Reducing Filamin A Restores Cortical Synaptic Connectivity and Early Social Communication Following Cellular Mosaicism in Autism Spectrum Disorder Pathways.

Communication in the form of nonverbal, social vocalization, or crying is evolutionary conserved in mammals and is impaired early in human infants that are later diagnosed with autism spectrum disorder (ASD). Defects in infant vocalization have been proposed as an early sign of ASD that may exacerbate ASD development. However, the neural mechanisms associated with early communicative deficits in ASD are not known. Here, we expressed a constitutively active mutant of Rheb (RhebS16H), which is known to upregulate two ASD core pathways, mTOR complex 1 (mTORC1) and ERK1/2, in Layer (L) 2/3 pyramidal neurons of the neocortex of mice of either sex. We found that cellular mosaic expression of RhebS16H in L2/3 pyramidal neurons altered the production of isolation calls from neonatal mice. This was accompanied by an expected misplacement of neurons and dendrite overgrowth, along with an unexpected increase in spine density and length, which was associated with increased excitatory synaptic activity. This contrasted with the known decrease in spine density in RhebS16H neurons of 1-month-old mice. Reducing the levels of the actin cross-linking and adaptor protein filamin A (FLNA), known to be increased downstream of ERK1/2, attenuated dendrite overgrowth and fully restored spine properties, synaptic connectivity, and the production of pup isolation calls. These findings suggest that upper-layer cortical pyramidal neurons contribute to communicative deficits in a condition known to affect two core ASD pathways and that these mechanisms are regulated by FLNA.

Prefrontal cortical network dysfunction from acute neurotoxicant exposure.

Prefrontal cortical (PFC) dysfunction has been linked to disorders exhibiting deficits in cognitive performance, attention, motivation, and impulse control. Neurons of the PFC are susceptible to glutamatergic excitotoxicity, an effect associated with cortical degeneration in frontotemporal disorders (FTDs). PFC susceptibility to environmental toxicant exposure, one possible contributor to sporadic FTD, has not been systematically studied. Here, we tested the ability of a well-known environmental neurotoxicant, methylmercury (MeHg), to induce hyperexcitability in medial prefrontal cortex (mPFC) excitatory pyramidal neurons, using whole cell patch-clamp recording. Acute MeHg exposure (20 µM) produced significant mPFC dysfunction, with a shift in the excitatory to inhibitory (E-I) balance toward increased excitability. Both excitatory postsynaptic current (EPSC) and inhibitory postsynaptic current (IPSC) charges were significantly increased after MeHg exposure. MeHg increased EPSC frequency, but there was no observable effect on IPSC frequency, EPSC amplitude or IPSC amplitude. Neither evoked AMPA receptor- nor NMDA receptor-mediated EPSC amplitudes were affected by MeHg. However, excitatory synapses experienced a significant reduction in paired-pulse depression and probability of release. In addition, MeHg induced temporal synchrony in spontaneous IPSCs, reflecting mPFC inhibitory network dysfunction. MeHg exposure also produced increased intrinsic excitability in mPFC neurons, with an increase in action potential firing rate. The observed effects of MeHg on mPFC reflect key potential mechanisms for neuropsychological symptoms from MeHg poisoning. Therefore, MeHg has a significant effect on mPFC circuits known to contribute to cognitive and emotional function and might contribute to etiology of neurodegenerative diseases, such as FTD.NEW & NOTEWORTHY Prefrontal cortical neurons are highly susceptible to glutamatergic excitotoxicity associated with neuronal degeneration in frontal dementia and to environmental toxicant exposure, one potential contributor to FTD. However, this has not been systematically studied. Our results demonstrate that methylmercury exposure leads to hyperexcitability of prefrontal cortical neurons by shifting excitatory to inhibitory (E-I) balance and raising sensitivity for spiking. Our results provide a mechanism by which environmental neurotoxicants may contribute to pathogenesis of diseases such as FTD.

Neuronal Nitric Oxide Synthase Regulates Cerebellar Parallel Fiber Slow EPSC in Purkinje Neurons by Modulating STIM1-Gated TRPC3-Containing Channels.

