Probability Of Glutamate Release Research Articles

Deficiency in transmitter release triggers homeostatic transcriptional changes that increase presynaptic excitability.

Weakening of synaptic transmission at the Drosophila larval neuromuscular junction triggers two forms of homeostatic compensation, one that increases the probability of glutamate release per action potential (Pr) and another that increases motoneuron (MN) activity. We investigated the molecular changes in MNs that underlie the increase in MN activity. RNA-seq analysis on MNs whose glutamate release is weakened by knockdown of components of the MN transmitter release machinery reveals a reduction in expression of a group of genes that encode potassium channels and their positive modulators. These results identify a mechanism of compensation for weakened synaptic transmission by MNs, which engages a transcriptional program in those cells to increase firing and, thereby, ensure sufficient locomotory drive.

Sex-specific behavioral and thalamo-accumbal circuit adaptations after oxycodone abstinence.

Opioid use disorder is marked by a progressive change in the motivation to administer the drug even in the presence of negative consequences. After long periods of abstinence, the urge to return to taking the drug intensifies over time, known as incubation of craving. Conditioned responses to drug-related stimuli, can acquire motivational properties and exert control over motivated behaviors leading to relapse. Although, preclinical data suggest that the behavioral expression of opioid use is similar between male and female rodents, we do not have conclusive results on sex differences on craving and relapse across abstinence periods. Here, we investigated the effects of abstinence from oxycodone self-administration on neurotransmission in the paraventricular thalamus (PVT) to nucleus accumbens shell (NAcSh) pathway in male and female rats. Using optogenetics and ex vivo electrophysiology, we assessed synaptic strength and glutamate release probability in this pathway, as well as NAcSh medium spiny neurons (MSN) intrinsic excitability, in slices from rats which were subjected to either 1 (acute) or 14 (prolonged) days of forced abstinence after self-administration. Our results revealed no sex differences in oxycodone self-administration or somatic withdrawal symptoms following acute abstinence. However, we found a sex-specific enhancement in cue-induced relapse after prolonged, but not acute, abstinence from oxycodone self-administration, with females exhibiting higher relapse rates. Notably, prolonged abstinence led to similar increases in synaptic strength at PVT-NAcSh inputs compared to saline controls in both sexes, which was not observed after acute abstinence. Thus, prolonged abstinence results in a time-dependent increase in PVT-NAcSh synaptic strength and sex-specific effects on cue-induced relapse rates. These findings suggest that prolonged abstinence leads to significant synaptic changes, contributing to heightened relapse vulnerability, highlighting the need for targeted therapeutic strategies in opioid use disorder.

Overwintering Induces Ischemia-Tolerance of Synaptic Function in the Forebrain of Bullfrogs

Healthy brain function requires the continuous delivery of oxygen and glucose to nerve cells. Interruptions result in rapid cessation of normal circuit function in most vertebrates. The American bullfrog ( Lithobates catesbeianus) is an interesting model, as we have shown that brainstem synapses increase their capacity to function during hypoxia and ischemia by roughly 20-30 fold following aquatic overwintering (cold-acclimated, CA) compared to control conditions (warm-acclimated, WA). Here, we tested the hypothesis that other synapses, specially those in the forebrain, also improve their function to determine if extreme metabolic plasticity is specific to the brainstem, or a generalized response throughout the central nervous system. We used the synapse that carries thalamic input to dorsal pallium, which is involved in sensory processing and associative memory. To measure synaptic function, we stimulated thalamic axons and recorded evoked excitatory postsynaptic currents (eEPSCs) in the medial dorsal pallium, the proposed homolog to the mammalian hippocampus, in normoxic conditions and in oxygen and glucose deprivation (OGD). eEPSC amplitude at a baseline frequency of 0.5 Hz was depressed at OGD onset in WA frogs (paired t-test, p=0.0002, n=12) but not in CA animals (paired t-test, p=0.1602, n=6). This suggests that unlike WA frogs, overwintering allows maintained synaptic function in OGD within the forebrain, as well as the brainstem. Mechanistically, the maintenance of synaptic function in OGD appeared to be associated with dynamic increase in the probability of neurotransmitter release. Paired-pulse experiments to assess dynamics in neurotransmitter release in response to high-frequency stimulus pairs showed that synapses depressed in normoxic conditions in both WA (9 of 11 cells) and CA (4 out of 6 cells) groups. In OGD, synaptic depression was maintained in WA frogs (paired t-test, p=0.9192); however, synaptic depression reversed, whereby paired-pulse stimulation instead led to synaptic facilitation in 5 out of 6 neurons (paired t-test, p=0.0113). This suggests that synapses may alter the probability of glutamate release to act as a countermeasure to maintain forebrain function during energetic stress associated with emergence. Overall, these results show that metabolic plasticity to restart critical behaviors in hypoxia following hibernation is preserved across the brain and that altered synaptic dynamics may play a role in this response. This research was funded by the National Institutes of Health (R01NS114514, R15NS112920-01A1). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Changes in neuronal excitability in the rat hippocampus in a prolonged febrile seizures model

