions, including metabolism. In a well-defined pathway to decrease appetite, hypothalamic proopiomelanocortin (POMC) neurons localized in the arcuate nucleus (Arc) project to MC4R neurons in the paraventricular nuclei (PVN) to release the natural MC4R agonist α-melanocyte-stimulating hormone (α-MSH). Arc neurons also project excitatory glutamatergic fibers to the MC4R neurons in the PVN for a fast synaptic transmission to regulate a satiety pathway potentiated by α-MSH. By using super-resolution microscopy, we found that in hypothalamic neurons in a primary culture, postsynaptic density protein 95 (PSD95) colocalizes with GluN1, a subunit of the ionotropic N-methyl-D-aspartate receptor (NMDAR). Thus, hypothalamic neurons form excitatory postsynaptic specializations. To study the MC4R distribution at these sites, tagged HA-MC4R under the synapsin promoter was expressed in neurons by adeno-associated virus (AAV) gene transduction. HA-MC4R immunofluorescence peaked at the center and in proximity to the PSD95- and NMDAR-expressing sites. These data provide morphological evidence that MC4R localizes together with glutamate receptors at postsynaptic and peri-postsynaptic sites.

Peptide and Peptidomimetic Inhibitors Targeting the Interaction of Collapsin Response Mediator Protein 2 with the N-Type Calcium Channel for Pain Relief.

Ion channels serve pleiotropic functions. Often found in complexes, their activities and functions are sculpted by auxiliary proteins. We discovered that collapsin response mediator protein 2 (CRMP2) is a binding partner and regulator of the N-type voltage-gated calcium channel (CaV2.2), a genetically validated contributor to chronic pain. Herein, we trace the discovery of a new peptidomimetic modulator of this interaction, starting from the identification and development of CBD3, a CRMP2-derived CaV binding domain peptide. CBD3 uncouples CRMP2-CaV2.2 binding to decrease CaV2.2 surface localization and calcium currents. These changes occur at presynaptic sites of nociceptive neurons and indeed, CBD3 ameliorates chronic pain in preclinical models. In pursuit of a CBD3 peptidomimetic, we exploited a unique approach to identify a dipeptide with low conformational flexibility and high solvent accessibility that anchors binding to CaV2.2. From a pharmacophore screen, we obtained CBD3063, a small-molecule that recapitulated CBD3's activity, reversing nociceptive behaviors in rodents of both sexes without sensory, affective, or cognitive effects. By disrupting the CRMP2-CaV2.2 interaction, CBD3063 exerts these effects indirectly through modulating CaV2.2 trafficking, supporting CRMP2 as an auxiliary subunit of CaV2.2. The parent peptide CBD3 was also found by us and others to have neuroprotective properties at postsynaptic sites, through N-methyl-d-aspartate receptor and plasmalemmal Na+/Ca2+ exchanger 3, potentially acting as an auxiliary subunit for these pathways as well. Our new compound is poised to address several open questions regarding CRMP2's role in regulating the CaV2.2 pathways to treat pain with the potential added benefit of neuroprotection.

Blockade of pre- and post-synaptic GABAB receptors in the anteroventral bed nucleus of stria terminalis produces anxiolytic-like and anxiety-like effects in parkinsonian rats respectively

The anteroventral bed nucleus of stria terminalis (avBNST) is a limbic forebrain region involved in the regulation of anxiety, and expresses GABAB receptors, which are located at both pre- and post-synaptic sites. However, it is unclear how blockade of these receptors affects anxiety-like behaviors, particularly in Parkinson's disease (PD)-related anxiety. In the present study, unilateral 6-hydroxydopamine (6-OHDA) lesions of the substantia nigra pars compacta in rats induced anxiety-like behaviors, and increased GABA release and decreased glutamate release in the avBNST, as well as decreased level of dopamine (DA) in the basolateral amygdala (BLA). Intra-avBNST injection of pre-synaptic GABAB receptor antagonist CGP36216 produced anxiolytic-like effects, while the injection of post-synaptic GABAB receptor antagonist CGP35348 induced anxiety-like responses in both sham and 6-OHDA rats. Intra-avBNST injection of CGP36216 inhibited the GABAergic neurons and increased GABA/glutamate ratio in the avBNST and increased levels of DA and serotonin (5-HT) in the BLA; conversely, CGP35348 produced opposite effects on the firing activity of avBNST GABAergic neurons and levels of the neurotransmitters in the avBNST and BLA. Moreover, the doses of the antagonists producing significant behavioral effects in 6-OHDA rats were lower than those in sham rats, and the duration of action of the antagonists on the firing rate of the neurons and release of the neurotransmitters was prolonged in 6-OHDA rats. Altogether, these findings suggest that pre- and post-synaptic GABAB receptors in the avBNST are implicated in PD-related anxiety-like behaviors, and degeneration of the nigrostriatal pathway enhances functions and/or upregulates expression of these receptors.

