Homeostatic Synaptic Plasticity of Miniature Excitatory Postsynaptic Currents in Mouse Cortical Cultures Requires Neuronal Rab3A.

Following prolonged activity blockade, amplitudes of miniature excitatory postsynaptic currents (mEPSCs) increase, a form of plasticity termed "homeostatic synaptic plasticity." We previously showed that a presynaptic protein, the small GTPase Rab3A, is required for full expression of the increase in miniature endplate current amplitudes following prolonged blockade of action potential activity at the mouse neuromuscular junction in vivo (Wang et al., 2011), but it is unknown whether this form of Rab3A-dependent homeostatic plasticity shares any characteristics with central synapses. We show here that homeostatic synaptic plasticity of mEPSCs is impaired in mouse cortical neuron cultures prepared from Rab3A-/- and mutant mice expressing a single point mutation of Rab3A, Rab3A Earlybird mice. To determine if Rab3A is involved in the well-established homeostatic increase in postsynaptic AMPA-type receptors (AMPARs), we performed a series of experiments in which electrophysiological recordings of mEPSCs and confocal imaging of synaptic AMPAR immunofluorescence were assessed within the same cultures. We found that Rab3A was required for the increase in synaptic AMPARs following prolonged activity blockade, but the increase in mEPSC amplitudes was not always accompanied by an increase in postsynaptic AMPAR levels, suggesting other factors may contribute. Finally, we demonstrate that Rab3A is acting in neurons because only selective loss of Rab3A in neurons, not glia, disrupted the homeostatic increase in mEPSC amplitudes. This is the first demonstration that neuronal Rab3A is required for homeostatic synaptic plasticity and that it does so partially through regulation of the surface expression of AMPA receptors.

Fundamental Sex Differences in Cocaine-Induced Plasticity of Dopamine D1 Receptor- and D2 Receptor-Expressing Medium Spiny Neurons in the Mouse Nucleus Accumbens Shell.

Background: Cocaine-induced plasticity in the nucleus accumbens shell of males occurs primarily in dopamine D1 receptor-expressing medium spiny neurons (D1R-MSNs), with little if any impact on dopamine D2 receptor-expressing medium spiny neurons (D2R-MSNs). In females, the effect of cocaine on accumbens shell D1R- and D2R-MSN neurophysiology has yet to be reported, nor have estrous cycle effects been accounted for. Methods: We used a 5-day locomotor sensitization paradigm followed by a 10- to 14-day drug-free abstinence period. We then obtained ex vivo whole-cell recordings from fluorescently labeled D1R-MSNs and D2R-MSNs in the nucleus accumbens shell of male and female mice during estrus and diestrus. We examined accumbens shell neuronal excitability as well as miniature excitatory postsynaptic currents (mEPSCs). Results: In females, we observed alterations in D1R-MSN excitability across the estrous cycle similar in magnitude to the effects of cocaine in males. Furthermore, cocaine shifted estrous cycle-dependent plasticity from intrinsic excitability changes in D1R-MSNs to D2R-MSNs. In males, cocaine treatment produced the anticipated drop in D1R-MSN excitability with no effect on D2R-MSN excitability. Cocaine increased mEPSC frequencies and amplitudes in D2R-MSNs from females in estrus and mEPSC amplitudes of D2R-MSNs from females in diestrus. In males, cocaine increased both D1R- and D2R-MSN mEPSC amplitudes with no effect on mEPSC frequencies. Conclusions: Overall, while there are similar cocaine-induced disparities regarding the relative excitability of D1R-MSNs versus D2R-MSNs between the sexes, this is mediated through reduced D1R-MSN excitability in males, whereas it is due to heightened D2R-MSN excitability in females.