Febrile seizures (FSs) are a prevalent neurological disorder among children aged 3 months to 5 years, with the highest incidence observed in the second year of life [1]. Considering that neuronal and glial cell development and synaptic contact formation are ongoing during this period [2, 3], FSs can potentially influence these processes. However, the present data on the effect of FSs on brain development remain inconsistent. This study aims to examine the impact of extended seizures on the attributes of hippocampal pyramidal neurons in rats of varying ages. Wistar rats were used in this study. FSs were induced on postnatal day (P)10 through placing pups onto the bottom of a glass chamber for 30 minutes and exposing them to a controlled stream of heated air, causing their body temperature to rise to 39 °C and trigger the occurrence of FSs. Only animals that underwent FSs that persisted for at least 15 minutes were included in the study. Littermates were employed as controls and were placed away from the nest for the same duration but kept at room temperature. At postnatal days 12, 21–23, and 51–55, the rats were decapitated, and their brains were extracted. Horizontal brain slices (400 micrometers) were sliced. The study examined the biophysical properties of CA1 pyramidal neurons using the whole-cell patch-clamp method. 1.5-second current pulses were injected, and subthreshold membrane properties such as resting membrane potential, input resistance, membrane time constant, and intrinsic firing properties, were assessed. Extracellular field excitatory postsynaptic potentials (fEPSPs) were recorded from the CA1 stratum radiatum of the hippocampus to assess the efficacy of synaptic neurotransmission at CA3-CA1 pyramidal neuron synapses. The amplitude and fiber volley (FV) amplitude of each fEPSP were measured by applying high amplitude currents to each slice. A sigmoidal Gompertz function was used to evaluate the efficacy of neurotransmission. Paired-pulse stimulation was used to examine potential alterations in short-term synaptic plasticity. Paired pulses were administered at intervals spanning from 10 to 500 ms, and the paired-pulse ratio (PPR) was assessed as the proportion of the amplitude of the second to the first fEPSP for each interval. Maximum electroshock seizure threshold (MEST) was determined two months after FS to assess the animals’ susceptibility to seizures. The lowest current at which tonic hind limb extension was exhibited was determined for every animal. No significant changes in subthreshold firing properties were observed in P12 or P21 rats following FSs. However, we did observe several changes in intrinsic firing properties. For example, the maximum firing frequency decreased by 23% in P12 rats exposed to FSs, relative to age-matched control rats. Additionally, we noticed that firing frequency adaptation was significantly less pronounced in P12 rats that underwent FSs, compared to control rats of the same age. Seizure-induced alterations in firing characteristics were absent in P21 rats. No significant variations in fEPSP or FV amplitudes were found among the different groups of P12 and P55 rats at different current intensities. However, a significant increase in FV amplitudes and decrease in neuronal input-output (I/O) relationships between fEPSP and FV amplitudes were observed in P21 rats following FSs. At P12, short-term synaptic plasticity was disrupted, evidenced by a significant increase in PPR in rats two days post-FS. However, at P21 and P55, experimental and control groups were not significantly different. MEST test displayed a significant rise in the hind limb extension threshold in rats two months following FS as compared to control animals. Overall, these findings suggest alterations in neuronal excitability after prolonged FS. Two days after FS, neuronal excitability transiently decreased while PPR increased, indicating a decrease in the probability of presynaptic glutamate release in hippocampal neurons. The efficiency of synaptic neurotransmission in CA3-CA1 was reduced in three-week-old animals. Additionally, the MEST test demonstrated that rats, two months following FS, exhibited a higher hind limb extension threshold in comparison to control animals.