Responding to burst stimulation of parallel fibers (PFs), cerebellar Purkinje neurons (PNs) generate a convolved synaptic response displaying a fast excitatory postsynaptic current (EPSCFast) followed by a slow EPSC (EPSCSlow). The latter is companied with a rise of intracellular Ca2+ and critical for motor coordination. The genesis of EPSCSlow in PNs results from activation of metabotropic type 1 glutamate receptor (mGluR1), oligomerization of stromal interaction molecule 1 (STIM1) on the membrane of endoplasmic reticulum (ER) and opening of transient receptor potential canonical 3 (TRPC3) channels on the plasma membrane. Neuronal nitric oxide synthase (nNOS) is abundantly expressed in PFs and granule neurons (GNs), catalyzing the production of nitric oxide (NO) hence regulating PF-PN synaptic function. We recently found that nNOS/NO regulates the morphological development of PNs through mGluR1-regulated Ca2+-dependent mechanism. This study investigated the role of nNOS/NO in regulating EPSCSlow. Electrophysiological analyses showed that EPSCSlow in cerebellar slices of nNOS knockout (nNOS-/-) mice was significantly larger than that in wildtype (WT) mice. Activation of mGluR1 in cultured PNs from nNOS-/- mice evoked larger TRPC3-channel mediated currents and intracellular Ca2+ rise than that in PNs from WT mice. In addition, nNOS inhibitor and NO-donor increased and decreased, respectively, the TRPC3-current and Ca2+ rise in PNs. Moreover, the NO-donor effectively decreased TRPC3 currents in HEK293 cells expressing WT STIM1, but not cells expressing a STIM1 with cysteine mutants. These novel findings indicate that nNOS/NO inhibits TRPC3-containig channel mediated cation influx during EPSCSlow, at least in part, by S-nitrosylation of STIM1.

Deficits in Behavioral and Neuronal Pattern Separation in Temporal Lobe Epilepsy.

In temporal lobe epilepsy, the ability of the dentate gyrus to limit excitatory cortical input to the hippocampus breaks down, leading to seizures. The dentate gyrus is also thought to help discriminate between similar memories by performing pattern separation, but whether epilepsy leads to a breakdown in this neural computation, and thus to mnemonic discrimination impairments, remains unknown. Here we show that temporal lobe epilepsy is characterized by behavioral deficits in mnemonic discrimination tasks, in both humans (females and males) and mice (C57Bl6 males, systemic low-dose kainate model). Using a recently developed assay in brain slices of the same epileptic mice, we reveal a decreased ability of the dentate gyrus to perform certain forms of pattern separation. This is because of a subset of granule cells with abnormal bursting that can develop independently of early EEG abnormalities. Overall, our results linking physiology, computation, and cognition in the same mice advance our understanding of episodic memory mechanisms and their dysfunction in epilepsy.SIGNIFICANCE STATEMENT People with temporal lobe epilepsy (TLE) often have learning and memory impairments, sometimes occurring earlier than the first seizure, but those symptoms and their biological underpinnings are poorly understood. We focused on the dentate gyrus, a brain region that is critical to avoid confusion between similar memories and is anatomically disorganized in TLE. We show that both humans and mice with TLE experience confusion between similar situations. This impairment coincides with a failure of the dentate gyrus to disambiguate similar input signals because of pathologic bursting in a subset of neurons. Our work bridges seizure-oriented and memory-oriented views of the dentate gyrus function, suggests a mechanism for cognitive symptoms in TLE, and supports a long-standing hypothesis of episodic memory theories.

Activation of microglial GPR109A alleviates thermal hyperalgesia in female lupus mice by suppressing IL-18 and glutamatergic synaptic activity.

Many patients with systemic lupus erythematosus (SLE) live with chronic pain despite advances in medical management in reducing mortality related to SLE. Few animal studies have addressed mechanisms and treatment for chronic pain caused by SLE. In this study, we provide the first evidence for the analgesic effects of a GPR109A specific agonist (MK1903) and its action mechanisms in thermal hyperalgesia in female MRL/lpr mice, an SLE mouse model. Specifically, we show that MRL/lpr mice had a higher sensitivity to thermal stimuli at age 11-16 weeks, which was accompanied with significantly microglial and astrocytic activation, increases in p38 MAPK and glutamatergic synaptic activities in the spinal dorsal horn. We demonstrate that thermal hyperalgesia in MRL/lpr mice was significantly attenuated by intrathecal injection of MK1903. GPR109A was expressed in spinal microglia but not astrocytes or neurons. Its expression was significantly increased in MRL/lpr mice with thermal hyperalgesia. Activation of GPR109A receptors in microglia attenuated glutamatergic synaptic activity via suppressing production of interleukin-18 (IL-18). We provide evidence that activation of GPR109A attenuated thermal hyperalgesia in the SLE animal model via suppressing p38 MAPK activity and production of IL-18. Our study suggests that targeting the microglial GPR109A is a potent approach for reversing spinal neuroinflammation, abnormal excitatory synaptic activity, and management of thermal hyperalgesia caused by SLE.

Serotonergic Modulation of Spontaneous and Evoked Transmitter Release in Layer II Pyramidal Cells of Rat Somatosensory Cortex.