Two-photon imaging of excitatory and inhibitory neural response to infrared neural stimulation.

Pulsed infrared neural stimulation (INS, 1875nm) is an emerging neurostimulation technology that delivers focal pulsed heat to activate functionally specific mesoscale networks and holds promise for clinical application. However, little is known about its effect on excitatory and inhibitory cell types in cerebral cortex. Estimates of summed population neuronal response time courses provide a potential basis for neural and hemodynamic signals described in other studies. Using two-photon calcium imaging in mouse somatosensory cortex, we have examined the effect of INS pulse train application on hSyn neurons and mDlx neurons tagged with GCaMP6s. We find that, in anesthetized mice, each INS pulse train reliably induces robust response in hSyn neurons exhibiting positive going responses. Surprisingly, mDlx neurons exhibit negative going responses. Quantification using the index of correlation illustrates responses are reproducible, intensity-dependent, and focal. Also, a contralateral activation is observed when INS applied. In sum, the population of neurons stimulated by INS includes both hSyn and mDlx neurons; within a range of stimulation intensities, this leads to overall excitation in the stimulated population, leading to the previously observed activations at distant post-synaptic sites.

Mapping proteomic composition of excitatory postsynaptic sites in the cerebellar cortex.

Functions of the cerebellar cortex, from motor learning to emotion and cognition, depend on the appropriate molecular composition at diverse synapse types. Glutamate receptor distributions have been partially mapped using immunogold electron microscopy. However, information is lacking on the distribution of many other components, such as Shank2, a postsynaptic scaffolding protein whose cerebellar dysfunction is associated with autism spectrum disorders. Here, we used an adapted Magnified Analysis of the Proteome, an expansion microscopy approach, to map multiple glutamate receptors, scaffolding and signaling proteins at single synapse resolution in the cerebellar cortex. Multiple distinct synapse-selective distribution patterns were observed. For example, AMPA receptors were most concentrated at synapses on molecular layer interneurons and at climbing fiber synapses, Shank1 was most concentrated at parallel fiber synapses on Purkinje cells, and Shank2 at both climbing fiber and parallel fiber synapses on Purkinje cells but little on molecular layer interneurons. Our results are consistent with gene expression data but also reveal input-selective targeting within Purkinje cells. In specialized glomerular structures of the granule cell layer, AMPA receptors as well as most other synaptic components preferentially targeted to synapses. However, NMDA receptors and the synaptic GTPase activating protein SynGAP preferentially targeted to extrasynaptic sites. Thus, glomeruli may be considered integrative signaling units through which mossy fibers differentially activate synaptic AMPA and extrasynaptic NMDA receptor complexes. Furthermore, we observed NMDA receptors and SynGAP at adherens junctions, suggesting a role in structural plasticity of glomeruli. Altogether, these data contribute to mapping the cerebellar 'synaptome'.

Predation cues induce predator specific changes in olfactory neurons encoding defensive responses in agile frog tadpoles.