CAMP-dependent protein kinase signaling is required for (2R,6R)-hydroxynorketamine to potentiate hippocampal glutamatergic transmission.

(2R,6R)-Hydroxynorketamine (HNK) is a ketamine metabolite that shows rapid antidepressant-like effects in preclinical studies and lacks the adverse N-methyl-d-aspartate receptor (NMDAR) inhibition-related properties of ketamine. Investigating how (2R,6R)-HNK exerts its antidepressant actions may be informative in the design of novel pharmacotherapies with improved safety and efficacy. We sought to identify the molecular substrates through which (2R,6R)-HNK induces functional changes at excitatory synapses, a prevailing hypothesis for how rapid antidepressant effects are initiated. We recorded excitatory postsynaptic potentials in hippocampal slices from male Wistar Kyoto rats, which have impaired hippocampal plasticity and are resistant to traditional antidepressants. (2R,6R)-HNK (10 µM) led to a rapid potentiation of electrically evoked excitatory postsynaptic potentials at Schaffer collateral CA1 stratum radiatum synapses. This potentiation was associated with a decrease in paired pulse facilitation, suggesting an increase in the probability of glutamate release. The (2R,6R)-HNK-induced potentiation was blocked by inhibiting either cyclic adenosine monophosphate (cAMP) or its downstream target, cAMP-dependent protein kinase (PKA). As cAMP is a potent regulator of brain-derived neurotrophic factor (BDNF) release, we assessed whether (2R,6R)-HNK exerts this acute potentiation through a rapid increase in cAMP-dependent BDNF-TrkB signaling. We found that the cAMP-PKA-dependent potentiation was not dependent on TrkB activation by BDNF, which functionally delimits the acute synaptic effects of (2R,6R)-HNK from its sustained BDNF-dependent actions in vivo. These results suggest that, by potentiating glutamate release via cAMP-PKA signaling, (2R,6R)-HNK initiates acute adaptations in fast excitatory synaptic transmission that promote structural plasticity leading to maintained antidepressant action.NEW & NOTEWORTHY Ketamine is a rapid-acting antidepressant and its preclinical effects are mimicked by its (2R,6R)-(HNK) metabolite. We found that (2R,6R)-HNK initiates acute adaptations in fast excitatory synaptic transmission by potentiating glutamate release via cAMP-PKA signaling at hippocampal Schaffer collateral synapses. This cAMP-PKA-dependent potentiation was not dependent on TrkB activation by BDNF, which functionally delimits the rapid synaptic effects of (2R,6R)-HNK from its sustained BDNF-dependent actions that are thought to maintain antidepressant action in vivo.

Alterations in Rat Hippocampal Glutamatergic System Properties after Prolonged Febrile Seizures

Febrile seizures during early childhood may result in central nervous system developmental disorders. However, the specific mechanisms behind the impact of febrile seizures on the developing brain are not well understood. To address this gap in knowledge, we employed a hyperthermic model of febrile seizures in 10-day-old rats and tracked their development over two months. Our objective was to determine the degree to which the properties of the hippocampal glutamatergic system are modified. We analyzed whether pyramidal glutamatergic neurons in the hippocampus die after febrile seizures. Our findings indicate that there is a reduction in the number of neurons in various regions of the hippocampus in the first two days after seizures. The CA1 field showed the greatest susceptibility, and the reduction in the number of neurons in post-FS rats in this area appeared to be long-lasting. Electrophysiological studies indicate that febrile seizures cause a reduction in glutamatergic transmission, leading to decreased local field potential amplitude. This impairment could be attributable to diminished glutamate release probability as evidenced by decreases in the frequency of miniature excitatory postsynaptic currents and increases in the paired-pulse ratio of synaptic responses. We also found higher threshold current causing hind limb extension in the maximal electroshock seizure threshold test of rats 2 months after febrile seizures compared to the control animals. Our research suggests that febrile seizures can impair glutamatergic transmission, which may protect against future seizures.