As axons from the raphe nuclei densely innervate the somatosensory cortex, we investigated how serotonin (5-HT) modulates transmitter release in layer II pyramidal cells of rat barrel cortex. In the presence of tetrodotoxin and gabazine, 10 µM 5-HT caused a waxing and waning in the frequency of miniature excitatory postsynaptic currents (mEPSC) with no effect on amplitude. Specifically, within 15 min of recording the mEPSC frequency initially increased by 28 ± 7%, then dropped to below control (-15 ± 3%), before resurging back to 27 ± 7% larger than control. These changes were seen in 47% of pyramidal cells (responders) and were mediated by 5-HT2C receptors (5-HT2CR). Waxing resulted from phospholipase C activation, IP3 production, and Ca2+ release from presynaptic stores. Waning was prevented if PKC was blocked. In contrast, in paired recordings, the unitary EPSC amplitude was reduced by 50 ± 3% after 5-HT exposure in almost all cases with no significant effect on paired-pulse ratio and synaptic dynamics. This sustained EPSC reduction was also caused by 5-HT2R, but was mediated by presynaptic Gßγ subunits likely limiting influx through CaV2 channels. EPSC reduction, together with enhanced spontaneous noise in a restricted subset of inputs, could temporarily diminish the signal-to-noise ratio and affect the computation in the neocortical microcircuit.

Methylphenidate Restores Behavioral and Neuroplasticity Impairments in the Prenatal Nicotine Exposure Mouse Model of ADHD: Evidence for Involvement of AMPA Receptor Subunit Composition and Synaptic Spine Morphology in the Hippocampus.

In ADHD treatment, methylphenidate (MPH) is the most frequently used medication. The present work provides evidence that MPH restored behavioral impairments and neuroplasticity due to changes in AMPAR subunit composition and distribution, as well as maturation of dendritic spines, in a prenatal nicotine exposure (PNE) ADHD mouse model. PNE animals and controls were given a single oral dose of MPH (1 mg/kg), and their behavior was tested for attention, hyperactivity, and working memory. Long-term potentiation (LTP) was induced and analyzed at the CA3/CA1 synapse in hippocampal slices taken from the same animals tested behaviorally, measuring fEPSPs and whole-cell patch-clamp EPSCs. By applying crosslinking and Western blots, we estimated the LTP effects on AMPAR subunit composition and distribution. The density and types of dendritic spines were quantified by using the Golgi staining method. MPH completely restored the behavioral impairments of PNE mice. Reduced LTP and AMPA-receptor-mediated EPSCs were also restored. EPSC amplitudes were tightly correlated with numbers of GluA1/GluA1 AMPA receptors at the cell surface. Finally, we found a lower density of dendritic spines in hippocampal pyramidal neurons in PNE mice, with a higher fraction of thin-type immature spines and a lower fraction of mushroom mature spines; the latter effect was also reversed by MPH.

Mechanisms Underlying Enhancement of Spontaneous Glutamate Release by Group I mGluRs at a Central Auditory Synapse.

One emerging concept in neuroscience states that synaptic vesicles and the molecular machinery underlying spontaneous transmitter release are different from those underlying action potential-driven synchronized transmitter release. Differential neuromodulation of these two distinct release modes by metabotropic glutamate receptors (mGluRs) constitutes critical supporting evidence. However, the mechanisms underlying such a differential modulation are not understood. Here, we investigated the mechanisms of the modulation by group I mGluRs (mGluR Is) on spontaneous glutamate release in the medial nucleus of the trapezoid body (MNTB), an auditory brainstem nucleus critically involved in sound localization. Whole-cell patch recordings from brainstem slices of mice of both sexes were performed. Activation of mGluR I by 3,5-dihydroxyphenylglycine (3,5-DHPG; 200 µm) produced an inward current at -60 mV and increased spontaneous glutamate release in MNTB neurons. Pharmacological evidence indicated involvement of both mGluR1 and mGluR5, which was further supported for mGluR5 by immunolabeling results. The modulation was eliminated by blocking NaV channels (tetrodotoxin, 1 µm), persistent Na+ current (INaP; riluzole, 10 µm), or CaV channels (CdCl2, 100 µm). Presynaptic calyx recordings revealed that 3,5-DHPG shifted the activation of INaP to more hyperpolarized voltages and increased INaP at resting membrane potential. Our data indicate that mGluR I enhances spontaneous glutamate release via regulation of INaP and subsequent Ca2+-dependent processes under resting condition.SIGNIFICANCE STATEMENT For brain cells to communicate with each other, neurons release chemical messengers, termed neurotransmitters, in response to action potential invasion (evoked release). Neurons also release neurotransmitters spontaneously. Recent work has revealed different release machineries underlying these two release modes, and their different roles in synaptic development and plasticity. Our recent work discovered differential neuromodulation of these two release modes, but the mechanisms are not well understood. The present study showed that activation of group I metabotropic glutamate receptors enhanced spontaneous glutamate release in an auditory brainstem nucleus, while suppressing evoked release. The modulation is dependent on a persistent Na+ current and involves subsequent Ca2+ signaling, providing insight into the mechanisms underlying the different release modes in auditory processing.

Resolution of subcomponents of synaptic release from postsynaptic currents in rat hair-cell/auditory-nerve fiber synapses.