The nucleus accumbens shell (NAcSh) is a key brain region involved in motivation and reward. It is primarily composed of dopamine D₁ and D₂ receptor­expressing medium spiny neurons (D1R and D2R neurons). Previous studies in males demonstrated that D1R neurons undergo intrinsic plasticity following cocaine exposure, believed to underlie aspects of drug addiction. We confirmed this effect. It has also been generally assumed that females would show similar responses. However, this does not appear to be true, and our data indicate 2 novel findings. First, under baseline conditions, the estrous cycle produces recurring changes in D1R neuron excitability, with no changes observed in D2R neurons. Second, following cocaine exposure, D1R neuron plasticity is arrested, and D2R neurons begin to show estrous cycle effects on intrinsic excitability. These results indicate profound sex differences in the neurophysiological underpinnings of motivational behaviors including drug addiction.

Metformin Alleviates Pain States by Regulating the Balance of Spinal Synaptic Transmission.

BACKGROUND: Metformin has been shown to have potent analgesic effects; however, the underlying mechanism of synaptic plasticity mediating analgesia remained ambiguous. METHODS: In this study, animal behavioral tests, whole-cell patch­clamp recording, immunofluorescence staining, and network pharmacology techniques were applied to elucidate the mechanisms and potential targets of metformin-induced analgesia. RESULTS: Single or consecutive injections of metformin significantly inhibited spinal nerve ligation (SNL)-induced neuropathic pain, and formalin-induced acute inflammatory pain. Network pharmacology analysis of metformin action targets in pain database-related targets revealed 25 targets, including five hub targets (nitric oxide synthase 1 (NOS1), NOS2, NOS3, epidermal growth factor receptor (EGFR), and plasminogen (PLG)). Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis demonstrated that metformin-induced analgesia was markedly correlated with calcium signaling and synaptic transmission. Intrathecal injection of metformin significantly reversed nerve injury-induced c-Fos (neural activity biomarker) mRNA and protein expression in neuropathic rats by regulating NOS2 expression. In addition, whole-cell recordings of isolated spinal neurons demonstrated that metformin dose-dependently inhibited the enhanced frequency and amplitude of miniature excitatory synaptic currents (mEPSCs) but did not affect those of miniature inhibitory synaptic currents (mIPSCs) in neuropathic pain. CONCLUSIONS: This study further demonstrated that metformin might inhibit spinal glutamatergic transmission and abnormal nociceptive circuit transduction by monitoring synaptic transmission in pain. Results of this work provide an in-depth understanding of metformin analgesia via synaptic plasticity.

Stimulation of Neurite Outgrowth in Cerebrocortical Neurons by Sodium Channel Activator Brevetoxin-2 Requires Both N-Methyl-D-aspartate Receptor 2B (GluN2B) and p21 Protein (Cdc42/Rac)-Activated Kinase 1 (PAK1).

N-methyl-D-aspartate (NMDA) receptors play a critical role in activity-dependent dendritic arborization, spinogenesis, and synapse formation by stimulating calcium-dependent signaling pathways. Previously, we have shown that brevetoxin 2 (PbTx-2), a voltage-gated sodium channel (VGSC) activator, produces a concentration-dependent increase in intracellular sodium [Na+]I and increases NMDA receptor (NMDAR) open probabilities and NMDA-induced calcium (Ca2+) influxes. The objective of this study is to elucidate the downstream signaling mechanisms by which the sodium channel activator PbTx-2 influences neuronal morphology in murine cerebrocortical neurons. PbTx-2 and NMDA triggered distinct Ca2+-influx pathways, both of which involved the NMDA receptor 2B (GluN2B). PbTx-2-induced neurite outgrowth in day in vitro 1 (DIV-1) neurons required the small Rho GTPase Rac1 and was inhibited by both a PAK1 inhibitor and a PAK1 siRNA. PbTx-2 exposure increased the phosphorylation of PAK1 at Thr-212. At DIV-5, PbTx-2 induced increases in dendritic protrusion density, p-cofilin levels, and F-actin throughout the dendritic arbor and soma. Moreover, PbTx-2 increased miniature excitatory post-synaptic currents (mEPSCs). These data suggest that the stimulation of neurite outgrowth, spinogenesis, and synapse formation produced by PbTx-2 are mediated by GluN2B and PAK1 signaling.