Increased Heat Pain Tolerance but Hyperalgesia to Tonic Inflammatory Pain in the CRND8 Mouse Model of Alzheimer's Disease.

The effects of Alzheimer's disease (AD) pathology on the experience of pain are poorly understood. To understand the pathophysiological mechanisms underlying pain sensory transmission in the transgenic mouse model of AD, CRND8. We explored AD-related pathology in the spinal cord and dorsal root ganglia of 18-week-old female CRND8 mice. We assessed nociceptive responses to both acute heat stimuli and persistent inflammatory pain in CRND8 mice and non-transgenic (non-Tg) littermates. In addition, we searched for differences in biochemical correlates of inflammatory pain between CRND8 and non-Tg mice. Finally, we investigated the excitability of dorsal horn noc iceptive neurons in spinal cord slices from CRND8 and non-Tg mice. We demonstrated the presence of intracellular AD-like pathology in the spinal cord and in the dorsal root ganglia nociceptive sensory neurons of CRND8 mice. We found that CRND8 mice had a reduced susceptibility to acute noxious heat stimuli and an increased sensitivity to tonic inflammatory pain. Tonic inflammatory pain correlated with a lack of induction of pro-opiomelanocortin in the spinal cord of CRND8 mice as compared to non-Tg mice. Electrophysiological recording in acute spinal cord slice preparations indicated an increased probability of glutamate release at the membrane of dorsal horn nociceptive neurons in CRND8 mice. This study suggests that an increased thermal tolerance and a facilitation of nociception by peripheral inflammation can coexist in AD.

Sex differences in pre- and post-synaptic glutamate signaling in the nucleus accumbens core

BackgroundGlutamate signaling within the nucleus accumbens underlies motivated behavior and is involved in psychiatric disease. Although behavioral sex differences in these processes are well-established, the neural mechanisms driving these differences are largely unexplored. In these studies, we examine potential sex differences in synaptic plasticity and excitatory transmission within the nucleus accumbens core. Further understanding of baseline sex differences in reward circuitry will shed light on potential mechanisms driving behavioral differences in motivated behavior and psychiatric disease.MethodsBehaviorally naïve adult male and female Long-Evans rats, C57Bl/6J mice, and constitutive PKMζ knockout mice were killed and tissue containing the nucleus accumbens core was collected for ex vivo slice electrophysiology experiments. Electrophysiology recordings examined baseline sex differences in synaptic plasticity and transmission within this region and the potential role of PKMζ in long-term depression.ResultsWithin the nucleus accumbens core, both female mice and rats exhibit higher AMPA/NMDA ratios compared to male animals. Further, female mice have a larger readily releasable pool of glutamate and lower release probability compared to male mice. No significant sex differences were detected in spontaneous excitatory postsynaptic current amplitude or frequency. Finally, the threshold for induction of long-term depression was lower for male animals than females, an effect that appears to be mediated, in part, by PKMζ.ConclusionsWe conclude that there are baseline sex differences in synaptic plasticity and excitatory transmission in the nucleus accumbens core. Our data suggest there are sex differences at multiple levels in this region that should be considered in the development of pharmacotherapies to treat psychiatric illnesses such as depression and substance use disorder.

SNAP25 differentially contributes to Gi/o-coupled receptor function at glutamatergic synapses in the nucleus accumbens.