The synapse between inner hair cells and auditory nerve fiber dendrites shows large excitatory postsynaptic currents (EPSCs), which are either monophasic or multiphasic. Multiquantal or uniquantal (flickering) release of neurotransmitter has been proposed to underlie the unusual multiphasic waveforms. Here the nature of multiphasic waveforms is analyzed using EPSCs recorded in vitro in rat afferent dendrites. Spontaneous EPSCs were deconvolved into a sum of presumed release events having monophasic EPSC waveforms. Results include, first, the charge of EPSCs is about the same for multiphasic versus monophasic EPSCs. Second, EPSC amplitudes decline with the number of release events per EPSC. Third, there is no evidence of a mini-EPSC. Most results can be accounted for by versions of either uniquantal or multiquantal release. However, serial neurotransmitter release in multiphasic EPSCs shows properties that are not fully explained by either model, especially that the amplitudes of individual release events are established at the beginning of a multiphasic EPSC, constraining possible models of vesicle release.NEW & NOTEWORTHY How do monophasic and multiphasic waveshapes arise in auditory-nerve dendrites; mainly are they uniquantal, arising from release of a single vesicle, or multiquantal, requiring several vesicles? The charge injected by excitatory postsynaptic currents (EPSCs) is the same for monophasic or multiphasic EPSCs, supporting uniquantal release. Serial adaptation of responses to sequential EPSCs favors a multiquantal model. Finally, neurotransmitter partitioning into similar sized release boluses occurs at the first bolus in the EPSC, not easily explained with either model.

Light stimulation during postnatal development is not determinant for glutamatergic neurotransmission from the retinohypothalamic tract to the suprachiasmatic nucleus in rats.

The hypothalamic suprachiasmatic nucleus (SCN) is the leading circadian pacemaker in mammals, which synchronizes with environmental light through the retinohypothalamic tract (RHT). Although the SCN regulates circadian rhythms before birth, postnatal synaptic changes are needed for the RHT-SCN pathway to achieve total functional development. However, it is unknown whether visual experience affects developmental maturation. Here, we studied the effects of constant darkness (DD) rearing on the physiology (at pre- and postsynaptic levels) of glutamatergic neurotransmission between RHT and SCN during postnatal development in rats. Upon recording spontaneous and evoked excitatory postsynaptic currents (EPSCs) by electrical stimulation of RHT fibers, we found that DD animals at early postnatal ages (P3-19) exhibited different frequencies of spontaneous EPSCs and lower synaptic performance (short-term depression, release sites, and recruitment of RHT fibers) when compared with their normal light/dark (LD) counterparts. At the oldest age evaluated (P30-35), there was a synaptic response strengthening (probability of release, vesicular re-filling rate, and reduced synaptic depression) in DD rats, which functionally equaled (or surmounted) that of LD animals. Control experiments evaluating EPSCs in ventral SCN neurons of LD rats during day and night revealed no significant differences in spontaneous or evoked EPSCs by high-frequency trains in the RHT at any postnatal age. Our results suggest that DD conditions induce a compensatory mechanism in the glutamatergic signaling of the circadian system to increase the chances of synchronization to light at adult ages, and that the synaptic properties of RHT terminals during postnatal development are not critically influenced by environmental light.

Presynaptic Diversity Revealed by Ca2+-Permeable AMPA Receptors at the Calyx of Held Synapse.

GluA2-lacking Ca2+-permeable AMPARs (CP-AMPARs) play integral roles in synaptic plasticity and can mediate excitotoxic cellular signaling at glutamatergic synapses. However, the developmental profile of functional CP-AMPARs at the auditory brainstem remains poorly understood. Through a combination of electrophysiological and live-cell Ca2+ imaging from mice of either sex, we show that the synaptic release of glutamate from the calyx of Held nerve terminal activates CP-AMPARs in the principal cells of the medial nucleus of the trapezoid body in the brainstem. This leads to significant Ca2+ influx through these receptors before the onset of hearing at postnatal day 12 (P12). Using a selective open channel blocker of CP-AMPARs, IEM-1460, we estimate that ∼80% of the AMPAR population are permeable to Ca2+ at immature P4-P5 synapses. However, after the onset of hearing, Ca2+ influx through these receptors was greatly reduced. We estimate that CP-AMPARs comprise approximately 40% and 33% of the AMPAR population at P18-P22 and P30-P34, respectively. By quantifying the rate of EPSC block by IEM-1460, we found an increased heterogeneity in glutamate release probability for adult-like calyces (P30-P34). Using tetraethylammonium (TEA), a presynaptic potassium channel blocker, we show that the apparent reduction of CP-AMPARs in more mature synapses is not a consequence of presynaptic action potential (AP) speeding. Finally, through postsynaptic AP recordings, we show that inhibition of CP-AMPARs reduces spike fidelity in juvenile synapses, but not in more mature synapses. We conclude that the expression of functional CP-AMPARs declines over early postnatal development in the calyx of Held synapse.SIGNIFICANCE STATEMENT The calyx of Held synapse is pivotal to the circuitry that computes sound localization. Postsynaptic Ca2+ influx via AMPARs may be critical for signaling the maturation of this brainstem synapse. The GluA4 subunit may dominate the AMPAR complex at mature synapses because of its fast gating kinetics and large unitary conductance. The expectation is that AMPARs dominated by GluA4 subunits should be highly Ca2+ permeable. However, we find that Ca2+-permeable AMPAR expression declines during postnatal development. Using the rate of EPSC block by IEM-1460, an open channel blocker of Ca2+-permeable AMPARs, we propose a novel method to determine glutamate release probability and uncover an increased heterogeneity in release probability for more mature calyces of Held nerve terminals.