Diverging from the Norm: Reevaluating What Miniature Excitatory Postsynaptic Currents Tell Us about Homeostatic Synaptic Plasticity.

The idea that the nervous system maintains a set point of network activity and homeostatically returns to that set point in the face of dramatic disruption-during development, after injury, in pathologic states, and during sleep/wake cycles-is rapidly becoming accepted as a key plasticity behavior, placing it alongside long-term potentiation and depression. The dramatic growth in studies of homeostatic synaptic plasticity of miniature excitatory synaptic currents (mEPSCs) is attributable, in part, to the simple yet elegant mechanism of uniform multiplicative scaling proposed by Turrigiano and colleagues: that neurons sense their own activity and globally multiply the strength of every synapse by a single factor to return activity to the set point without altering established differences in synaptic weights. We have recently shown that for mEPSCs recorded from control and activity-blocked cultures of mouse cortical neurons, the synaptic scaling factor is not uniform but is close to 1 for the smallest mEPSC amplitudes and progressively increases as mEPSC amplitudes increase, which we term divergent scaling. Using insights gained from simulating uniform multiplicative scaling, we review evidence from published studies and conclude that divergent synaptic scaling is the norm rather than the exception. This conclusion has implications for hypotheses about the molecular mechanisms underlying synaptic scaling.

Early synaptic deficits in GSK-3ß overexpressing mice.

Synaptic dysfunction is the prominent feature of many neuropsychiatric and neurological diseases, in which glycogen synthase kinase 3ß (GSK-3ß) has been shown to play a role. Overexpression of constitutively active form of GSK-3ß (GSK-3ß[S9A]) in mice recapitulates the cognitive and structural brain deficits characteristic for manic phase of bipolar disorder (BD). Yet, the mechanisms underlying GSK-3ß-induced synaptic dysfunction have not been fully elucidated. The aim of the present study was to dissect the effect of GSK-3ß overactivity on synaptic function in adolescent (3-week-old) mice. We found that overactivity of GSK-3ß in adolescent transgenic mice leads to an alteration in dendritic spines morphology of granule cells in dentate gyrus (DG) without changes in overall spine density. There was an increase in the number of thin, presumably immature dendritic spines in GSK-3ß[S9A] mice. Subsequent electrophysiological analysis showed changes in excitatory synaptic transmission manifested by an increase of inter-event intervals of miniature excitatory postsynaptic currents (mEPSCs) in DG granule cells and an increase in the number of silent (unfunctional) synapses at the perforant path-DG pathway in GSK-3ß[S9A] mice. Altogether, our data indicate that GSK-3ß overactivity leads to synaptic deficits in adolescent, GSK-3ß[S9A] mice. These data might provide potential mechanisms underlying GSK-3ß-induced synaptic dysfunction in psychiatric disorders.

Rapamycin Pretreatment Attenuates High Glucose-induced Alteration of Synaptic Transmission in Hippocampal Dentate Gyrus Neurons.

Diabetic neuropathy is one of the most common complications in patients with diabetes and leads to cognitive impairment. It is suggested that protracted hyperglycemia is the main trigger of cognitive deficits in diabetes and causes hippocampal abnormalities. Rapamycin, an inhibitor of mammalian target of rapamycin complex 1 (mTORC1), can significantly ameliorate cognitive deficits in neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease and Huntington disease. We employed whole-cell patch clamping to examine the effects of rapamycin on neuronal electrophysiological characteristics of the hippocampal dentate gyrus in mice under the condition of high glucose. We recorded the action potentials (APs) and the miniature excitatory postsynaptic currents (mEPSCs) of dentate gyrus neurons. We found that high glucose increased the half-width, the duration and decreased the peak amplitude of Aps as well as the inter-event interval (IEI) of mEPSCs in hippocampal dentate gyrus neurons. However, rapamycin pre-treatment reversed the changes induced by high glucose. Moreover, we demonstrated that rapamycin pre-treatment reversed the down-regulation of postsynaptic density protein 95 (PSD-95) expression caused by high glucose. Therefore, pre-treatment with rapamycin could ameliorate high glucose-induced alteration of synaptic transmission in the hippocampal dentate gyrus.