The nucleus accumbens (NAc) guides reward-related motivated behavior implicated in pathological behavioral states, including addiction and depression. These behaviors depend on the precise neuromodulatory actions of Gi/o-coupled G-protein-coupled receptors (GPCRs) at glutamatergic synapses onto medium spiny projection neurons (MSNs). Previous work has shown that discrete classes of Gi/o-coupled GPCR mobilize Gβγ to inhibit vesicular neurotransmitter release via t-SNARE protein, SNAP25. However, it remains unknown which Gαi/o systems in the NAc utilize Gβγ-SNARE signaling to dampen glutamatergic transmission. Utilizing patch-clamp electrophysiology and pharmacology in a transgenic mouse line with a C-terminal three-residue deletion of SNAP25 (SNAP25Δ3) weaking the Gβγ-SNARE interaction, we surveyed a broad cohort of Gi/o-coupled GPCRs with robust inhibitory actions at glutamatergic synapses in the NAc. We find that basal presynaptic glutamate release probability is reduced in SNAP25Δ3 mice. While κ opioid, CB1, adenosine A1, group II metabotropic glutamate receptors, and histamine H3 receptors inhibit glutamatergic transmission onto MSNs independent of SNAP25, we report that SNAP25 contributes significantly to the actions of GABAB, 5-HT1B/D, and μ opioid receptors. These findings demonstrate that presynaptic Gi/o-coupled GPCRs recruit heterogenous effector mechanisms at glutamatergic synapses in the NAc, with a subset requiring SNA25-dependent Gβγ signaling.

Insulin modulates the paired-pulse plasticity at glutamatergic synapses of hippocampal neurons under hypoinsulinemia.

Hypoinsulinemia is a pathological consequence of diabetes mellitus that can cause a number of complications of the central and peripheral nervous system. Dysfunction of signaling cascades of insulin receptors under insulin deficiency can contribute to the development of cognitive disorders associated with impaired synaptic plasticity properties. Earlier we have shown that hypoinsulinemia causes a shift of short-term plasticity in glutamatergic hippocampal synapses from facilitation to depression and apparently involves mechanisms of glutamate release probability reduction. Here we used the whole cell patch-clamp recording of evoked glutamatergic excitatory postsynaptic currents (eEPSCs) and the method of local extracellular electrical stimulation of a single presynaptic axon to investigate the effect of insulin (100 nM) on the paired-pulse plasticity at glutamatergic synapses of cultured hippocampal neurons under hypoinsulinemia. Our data indicate that under normoinsulinemia additional insulin enhances the paired-pulse facilitation (PPF) of eEPSCs in hippocampal neurons by stimulating the glutamate release in their synapses. Under hypoinsulinemia, insulin did not have a significant effect on the parameters of paired-pulse plasticity on neurons of PPF subgroup, which may indicate the development of insulin resistance, while the effect of insulin on PPD neurons indicates its ability to recover the form normoinsulinemia, including the increasing probability of plasticity to the control level in of glutamate release in their synapses.

Neuronal Firing and Glutamatergic Synapses in the Substantia Nigra Pars Reticulata of LRRK2-G2019S Mice.

Pathogenic mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are frequent causes of familial Parkinson's Disease (PD), an increasingly prevalent neurodegenerative disease that affects basal ganglia circuitry. The cellular effects of the G2019S mutation in the LRRK2 gene, the most common pathological mutation, have not been thoroughly investigated. In this study we used middle-aged mice carrying the LRRK2-G2019S mutation (G2019S mice) to identify potential alterations in the neurophysiological properties and characteristics of glutamatergic synaptic transmission in basal ganglia output neurons, i.e., substantia nigra pars reticulata (SNr) GABAergic neurons. We found that the intrinsic membrane properties and action potential properties were unaltered in G2019S mice compared to wild-type (WT) mice. The spontaneous firing frequency was similar, but we observed an increased regularity in the firing of SNr neurons recorded from G2019S mice. We examined the short-term plasticity of glutamatergic synaptic transmission, and we found an increased paired-pulse depression in G2019S mice compared to WT mice, indicating an increased probability of glutamate release in SNr neurons from G2019S mice. We measured synaptic transmission mediated by NMDA receptors and we found that the kinetics of synaptic responses mediated by these receptors were unaltered, as well as the contribution of the GluN2B subunit to these responses, in SNr neurons of G2019S mice compared to WT mice. These results demonstrate an overall maintenance of basic neurophysiological and synaptic characteristics, and subtle changes in the firing pattern and in glutamatergic synaptic transmission in basal ganglia output neurons that precede neurodegeneration of dopaminergic neurons in the LRRK2-G2019S mouse model of late-onset PD.

Enhanced Feedback Inhibition Due to Increased Recruitment of Somatostatin-Expressing Interneurons and Enhanced Cortical Recurrent Excitation in a Genetic Mouse Model of Migraine.