Hippocampal microglial activation triggers a neurotoxic-specific astrocyte response and mediates etomidate-induced long-term synaptic inhibition.

BACKGROUND: Accumulating evidence has highlighted the importance of microglial and astrocyte responses in the pathological development of postoperative cognitive dysfunction (POCD). However, the mechanisms involved are not well understood. METHODS: A perioperative neurocognitive disorders (PND) mouse model was generated by administering etomidate, and cognitive function was assessed using the Morris water maze and novel object recognition tests. Excitatory and inhibitory postsynaptic currents were recorded to analyze neuronal activity. In addition, microglia and astrocytes were isolated by magnetic-activated cell sorting, and genes that were activated in these cells were identified using quantitative polymerase chain reaction. RESULTS: We observed dramatic cognitive impairment at 1 and 3 weeks after etomidate was administered to 18 month-old mice. Microglia and astrocytes isolated from the hippocampus showed significant microglial activation during the early pathological stage (i.e., 1 week after etomidate injection) and an A1-specific astrocyte response during the late pathological stage (i.e., 3 weeks after etomidate injection). Furthermore, when microglia were eliminated before etomidate was injected, the A1-specific astrocyte activation response was significantly reduced, and cognitive function improved. However, when microglia were eliminated after etomidate application, astrocyte activation and cognitive function were not significantly altered. In addition, activating microglia immediately after a sedative dose of etomidate was injected markedly increased A1-specific astrocyte activation and cognitive dysfunction. CONCLUSIONS: A1-specific astrocyte activation is triggered by activated microglia during the initial pathological stage of PND and induces long-term synaptic inhibition and cognitive deficiencies. These results improve our understanding of how PND develops and may suggest therapeutic targets.

Xenon modulates synaptic transmission to rat hippocampal CA3 neurons at both pre- and postsynaptic sites.

KEY POINTS: Xenon (Xe) non-competitively inhibited whole-cell excitatory glutamatergic current (IGlu ) and whole-cell currents gated by ionotropic glutamate receptors (IAMPA , IKA , INMDA ), but had no effect on inhibitory GABAergic whole-cell current (IGABA ). Xe decreased only the frequency of glutamatergic spontaneous and miniature excitatory postsynaptic currents and GABAergic spontaneous inhibitory postsynaptic currents without changing the amplitude or decay times of these synaptic responses. Xe decreased the amplitude of both the action potential-evoked excitatory and the action potential-evoked inhibitory postsynaptic currents (eEPSCs and eIPSCs, respectively) via a presynaptic inhibition in transmitter release. We conclude that the main site of action of Xe is presynaptic in both excitatory and inhibitory synapses, and that the Xe inhibition is much greater for eEPSCs than for eIPSCs. ABSTRACT: To clarify how xenon (Xe) modulates excitatory and inhibitory whole-cell and synaptic responses, we conducted an electrophysiological experiment using the 'synapse bouton preparation' dissociated mechanically from the rat hippocampal CA3 region. This technique can evaluate pure single- or multi-synapse responses and enabled us to accurately quantify how Xe influences pre- and postsynaptic aspects of synaptic transmission. Xe inhibited whole-cell glutamatergic current (IGlu ) and whole-cell currents gated by the three subtypes of glutamate receptor (IAMPA , IKA and INMDA ). Inhibition of these ionotropic currents occurred in a concentration-dependent, non-competitive and voltage-independent manner. Xe markedly depressed the slow steady current component of IAMPA almost without altering the fast phasic IAMPA component non-desensitized by cyclothiazide. It decreased current frequency without affecting the amplitude and current kinetics of glutamatergic spontaneous excitatory postsynaptic currents and miniature excitatory postsynaptic currents. It decreased the amplitude, increasing the failure rate (Rf) and paired-pulse rate (PPR) without altering the current kinetics of glutamatergic action potential-evoked excitatory postsynaptic currents. Thus, Xe has a clear presynaptic effect on excitatory synaptic transmission. Xe did not alter the GABA-induced whole-cell current (IGABA ). It decreased the frequency of GABAergic spontaneous inhibitory postsynaptic currents without changing the amplitude and current kinetics. It decreased the amplitude and increased the PPR and Rf of the GABAergic action potential-evoked inhibitory postsynaptic currents without altering the current kinetics. Thus, Xe acts exclusively at presynaptic sites at the GABAergic synapse. In conclusion, our data indicate that a presynaptic decrease of excitatory transmission is likely to be the major mechanism by which Xe induces anaesthesia, with little contribution of effects on GABAergic synapses.