Protein Expression Landscape Defines the Formation Potential of Mouse Blastoids From EPSCs.

Preimplantation embryo development is a precisely regulated process organized by maternally inherited and newly synthesized proteins. Recently, some studies have reported that blastocyst-like structures, named blastoids, can be generated from mouse ESCs (embryonic stem cells) or EPSCs (extended pluripotent stem cells). In this study, to explore the dynamic expression characteristics of proteins and their PTMs in mouse EPS blastoids, we revealed the protein expression profile of EPS blastoids and metabolite characteristics by TMT-based quantitative mass spectrometry (MS) strategy. Furthermore, the protein phosphorylation sites were identified to show the phosphoproteomic analysis in blastoids compared with mouse early embryos. Above all, our study revealed the protein expression profile of EPS blastoids compared with mouse embryos during preimplantation development and indicated that glucose metabolism is key to blastoid formation.

Spinal microglial ß-endorphin signaling mediates IL-10 and exenatide-induced inhibition of synaptic plasticity in neuropathic pain.

AIM: This study aimed to investigate the regulation of pain hypersensitivity induced by the spinal synaptic transmission mechanisms underlying interleukin (IL)-10 and glucagon-like peptide 1 receptor (GLP-1R) agonist exenatide-induced pain anti-hypersensitivity in neuropathic rats through spinal nerve ligations. METHODS: Neuropathic pain model was established by spinal nerve ligation of L5/L6 and verified by electrophysiological recording and immunofluorescence staining. Microglial expression of ß-endorphin through autocrine IL-10- and exenatide-induced inhibition of glutamatergic transmission were performed by behavioral tests coupled with whole-cell recording of miniature excitatory postsynaptic currents (mEPSCs) and miniature inhibitory postsynaptic currents (mIPSCs) through application of endogenous and exogenous IL-10 and ß-endorphin. RESULTS: Intrathecal injections of IL-10, exenatide, and the µ-opioid receptor (MOR) agonists ß-endorphin and DAMGO inhibited thermal hyperalgesia and mechanical allodynia in neuropathic rats. Whole-cell recordings of bath application of exenatide, IL-10, and ß-endorphin showed similarly suppressed enhanced frequency and amplitude of the mEPSCs in the spinal dorsal horn neurons of laminae II, but did not reduce the frequency and amplitude of mIPSCs in neuropathic rats. The inhibitory effects of IL-10 and exenatide on pain hypersensitive behaviors and spinal synaptic plasticity were totally blocked by pretreatment of IL-10 antibody, ß-endorphin antiserum, and MOR antagonist CTAP. In addition, the microglial metabolic inhibitor minocycline blocked the inhibitory effects of IL-10 and exenatide but not ß-endorphin on spinal synaptic plasticity. CONCLUSION: This suggests that spinal microglial expression of ß-endorphin mediates IL-10- and exenatide-induced inhibition of glutamatergic transmission and pain hypersensitivity via presynaptic and postsynaptic MORs in spinal dorsal horn.

Naturalistic Spike Trains Drive State-Dependent Homeostatic Plasticity in Superficial Layers of Visual Cortex.

The history of neural activity determines the synaptic plasticity mechanisms employed in the brain. Previous studies report a rapid reduction in the strength of excitatory synapses onto layer 2/3 (L2/3) pyramidal neurons of the primary visual cortex (V1) following two days of dark exposure and subsequent re-exposure to light. The abrupt increase in visually driven activity is predicted to drive homeostatic plasticity, however, the parameters of neural activity that trigger these changes are unknown. To determine this, we first recorded spike trains in vivo from V1 layer 4 (L4) of dark exposed (DE) mice of both sexes that were re-exposed to light through homogeneous or patterned visual stimulation. We found that delivering the spike patterns recorded in vivo to L4 of V1 slices was sufficient to reduce the amplitude of miniature excitatory postsynaptic currents (mEPSCs) of V1 L2/3 neurons in DE mice, but not in slices obtained from normal reared (NR) controls. Unexpectedly, the same stimulation pattern produced an up-regulation of mEPSC amplitudes in V1 L2/3 neurons from mice that received 2 h of light re-exposure (LE). A Poisson spike train exhibiting the same average frequency as the patterns recorded in vivo was equally effective at depressing mEPSC amplitudes in L2/3 neurons in V1 slices prepared from DE mice. Collectively, our results suggest that the history of visual experience modifies the responses of V1 neurons to stimulation and that rapid homeostatic depression of excitatory synapses can be driven by non-patterned input activity.