Migraine is a complex brain disorder, characterized by attacks of unilateral headache and global dysfunction in multisensory information processing, whose underlying cellular and circuit mechanisms remain unknown. The finding of enhanced excitatory, but unaltered inhibitory, neurotransmission at cortical synapses between pyramidal cells (PCs) and fast-spiking interneurons (FS INs) in mouse models of familial hemiplegic migraine (FHM) suggested the hypothesis that dysregulation of the excitatory-inhibitory (E/I) balance in specific circuits is a key pathogenic mechanism. Here, we investigated the cortical layer 2/3 (L2/3) feedback inhibition microcircuit involving somatostatin-expressing (SOM) INs in FHM1 mice of both sexes carrying a gain-of-function mutation in CaV2.1. Unitary inhibitory neurotransmission at SOM IN-PC synapses was unaltered while excitatory neurotransmission at both PC-SOM IN and PC-PC synapses was enhanced, because of increased probability of glutamate release, in FHM1 mice. Short-term synaptic depression was enhanced at PC-PC synapses while short-term synaptic facilitation was unaltered at PC-SOM IN synapses during 25-Hz repetitive activity. The frequency-dependent disynaptic inhibition (FDDI) mediated by SOM INs was enhanced, lasted longer and required shorter high-frequency bursts to be initiated in FHM1 mice. These findings, together with previous evidence of enhanced disynaptic feedforward inhibition by FS INs, suggest that the increased inhibition may effectively counteract the increased recurrent excitation in FHM1 mice and may even prevail in certain conditions. Considering the involvement of SOM INs in γ oscillations, surround suppression and context-dependent sensory perception, the facilitated recruitment of SOM INs, together with the enhanced recurrent excitation, may contribute to dysfunctional sensory processing in FHM1 and possibly migraine.SIGNIFICANCE STATEMENT Migraine is a complex brain disorder, characterized by attacks of unilateral headache and global dysfunction in multisensory information processing, whose underlying cellular and circuit mechanisms remain unknown, although dysregulation of the excitatory-inhibitory (E/I) balance in specific circuits could be a key pathogenic mechanism. Here, we provide insights into these mechanisms by investigating the cortical feedback inhibition microcircuit involving somatostatin-expressing interneurons (SOM INs) in a mouse model of a rare monogenic migraine. Despite unaltered inhibitory synaptic transmission, the disynaptic feedback inhibition mediated by SOM INs was enhanced in the migraine model because of enhanced recruitment of the INs. Recurrent cortical excitation was also enhanced. These alterations may contribute to context-dependent sensory processing dysfunctions in migraine.

(2R,6R)-hydroxynorketamine rapidly potentiates optically-evoked Schaffer collateral synaptic activity

(2R,6R)-hydroxynorketamine (HNK) is a metabolite of ketamine that exerts rapid and sustained antidepressant-like effects in preclinical studies. We hypothesize that the rapid antidepressant actions of (2R,6R)-HNK involve an acute increase in glutamate release at Schaffer collateral synapses. Here, we used an optogenetic approach to assess whether (2R,6R)-HNK promotes glutamate release at CA1-projecting Schaffer collateral terminals in response to select optical excitation of CA3 afferents. The red-shifted channelrhodopsin, ChrimsonR, was expressed in dorsal CA3 neurons of adult male Sprague Dawley rats. Transverse slices were collected four weeks later to determine ChrimsonR expression and to assess the acute synaptic effects of an antidepressant-relevant concentration of (2R,6R)-HNK (10 μM). (2R,6R)-HNK led to a rapid potentiation of CA1 field excitatory postsynaptic potentials evoked by recurrent optical stimulation of ChrimsonR-expressing CA3 afferents. This potentiation is mediated in part by an increase in glutamate release probability, as (2R,6R)-HNK suppressed paired-pulse facilitation at CA3 projections, an effect that correlated with the magnitude of the (2R,6R)-HNK-induced potentiation of CA1 activity. These results demonstrate that (2R,6R)-HNK increases the probability of glutamate release at CA1-projecting Schaffer collateral afferents, which may be involved in the antidepressant-relevant behavioral adaptations conferred by (2R,6R)-HNK in vivo. The current study also establishes proof-of-principle that genetically-encoded light-sensitive proteins can be used to investigate the synaptic plasticity induced by novel antidepressant compounds in neuronal subcircuits.This article is part of the Special Issue on ‘Ketamine and its Metabolites’.