2-Deoxy-d-glucose reduces epileptiform activity by presynaptic mechanisms.

2-Deoxy-d-glucose (2DG), a glucose analog that inhibits glycolysis, has acute and chronic antiepileptic effects. We evaluated 2DG's acute effects on synaptic and membrane properties of CA3 pyramidal neurons in vitro. 2DG (10 mM) had no effects on spontaneously occurring postsynaptic currents (PSCs) in 3.5 mM extracellular potassium concentration ([K+]o). In 7.5 mM [K+]o, 2DG significantly reduced the frequency of epileptiform bursting and the charge carried by postsynaptic currents (PSCs) with a greater effect on inward excitatory compared with outward inhibitory charge (71% vs. 40%). In 7.5 mM [K+]o and bicuculline, 2DG reduced significantly the excitatory charge by 67% and decreased the frequency but not amplitude of excitatory PSCs between bursts. In 7.5 mM [K+]o, 2DG reduced pharmacologically isolated inhibitory PSC frequency without a change in amplitude. The frequency but not amplitude of inward miniature PSCs was reduced when 2DG was applied in 7.5 mM [K+]o before bath application of TTX, but there was no effect when 2DG was applied after TTX, indicating a use-dependent uptake of 2DG was required for its actions at a presynaptic locus. 2DG did not alter membrane properties of CA3 neurons except for reducing the slow afterhyperpolarization in 3.5 but not 7.5 mM [K+]o. The reduction in frequency of spontaneous and inward miniature PSCs in elevated [K+]o indicates a presynaptic mechanism of action. 2DG effects required use-dependent uptake and suggest an important role for glycolysis in neuronal metabolism and energetics in states of high neural activity as occur during abnormal network synchronization and seizures. NEW & NOTEWORTHY 2-Deoxy-d-glucose (2DG) is a glycolytic inhibitor and suppresses epileptiform activity acutely and has chronic antiepileptic effects. The mechanisms of the acute effects are not well delineated. In this study, we show 2DG suppressed abnormal network epileptiform activity without effecting normal synaptic network activity or membrane properties. The effects appear to be use dependent and have a presynaptic locus of action. Inhibition of glycolysis is a novel presynaptic mechanism to limit abnormal neuronal network activity and seizures.

Diversity of dendritic morphology and entorhinal cortex synaptic effectiveness in mouse CA2 pyramidal neurons.

Excitatory synaptic inputs from specific brain regions are often targeted to distinct dendritic arbors on hippocampal pyramidal neurons. Recent work has suggested that CA2 pyramidal neurons respond robustly and preferentially to excitatory input into the stratum lacunosum moleculare (SLM), with a relatively modest response to Schaffer collateral excitatory input into stratum radiatum (SR) in acute mouse hippocampal slices, but the extent to which this difference may be explained by morphology is unknown. In an effort to replicate these findings and to better understand the role of dendritic morphology in shaping responses from proximal and distal synaptic sites, we measured excitatory postsynaptic currents and action potentials in CA2 pyramidal cells in response to SR and SLM stimulation and subsequently analyzed confocal images of the filled cells. We found that, in contrast to previous reports, SR stimulation evoked substantial responses in all recorded CA2 pyramidal cells. Strikingly, however, we found that not all neurons responded to SLM stimulation, and in those neurons that did, responses evoked by SLM and SR were comparable in size and effectiveness in inducing action potentials. In a comprehensive morphometric analysis of CA2 pyramidal cell apical dendrites, we found that the neurons that were unresponsive to SLM stimulation were the same ones that lacked substantial apical dendritic arborization in the SLM. Neurons responsive to both SR and SLM stimulation had roughly equal amounts of dendritic branching in each layer. Remarkably, our study in mouse CA2 generally replicates the work characterizing the diversity of CA2 pyramidal cells in the guinea pig hippocampus. We conclude, then, that like in guinea pig, mouse CA2 pyramidal cells have a diverse apical dendrite morphology that is likely to be reflective of both the amount and source of excitatory input into CA2 from the entorhinal cortex and CA3.

Activity-dependent synaptic integration and modulation of bilateral excitatory inputs in an auditory coincidence detection circuit.