Excitatory synaptic transmission in hippocampal area CA1 is enhanced then reduced as chronic epilepsy progresses.

This study examines changes in synaptic transmission with progression of the chronic epileptic state. Male Sprague-Dawley rats (P40-45) were injected with either saline or pilocarpine. In rats injected with pilocarpine, status epilepticus ensued. Hippocampal slices were cut 20-60 days or 80-110 days post-treatment. Evoked and miniature EPSCs (mEPSCs) were recorded from CA1 pyramidal neurons using whole-cell voltage-clamp. Fiber volleys were also recorded from stratum radiatum. Evoked EPSCs from the pilocarpine-treated cohort showed enhanced amplitudes 20-60 days post-treatment compared to the saline-treated cohort, whereas mEPSCs recorded from the same age group showed no change in event frequency and a slight but significant decrease in mEPSC amplitude distribution. In contrast, comparing evoked EPSCs and mEPSCs recorded 80-110 days after treatment indicated reduced amplitudes from pilocarpine-treated animals compared to controls. mEPSC inter-event interval decreased. This could be explained by a partial depletion of the ready releasable pool of neurotransmitter vesicles in Schaffer collateral presynaptic terminals of the pilocarpine-treated rats. In both saline- and pilocarpine-treated cohorts, concomitant decreases in mEPSC amplitudes as time after treatment progressed suggest that age-related changes in CA1 circuitry may be partially responsible for changes in synaptic transmission that may influence the chronic epileptic state.

Orexin-A differentially modulates inhibitory and excitatory synaptic transmission in rat inner retina.

In this work, modulation by orexin-A of the release of glutamate and GABA from bipolar and amacrine cells respectively was studied by examining the effects of the neuropeptide on miniature excitatory postsynaptic currents (mEPSCs) and miniature inhibitory postsynaptic currents (mIPSCs) of rat retinal ganglion cells (GCs). Using RNAscope in situ hybridization in combination with immunohistochemistry, we showed positive signals for orexin receptor-1 (OX1R) mRNA in the bipolar cell terminals and those for orexin receptor-2 (OX2R) mRNA in the amacrine cell terminals. With whole-cell patch-clamp recordings in rat retinal slices, we demonstrated that application of orexin-A reduced the interevent interval of mEPSCs of GCs through OX1R. However, it increased the interevent interval of mIPSCs, mediated by GABAA receptors, through OX2R. Furthermore, orexin-A-induced reduction of mEPSC interevent interval was abolished by the application of PI-PLC inhibitors or PKC inhibitors. In contrast, orexin-A-induced increase of GABAergic mIPSC interevent interval was mimicked by 8-Br-cAMP or an adenylyl cyclase activator, but was eliminated by PKA antagonists. Finally, application of nimodipine, an L-type Ca2+ channel blocker, increased both mEPSC and mIPSC interevent interval, and co-application of orexin-A no longer changed the mEPSCs and mIPSCs. We conclude that orexin-A increases presynaptic glutamate release onto GCs by activating L-type Ca2+ channels in bipolar cells, a process that is mediated by an OX1R/PI-PLC/PKC signaling pathway. However, orexin-A decreases presynaptic GABA release onto GCs by inhibiting L-type Ca2+ channels in amacrine cells, a process that is mediated by an OX2R/cAMP-PKA signaling pathway.

Discovery and characterization of the novel conotoxin Lv1d from Conus lividus that presents analgesic activity.