GABAB receptors constrain glutamate presynaptic release and postsynaptic actions in substantia gelatinosa of rat spinal cord.

The substantia gelatinosa (SG, lamina II of spinal cord gray matter) is pivotal for modulating nociceptive information from the peripheral to the central nervous system. γ-Aminobutyric acid type B receptors (GABABRs), the metabotropic GABA receptor subtype, are widely expressed in pre- and postsynaptic structures of the SG. Activation of GABABRs by exogenous agonists induces both pre- and postsynaptic inhibition. However, the actions of endogenous GABA via presynaptic GABABRs on glutamatergic synapses, and the postsynaptic GABABRs interaction with glutamate, remain elusive. In the present study, first, using in vitro whole-cell recordings and taking minimal stimulation strategies, we found that in rat spinal cord glutamatergic synapses, blockade of presynaptic GABABRs switched "silent" synapses into active ones and increased the probability of glutamate release onto SG neurons; increasing ambient GABA concentration mimicked GABABRs activation on glutamatergic terminals. Next, using holographic photostimulation to uncage glutamate on postsynaptic SG neurons, we found that postsynaptic GABABRs modified glutamate-induced postsynaptic potentials. Taken together, our data identify that endogenous GABA heterosynaptically constrains glutamate release via persistently activating presynaptic GABABRs; and postsynaptically, GABABRs modulate glutamate responses. The results give new clues for endogenous GABA in modulating the nociception circuit of the spinal dorsal horn and shed fresh light on the postsynaptic interaction of glutamate and GABA.

LRRK2-G2019S mice display alterations in glutamatergic synaptic transmission in midbrain dopamine neurons.

The progressive degeneration of dopamine (DA) neurons in the substantia nigra compacta (SNc) leads to the emergence of motor symptoms in patients with Parkinson's disease (PD). To propose neuroprotective therapies able to slow or halt the progression of the disease, it is necessary to identify cellular alterations that occur before DA neurons degenerate and before the onset of the motor symptoms that characterize PD. Using electrophysiological, histochemical, and biochemical approaches, we have examined if glutamatergic synaptic transmission in DA neurons in the SNc and in the adjacent ventral tegmental area (VTA) was altered in middle‐aged (10–12 months old) mice with the hG2019S point mutation (G2019S) in the leucine‐rich repeat kinase 2 (LRRK2) gene. G2019S mice showed increased locomotion and exploratory behavior compared with wildtype (WT) littermates, and intact DA neuron integrity. The intrinsic membrane properties and action potential characteristics of DA neurons recorded in brain slices were similar in WT and G2019S mice. Initial glutamate release probability onto SNc‐DA neurons, but not VTA‐DA neurons, was reduced in G2019S mice. We also found reduced protein amounts of the presynaptic marker of glutamatergic terminals, VGLUT1, and of the GluA1 and GluN1 subunits of AMPA and NMDA receptors, respectively, in the ventral midbrain of G2019S mice. These results identify alterations in glutamatergic synaptic transmission in DA neurons of the SNc and VTA before the onset of motor impairments in the LRRK2‐G2019S mouse model of PD.

All-optical monitoring of excitation-secretion coupling demonstrates that SV2A functions downstream of evoked Ca2+ entry.