KEY POINTS: Binaural excitatory inputs to coincidence detection neurons in nucleus laminaris (NL) play essential roles in interaural time difference coding for sound localization. Here, we show that the two excitatory inputs are physiologically nearly completely segregated. Synaptic integration shows linear summation of EPSPs, ensuring high efficiency of coincidence detection of the bilateral excitatory inputs. We further show that the two excitatory inputs to single NL neurons are symmetrical in synaptic strength, kinetics and short-term plasticity. Modulation of the EPSCs by metabotropic glutamate receptors (mGluRs) is identical between the two excitatory inputs, maintaining balanced bilateral excitation under neuromodulatory conditions. Unilateral hearing deprivation reduces synaptic excitation and paradoxically strengthens mGluR modulation of EPSCs, suggesting activity-dependent anti-homeostatic regulation, a novel synaptic plasticity in response to sensory manipulations. ABSTRACT: Neurons in the avian nucleus laminaris (NL) receive bilateral excitatory inputs from the cochlear nucleus magnocellularis, via morphologically symmetrical dorsal (ipsilateral) and ventral (contralateral) dendrites. Using in vitro whole-cell patch recordings in chicken brainstem slices, we investigated synaptic integration and modulation of the bilateral inputs to NL under normal and hearing deprivation conditions. We found that the two excitatory inputs onto single NL neurons were nearly completely segregated, and integration of the two inputs was linear for EPSPs. The two inputs had similar synaptic strength, kinetics and short-term plasticity. EPSCs in low but not middle and high frequency neurons were suppressed by activation of group I and II metabotropic glutamate receptors (mGluR I and II), with similar modulatory strength between the ipsilateral and contralateral inputs. Unilateral hearing deprivation by cochlea removal reduced the excitatory transmission on the deprived dendritic domain of NL. Interestingly, EPSCs evoked at the deprived domain were modulated more strongly by mGluR II than at the counterpart domain that received intact input in low frequency neurons, suggesting anti-homeostatic regulation. This was supported by a stronger expression of mGluR II protein on the deprived neuropils of NL. Under mGluR II modulation, EPSCs on the deprived input show transient synaptic facilitation, forming a striking contrast with normal hearing conditions under which pure synaptic depression is observed. These results demonstrate physiological symmetry and thus balanced bilateral excitatory inputs to NL neurons. The activity-dependent anti-homeostatic plasticity of mGluR modulation constitutes a novel mechanism regulating synaptic transmission in response to sensory input manipulations.

Epsc Involved in the Encoding of Exopolysaccharides Produced by Bacillus amyloliquefaciens FZB42 Act to Boost the Drought Tolerance of Arabidopsis thaliana.

Bacillus amyloliquefaciens FZB42 is a plant growth-promoting rhizobacteria that stimulates plant growth, and enhances resistance to pathogens and tolerance of salt stress. Instead, the mechanistic basis of drought tolerance in Arabidopsis thaliana induced by FZB42 remains unexplored. Here, we constructed an exopolysaccharide-deficient mutant epsC and determined the role of epsC in FZB42-induced drought tolerance in A. thaliana. Results showed that FZB42 significantly enhanced growth and drought tolerance of Arabidopsis by increasing the survival rate, fresh and dry shoot weights, primary root length, root dry weight, lateral root number, and total lateral root length. Coordinated changes were also observed in cellular defense responses, including elevated concentrations of proline and activities of superoxide dismutase and peroxidase, decreased concentrations of malondialdehyde, and accumulation of hydrogen peroxide in plants treated with FZB42. The relative expression levels of drought defense-related marker genes, such as RD29A, RD17, ERD1, and LEA14, were also increased in the leaves of FZB42-treated plants. In addition, FZB42 induced the drought tolerance in Arabidopsis by the action of both ethylene and jasmonate, but not abscisic acid. However, plants inoculated with mutant strain epsC were less able to resist drought stress with respect to each of these parameters, indicating that epsC are required for the full benefit of FZB42 inoculation to be gained. Moreover, the mutant strain was less capable of supporting the formation of a biofilm and of colonizing the A. thaliana root. Therefore, epsC is an important factor that allows FZB42 to colonize the roots and induce systemic drought tolerance in Arabidopsis.

Comparative Proteomic Analysis of Two Ralstonia solanacearum Isolates Differing in Aggressiveness.

Ralstonia solanacearum is a soil-borne, plant xylem-infecting pathogen that causes the devastating bacterial wilt (BW) disease in a number of plant species. In the present study, two R. solanacearum strains with different degrees of aggressiveness-namely RsH (pathogenic to Hawaii 7996, a tomato cultivar resistant against most strains) and RsM (non-pathogenic to Hawaii 7996) were identified. Phylogenetic analysis revealed that both RsM and RsH belonged to phylotype I. To further elucidate the underlying mechanism of the different pathotypes between the two strains, we performed a comparative proteomics study on RsM and RsH in rich and minimal media to identify the change in the level of protein abundance. In total, 24 differential proteins were identified, with four clusters in terms of protein abundance. Further bioinformatics exploration allowed us to classify these proteins into five functional groups. Notably, the pathogenesis of RsM and RsH was particularly characterized by a pronounced difference in the abundance of virulence- and metabolism-related proteins, such as UDP-N-acetylglucosamine 2-epimerase (epsC) and isocitrate lyase (ICL), which were more abundant in the high pathogenicity strain RsH. Thus, we propose that the differences in pathogenicity between RsM and RsH can possibly be partially explained by differences in extracellular polysaccharide (EPS) and glyoxylate metabolism-related proteins.

Synaptic excitation by climbing fibre collaterals in the cerebellar nuclei of juvenile and adult mice.