Although behavioural defensive responses have been recorded several times in both laboratory and natural habitats, their neural mechanisms have seldom been investigated. To explore how chemical, water-borne cues are conveyed to the forebrain and instruct behavioural responses in anuran larvae, we conditioned newly hatched agile frog tadpoles using predator olfactory cues, specifically either native odonate larvae or alien crayfish kairomones. We expected chronic treatments to influence the basal neuronal activity of the tadpoles' mitral cells and alter their sensory neuronal connections, thereby impacting information processing. Subsequently, these neurons were acutely perfused, and their responses were compared with the defensive behaviour of tadpoles previously conditioned and exposed to the same cues. Tadpoles conditioned with odonate cues differed in both passive and active cell properties compared to those exposed to water (controls) or crayfish cues. The observed upregulation of membrane conductance and increase in both the number of active synapses and receptor density at the postsynaptic site are believed to have enhanced their responsiveness to external stimuli. Odonate cues also affected the resting membrane potential and firing rate of mitral cells during electrophysiological patch-clamp recordings, suggesting a rearrangement of the repertoire of voltage-dependent conductances expressed in cell membranes. These recorded neural changes may modulate the induction of an action potential and transmission of information. Furthermore, the recording of neural activity indicated that the lack of defensive responses towards non-native predators is due to the non-recognition of their olfactory cues.

Rapid sequential clustering of NMDARs, CaMKII, and AMPARs upon activation of NMDARs at developing synapses.

Rapid, synapse-specific neurotransmission requires the precise alignment of presynaptic neurotransmitter release and postsynaptic receptors. How postsynaptic glutamate receptor accumulation is induced during maturation is not well understood. We find that in cultures of dissociated hippocampal neurons at 11 days in vitro (DIV) numerous synaptic contacts already exhibit pronounced accumulations of the pre- and postsynaptic markers synaptotagmin, synaptophysin, synapsin, bassoon, VGluT1, PSD-95, and Shank. The presence of an initial set of AMPARs and NMDARs is indicated by miniature excitatory postsynaptic currents (mEPSCs). However, AMPAR and NMDAR immunostainings reveal rather smooth distributions throughout dendrites and synaptic enrichment is not obvious. We found that brief periods of Ca2+ influx through NMDARs induced a surprisingly rapid accumulation of NMDARs within 1 min, followed by accumulation of CaMKII and then AMPARs within 2-5 min. Postsynaptic clustering of NMDARs and AMPARs was paralleled by an increase in their mEPSC amplitudes. A peptide that blocked the interaction of NMDAR subunits with PSD-95 prevented the NMDAR clustering. NMDAR clustering persisted for 3 days indicating that brief periods of elevated glutamate fosters permanent accumulation of NMDARs at postsynaptic sites in maturing synapses. These data support the model that strong glutamatergic stimulation of immature glutamatergic synapses results in a fast and substantial increase in postsynaptic NMDAR content that required NMDAR binding to PSD-95 or its homologues and is followed by recruitment of CaMKII and subsequently AMPARs.

Tau regulates Arc stability in neuronal dendrites via a proteasome-sensitive but ubiquitin-independent pathway

Tauopathies are neurodegenerative disorders characterized by the deposition of aggregates of the microtubule-associated protein tau, a main component of neurofibrillary tangles. Alzheimer’s disease (AD) is the most common type of tauopathy and dementia, with amyloid-beta pathology as an additional hallmark feature of the disease. Besides its role in stabilizing microtubules, tau is localized at postsynaptic sites and can regulate synaptic plasticity. The activity-regulated cytoskeleton-associated protein (Arc), is an immediate early gene that plays a key role in synaptic plasticity, learning and memory. Arc has been implicated in AD pathogenesis and regulates the release of amyloid-beta (Aβ). We found that decreased Arc levels correlate with AD status and disease severity. Importantly, Arc protein was upregulated in the hippocampus of Tau knockout (Tau KO) mice and dendrites of Tau KO primary hippocampal neurons. Overexpression of tau decreased Arc stability in an activity-dependent manner, exclusively in neuronal dendrites, which was coupled to an increase in the expression of dendritic and somatic surface GluA1-containing α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptors. The tau-dependent decrease in Arc was found to be proteasome-sensitive, yet independent of Arc ubiquitination and required the endophilin-binding domain of Arc. Importantly, these effects on Arc stability and GluA1 localization were not observed in the commonly studied tau mutant, P301L. These observations provide a potential molecular basis for synaptic dysfunction mediated through the accumulation of tau in dendrites. Our findings confirm that Arc is misregulated in AD, and further show a physiological role for tau in regulating Arc stability and AMPA receptor targeting.

Molecular Organization and Regulation of the Mammalian Synapse by the Post-Translational Modification SUMOylation.