Cone snails are predatory gastropod mollusks that are distributed in all tropical marine environments and contain small peptides (conotoxins) in their venom to capture prey. However, the biochemical and molecular aspects of conotoxins remain poorly understood. In this article, a novel α4/7-conotoxin, Lv1d, was obtained from the venom duct cDNA library of the worm-hunting Conus lividus collected from the South China Sea. The cDNA of Lv1c encodes a 65 residue conopeptide precursor, which consists of a 21 residue signal peptide, a 27 residue Pro region, and 17 residues of mature peptide. The mature peptide Lv1d was chemically synthesized according to the sequence GCCSDPPCRHKHQDLCG. It was found that 10 µM Lv1d can completely inhibit frog sciatic nerve-gastrocnemius muscle contractility within 60 min. Moreover, 100 µg/kg Lv1d showed good analgesic effects in mouse hot plate model and formalin test. Patch clamp experiments showed that 5 µM Lv1d can inhibit the cholinergic microexcitatory postsynaptic currents (mEPSCs) requency and amplitude of projection neurons in Drosophila. In conclusion, the synthesis of Lv1d and its biological and physiological data might contribute to the development of this peptide as a novel potential drug for therapeutic applications. This finding also expands the knowledge of the targeting mechanism of the α4/7-subfamily conotoxins.

The role of p21-activated kinase in maintaining the fear learning-induced modulation of excitation/inhibition ratio in lateral amygdala.

We study the relations between different learning paradigms and enduring changes in excitatory synaptic transmission. Here we show that auditory fear conditioning (AFC), but not olfactory fear conditioning (OFC) training, led to enduring enhancement in AMPA-mediated miniature EPSCs (mEPSCs). Moreover, olfactory unpaired training led to a stable significant reduction in excitatory synaptic transmission. However, olfactory discrimination learning (OD) did not modulate postsynaptic AMPA-mediated mEPSCs in LA. The p21-activated kinase (PAK) activity, previously shown to have a key role in maintaining persistent long-lasting enhancement in synaptic inhibition after OFC, has an opposing effect on excitatory synaptic transmission. PAK maintained the level of excitatory synaptic transmission in the amygdala in all experimental groups, except in neurons in the OFC trained rats. PAK also maintained excitatory synaptic transmission in all neurons of auditory fear conditioning and naïve training groups except in neurons of the auditory safety learning. Safety learning was previously shown in our study to enhance synaptic inhibition. We thus suggest that PAK maintains inhibitory synaptic transmission in a learning-dependent manner and on the other hand affects excitatory synaptic transmission only in groups where learning has not affected inhibitory transmission. Thus, PAK controls learning-induced changes in the excitation/inhibition balance.

A GLP-1/GIP/Gcg receptor triagonist improves memory behavior, as well as synaptic transmission, neuronal excitability and Ca2+ homeostasis in 3xTg-AD mice.

Alzheimer's disease (AD) is a progressively neurodegenerative disorder, which seriously affects human health and cannot be stopped by current treatments. Type 2 diabetes mellitus (T2DM) is a risk factor for AD. Our recent studies reported the neuroprotective effects of a GLP-1/GIP/Glucagon receptor triagonist (Triagonist), a novel unimolecular anti-diabetic drug, in cognitive and pathological improvements of 3xTg-AD mice. However, the detailed electrophysiological and molecular mechanisms underlying neuroprotection remain unexplored. The present study investigated the underlying electrophysiological and molecular mechanisms further by using whole-cell patch clamp techniques. Our results revealed that chronic Triagonist treatment effectively reduced working memory and reference memory errors of 3xTg-AD mice in a radial maze test. In addition, the Triagonist increased spontaneous excitatory synaptic activities, differentially modulated voltage- and chemically-gated Ca2+ flux, and reduced the over-excitation of pyramidal neurons in hippocampal slices of 3xTg-AD mice. In addition, chronic Triagonist treatment also up-regulated the expression levels of synaptophysin and PSD-95 in the hippocampus of 3xTg-AD mice. These results indicate that the Triagonist could improve memory formation, as well as synaptic transmission, Ca2+ balance, and neuronal excitability in 3xTg-AD mice. These neuroprotective effects of Triagonist may be involved in the up-regulation of synaptophysin and PSD-95. Therefore, the study suggests that multi-receptor agonists might be a novel therapeutic strategy for the treatment of AD.