KEY POINTS: The inferior olive sends instructive motor signals to the cerebellum via the climbing fibre projection, which sends collaterals directly to large premotor neurons of the mouse cerebellar nuclei (CbN cells). Optogenetic activation of inferior olivary axons in vitro evokes EPSCs in CbN cells of several hundred pA to more than 1 nA. The inputs are three-fold larger at younger ages, 12 to 14 days old, than at 2 months old, suggesting a strong functional role for this pathway earlier in development. The EPSCs are multipeaked, owing to burst firing in several olivary afferents that fire asynchronously. The convergence of climbing fibre collaterals onto CbN cells decreases from ∼40 to ∼8, which is consistent with the formation of closed-loop circuits in which each CbN neuron receives input from 4-7 collaterals from inferior olivary neurons as well as from all 30-50 Purkinje cells that are innervated by those olivary neurons. ABSTRACT: The inferior olive conveys instructive signals to the cerebellum that drive sensorimotor learning. Inferior olivary neurons transmit their signals via climbing fibres, which powerfully excite Purkinje cells, evoking complex spikes and depressing parallel fibre synapses. Additionally, however, these climbing fibres send collaterals to the cerebellar nuclei (CbN). In vivo and in vitro data suggest that climbing fibre collateral excitation is weak in adult mice, raising the question of whether the primary role of this pathway may be developmental. We therefore examined climbing fibre collateral input to large premotor CbN cells over development by virally expressing channelrhodopsin in the inferior olive. In acute cerebellar slices from postnatal day (P)12-14 mice, light-evoked EPSCs were large (> 1 nA at -70 mV). The amplitude of these EPSCs decreased over development, reaching a plateau of ∼350 pA at P20-60. Trains of EPSCs (5 Hz) depressed strongly throughout development, whereas convergence estimates indicated that the total number of functional afferents decreased with age. EPSC waveforms consisted of multiple peaks, probably resulting from action potential bursts in single collaterals and variable times to spike threshold in converging afferents. Activating climbing fibre collaterals evoked well-timed increases in firing probability in CbN neurons, especially in younger mice. The initially strong input, followed by the decrement in synaptic strength coinciding with the pruning of climbing fibres in the cerebellar cortex, implicates the climbing fibre collateral pathway in early postnatal development. Additionally, the persistence of substantial synaptic input at least to P60 suggests that this pathway may function in cerebellar processing into adulthood.

The α2A -adrenoceptor suppresses excitatory synaptic transmission to both excitatory and inhibitory neurons in layer 4 barrel cortex.

KEY POINTS: The effects of noradrenaline on excitatory synaptic transmission to regular spiking (excitatory) cells as well as regular spiking non-pyramidal and fast spiking (both inhibitory) cells in cortical layer 4 were studied in thalamocortical slice preparations, focusing on vertical input from thalamus and layer 2/3 in the mouse barrel cortex. Excitatory synaptic responses were suppressed by noradrenaline. However, currents induced by iontophoretically applied glutamate were not suppressed. Further, paired pulse ratio and coefficient of variation analysis indicated the site of action was presynaptic. Pharmacological studies indicated that the suppression was mediated by the α2- adrenoceptor. Consistent with this, involvement of α2A -adrenoceptor activation in the synaptic suppression in excitatory and inhibitory cells was confirmed by the use of α2A -adrenoceptor knockout mice. ABSTRACT: The mammalian neocortex is widely innervated by noradrenergic (NA) fibres from the locus coeruleus. To determine the effects of NA on vertical synaptic inputs to layer 4 (L4) cells from the ventrobasal thalamus and layer 2/3 (L2/3), thalamocortical slices were prepared and whole-cell recordings were made from L4 cells. Excitatory synaptic responses were evoked by electrical stimulation of the thalamus or L2/3 immediately above. Recorded cells were identified as regular spiking, regular spiking non-pyramidal or fast spiking cells through their firing patterns in response to current injections. NA suppressed (∼50% of control) excitatory vertical inputs to all cell types in a dose-dependent manner. The presynaptic site of action of NA was suggested by three independent studies. First, responses caused by iontophoretically applied glutamate were not suppressed by NA. Second, the paired pulse ratio was increased during NA suppression. Finally, a coefficient of variation (CV) analysis was performed and the resultant diagonal alignment of the ratio of CV-2 plotted against the ratio of the amplitude of postsynaptic responses suggests a presynaptic mechanism for the suppression. Experiments with phenylephrine (an α1 -agonist), prazosin (an α1 -antagonist), yohimbine (an α2 -antagonist) and propranolol (a ß-antagonist) indicated that suppression was mediated by the α2 -adrenoceptor. To determine whether the α2A -adrenoceptor subtype was involved, α2A -adrenoceptor knockout mice were used. NA failed to suppress EPSCs in all cell types, suggesting an involvement of the α2A -adrenoceptor. Altogether, we concluded that NA suppresses vertical excitatory synaptic connections in L4 excitatory and inhibitory cells through the presynaptic α2A -adrenoceptor.