Neurotransmission occurs within highly specialized compartments forming the active synapse where the complex organization and dynamics of the interactions are tightly orchestrated both in time and space. Post-translational modifications (PTMs) are central to these spatiotemporal regulations to ensure an efficient synaptic transmission. SUMOylation is a dynamic PTM that modulates the interactions between proteins and consequently regulates the conformation, the distribution and the trafficking of the SUMO-target proteins. SUMOylation plays a crucial role in synapse formation and stabilization, as well as in the regulation of synaptic transmission and plasticity. In this review, we summarize the molecular consequences of this protein modification in the structural organization and function of the mammalian synapse. We also outline novel activity-dependent regulation and consequences of the SUMO process and explore how this protein modification can functionally participate in the compartmentalization of both pre- and post-synaptic sites.

Biophysical Modeling of Synaptic Plasticity.

Dendritic spines are small, bulbous compartments that function as postsynaptic sites and undergo intense biochemical and biophysical activity. The role of the myriad signaling pathways that are implicated in synaptic plasticity is well studied. A recent abundance of quantitative experimental data has made the events associated with synaptic plasticity amenable to quantitative biophysical modeling. Spines are also fascinating biophysical computational units because spine geometry, signal transduction, and mechanics work in a complex feedback loop to tune synaptic plasticity. In this sense, ideas from modeling cell motility can inspire us to develop multiscale approaches for predictive modeling of synaptic plasticity. In this article, we review the key steps in postsynaptic plasticity with a specific focus on the impact of spine geometry on signaling, cytoskeleton rearrangement, and membrane mechanics. We summarize the main experimental observations and highlight how theory and computation can aid our understanding of these complex processes.

The gephyrin scaffold modulates cortical layer 2/3 pyramidal neuron responsiveness to single whisker stimulation

Gephyrin is the main scaffolding protein at inhibitory postsynaptic sites, and its clusters are the signaling hubs where several molecular pathways converge. Post-translational modifications (PTMs) of gephyrin alter GABAA receptor clustering at the synapse, but it is unclear how this affects neuronal activity at the circuit level. We assessed the contribution of gephyrin PTMs to microcircuit activity in the mouse barrel cortex by slice electrophysiology and in vivo two-photon calcium imaging of layer 2/3 (L2/3) pyramidal cells during single-whisker stimulation. Our results suggest that, depending on the type of gephyrin PTM, the neuronal activities of L2/3 pyramidal neurons can be differentially modulated, leading to changes in the size of the neuronal population responding to the single-whisker stimulation. Furthermore, we show that gephyrin PTMs have their preference for selecting synaptic GABAA receptor subunits. Our results identify an important role of gephyrin and GABAergic postsynaptic sites for cortical microcircuit function during sensory stimulation.

Activation of innate immune receptor TLR9 by mitochondrial DNA plays essential roles in the chemical long-term depression of hippocampal neurons

Synaptic plasticity is believed to be the cellular basis for experience-dependent learning and memory. Although long-term depression (LTD), a form of synaptic plasticity, is caused by the activity-dependent reduction of cell surface α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type glutamate receptors (AMPA receptors) at postsynaptic sites, its regulation by neuronal activity is not completely understood. In this study, we showed that the inhibition of toll-like receptor-9 (TLR9), an innate immune receptor, suppresses N-methyl-d-aspartate (NMDA)-induced reduction of cell surface AMPA receptors in cultured hippocampal neurons. We found that inhibition of TLR9 also blocked NMDA-induced activation of caspase-3, which plays an essential role in the induction of LTD. siRNA-based knockdown of TLR9 also suppressed the NMDA-induced reduction of cell surface AMPA receptors, although the scrambled RNA had no effect on the NMDA-induced trafficking of AMPA receptors. Overexpression of the siRNA-resistant form of TLR9 rescued the AMPA receptor trafficking abolished by siRNA. Furthermore, NMDA stimulation induced rapid mitochondrial morphological changes, mitophagy, and the binding of mitochondrial DNA (mtDNA) to TLR9. Treatment with dideoxycytidine and mitochondrial division inhibitor-1, which block mtDNA replication and mitophagy, respectively, inhibited NMDA-dependent AMPA receptor internalization. These results suggest that mitophagy induced by NMDA receptor activation releases mtDNA and activates TLR9, which plays an essential role in the trafficking of AMPA receptors during the induction of LTD.