Ultrastructural and functional changes at the tripartite synapse during epileptogenesis in a model of temporal lobe epilepsy.

The persistent unresponsiveness of many of the acquired epilepsies to traditional antiseizure medications has motivated the search for prophylactic drug therapies that could reduce the incidence of epilepsy in this at risk population. These studies are based on the idea of a period of epileptogenesis that can follow a wide variety of brain injuries. Epileptogenesis is hypothesized to involve changes in the brain not initially associated with seizures, but which result finally in seizure prone networks. Understanding these changes will provide crucial clues for the development of prophylactic drugs. Using the repeated low-dose kainate rat model of epilepsy, we have studied the period of epileptogenesis following status epilepticus, verifying the latent period with continuous EEG monitoring. Focusing on ultrastructural properties of the tripartite synapse in the CA1 region of hippocampus we found increased astrocyte ensheathment around both the presynaptic and postsynaptic elements, reduced synaptic AMPA receptor subunit and perisynaptic astrocyte GLT-1 expression, and increased number of docked vesicles at the presynaptic terminal. These findings were associated with an increase in frequency of the mEPSCs observed in patch clamp recordings of CA1 pyramidal cells. The results suggest a complex set of changes, some of which have been associated with increasingly excitable networks such as increased vesicles and mEPSC frequency, and some associated with compensatory mechanisms, such as increased astrocyte ensheathment. The diversity of ultrastructural and electrophysiological changes observed during epileptogeneiss suggests that potential drug targets for this period should be broadened to include all components of the tripartite synapse.

Ketamine Regulates Phosphorylation of CRMP2 To Mediate Dendritic Spine Plasticity.

Ketamine is widely used in infants and young children for anesthesia, and subanesthetic doses of ketamine make neurons form new protrusions and promote synapse formation. However, the precise pathological mechanisms remain to be elucidated. In this study, we demonstrated that ketamine administration significantly increased dendritic spine density and maturity in rat cortical neurons in vivo and in vitro. Western blot analysis showed that CRMP2 protein expression was significantly increased in cerebral cortex of ketamine group, and phosphorylation levels of CRMP at Thr514 and Ser522 were significantly reduced. Furthermore, overexpression of CRMP2 promoted the growth of cortical neuron processes and dendritic spines. Although the dendritic field was more complex after adding ketamine and the density of dendritic spines increased, there was no statistical difference and no obvious superposition effect was observed. Moreover, both Ser522 mutant construction of CRMP2, GFP-CRMP2-522D, and mcherry-CDK5 showed similar inhibitory effects on neurite outgrowth, which could be rescued by ketamine. The frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs) were significantly inhibited when GFP-CRMP2-522D and mCherry-CDK5 were transfected into cortical neurons and this trend could also be rescued by ketamine. In general, this study reveals a new mechanism by which ketamine promotes the growth and development of dendritic spines in developing cortical neurons.

Approaches and Limitations in the Investigation of Synaptic Transmission and Plasticity.

The numbers and strengths of synapses in the brain change throughout development, and even into adulthood, as synaptic inputs are added, eliminated, and refined in response to ongoing neural activity. A number of experimental techniques can assess these changes, including single-cell electrophysiological recording which offers measurements of synaptic inputs with high temporal resolution. Coupled with electrical stimulation, photoactivatable opsins, and caged compounds, to facilitate fine spatiotemporal control over release of neurotransmitters, electrophysiological recordings allow for precise dissection of presynaptic and postsynaptic mechanisms of action. Here, we discuss the strengths and pitfalls of various techniques commonly used to analyze synapses, including miniature excitatory/inhibitory (E/I) postsynaptic currents, evoked release, and optogenetic stimulation. Together, these techniques can provide multiple lines of convergent evidence to generate meaningful insight into the emergence of circuit connectivity and maturation. A full understanding of potential caveats and alternative explanations for findings is essential to avoid data misinterpretation.

Aerobic exercise regulates synaptic transmission and reactive oxygen species production in the paraventricular nucleus of spontaneously hypertensive rats.

Aerobic exercise lowers blood pressure in patients with hypertension, but the underlying mechanisms remain incompletely understood. The hypothalamic paraventricular nucleus (PVN) plays a key role in the control of sympathetic outflow and cardiovascular tone. We examined whether chronic aerobic exercise altered synaptic transmission and reactive oxygen species (ROS) production in the PVN. In the present study, spontaneously hypertensive rats (SHRs) were subjected to exercise training for 8 weeks, five times per week, with Wistar Kyoto (WKY) rats as the cohort control. Miniature excitatory and inhibitory postsynaptic currents (mEPSCs and mIPSCs) were recorded from the PVN in ex vivo hypothalamic slice preparations obtained after the last training, and biomarkers of oxidative stress and physical indexes were observed. The mean frequency and amplitude, as well as the rise time and the decay time constant of mIPSCs, significantly decreased in 20-wk-old SHRs compared to WKY 20-wk-old controls. In contrast to mIPSCs, only the mean mEPSC frequency was higher, and there were no other changes in mEPSCs in comparison to the control group. SHRs exhibited higher ROS, 8-OHdG, and MDA; and lower SOD1, SOD2, CAT, Ogg1, and SOD and CAT activity in the PVN. These SHRs also had a significant increase in heart rate, blood pressure and sympathetic nerve activity, and higher levels of norepinephrine (NE). Exercise training ameliorated all these abnormalities, resulting in an increase in the mean frequency, amplitude and kinetics of mIPSCs, accompanied by a decrease in the mean frequency of mEPSCs in the PVN. This study demonstrates that moderate intensity, high frequency exercise training induces a selective enhancement of inhibitory synaptic transmission in the PVN, which may dampen sympathetic activity and reduce blood pressure in hypertension. These changes may be due to antioxidant-related adaptations in the PVNs of SHRs.

The dual role of KCNQ/M channels upon OGD or OGD/R insults in cultured cortical neurons of mice: Timing is crucial in targeting M-channels against ischemic injur ies.

KCNQ/M potassium channels play a vital role in neuronal excitability; however, it is required to explore their pharmacological modulation on N-Methyl- d-aspartic acid receptors (NMDARs)-mediated glutamatergic transmission of neurons upon ischemic insults. In the current study, both presynaptic glutamatergic release and activities of NMDARs were measured by NMDAR-induced miniature excitatory postsynaptic currents (mEPSCs) in cultured cortical neurons of C57 mice undergoing oxygen and glucose deprivation (OGD) or OGD/reperfusion (OGD/R). The KCNQ/M-channel opener, retigabine (RTG), suppressed the overactivation of postsynaptic NMDARs induced by OGD and then NO transient; RTG also decreased OGD-induced neuronal death measured with MTT assay, suggesting the beneficial role of KCNQ/M-channels for the neurons exposed to ischemic insults. However, when the neurons exposed to the subsequent reperfusion, KCNQ/M-channels played a differential role from its protective effect. OGD/R increased presynaptic glutamatergic release, which was further augmented by RTG or decreased by KCNQ/M-channel blocker, XE991. Reactive oxygen species (ROS) were produced partly in a NO-dependent manner. In addition, XE991 decreased neuronal injuries upon reperfusion measured with DCF and PI staining. Meanwhile, the addition of RTG upon OGD or XE991 upon reperfusion can reverse OGD or OGD/R-reduced mitochondrial membrane potential. Our present study indicates the dual role of KCNQ/M-channels in OGD and OGD/R, which will decide the fate of neurons. Provided that activation of KCNQ/M-channels has differential effects on neuronal injuries during OGD or OGD/R, we propose that therapy targeting KCNQ/M-channels may be effective for ischemic injuries but the proper timing is so crucial for the corresponding treatment.