SV2A, an essential transporter-like synaptic vesicle protein, is a major target for antiepileptic drugs and a receptor for clostridial neurotoxins including Botox. While SV2A is required for normal levels of evoked neurotransmitter release, the mechanism underlying this role remains unclear. Here, we introduce a new chemogenetic approach for all-optical monitoring of excitation-secretion coupling, and we demonstrate its use in characterizing the SV2A knockout (KO) phenotype in cultured hippocampal neurons. This method employs the HaloTag system to target a robust small-molecule Ca2+ indicator, JF646 -BAPTA, to the presynaptic compartment. The far-red fluorescence of this indicator enables multiplexing with the fluorescent glutamate sensor iGluSnFR for detection of presynaptic Ca2+ influx and glutamate release at the same axonal boutons. Evoked glutamate release probability was reduced in SV2A KO neurons without a change in presynaptic Ca2+ entry, suggesting that SV2A supports vesicle fusion by increasing the functional availability, or efficiency, of the Ca2+ -regulated membrane fusion machinery. KEY POINTS: One of the most prescribed antiepileptic medications, levetiracetam, acts by binding a protein of uncertain molecular function. This transporter-like protein, SV2A, is trafficked to synaptic vesicles and acts to support neurotransmitter release, but the mechanism underlying this function has not been determined In this study, we sought to establish whether SV2A changes Ca2+ signalling at nerve terminals, which is a key regulatory system for synaptic vesicle exocytosis. To do so, we adapted new chemogenetic tools to perform all-optical measurements of presynaptic Ca2+ and glutamate release in neurons lacking SV2A. Our measurements showed that loss of SV2A reduces glutamate release without reducing Ca2+ influx at hippocampal nerve terminals, demonstrating that SV2A increases the likelihood that Ca2+ will trigger synaptic vesicle fusion.

Early Life Stress Induces Different Behaviors in Adolescence and Adulthood May Related With Abnormal Medial Prefrontal Cortex Excitation/Inhibition Balance.

Early life stress is thought to be a risk factor for emotional disorders, particularly depression and anxiety. Although the excitation/inhibition (E/I) imbalance has been implicated in neuropsychiatric disorders, whether early life stress affects the E/I balance in the medial prefrontal cortex at various developmental stages is unclear. In this study, rats exposed to maternal separation (MS) that exhibited a well-established early life stress paradigm were used to evaluate the E/I balance in adolescence (postnatal day P43–60) and adulthood (P82–100) by behavior tests, whole-cell recordings, and microdialysis coupled with high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis. First, the behavioral tests revealed that MS induced both anxiety- and depressive-like behaviors in adolescent rats but only depressive-like behavior in adult rats. Second, MS increased the action potential frequency and E/I balance of synaptic transmission onto L5 pyramidal neurons in the prelimbic (PrL) brain region of adolescent rats while decreasing the action potential frequency and E/I balance in adult rats. Finally, MS increases extracellular glutamate levels and decreased the paired-pulse ratio of evoked excitatory postsynaptic currents (EPSCs) of pyramidal neurons in the PrL of adolescent rats. In contrast, MS decreased extracellular glutamate levels and increased the paired-pulse ratio of evoked EPSCs of pyramidal neurons in the PrL of adult rats. The present results reveal a key role of E/I balance in different MS-induced disorders may related to the altered probability of presynaptic glutamate release at different developmental stages.

Estrogen signaling modulates behavioral selection toward pups and amygdalohippocampal area in the rhomboid nucleus of the bed nucleus of the stria terminalis circuit

Gonadal steroid hormone influences behavioral choice of adult animals toward pups, parental or aggressive. We previously reported that long-term administration of 17β-estradiol (E2) to male mice during sexual maturation induces aggressive behavior toward conspecific pups, which is called “infanticide,” and significantly enhanced excitatory synaptic transmission in the rhomboid nucleus of bed nucleus of the stria terminalis (BSTrh), which is an important brain region for infanticide. However, it is unclear how estrogen receptor–dependent signaling after sexual maturity regulates neural circuits including the BSTrh. Here we revealed that E2 administration to gonadectomized mice in adulthood elicited infanticidal behavior and enhanced excitatory synaptic transmission in the BSTrh by increasing the probability of glutamate release from the presynaptic terminalis. Next, we performed whole-brain mapping of E2-sensitive brain regions projecting to the BSTrh and found that amygdalohippocampal area (AHi) neurons that project to the BSTrh densely express estrogen receptor 1 (Esr1). Moreover, E2 treatment enhanced synaptic connectivity in the AHi-BSTrh pathway. Together, these results suggest that reinforcement of excitatory inputs from AHi neurons into the BSTrh by estrogen receptor–dependent signaling may contribute to the expression of infanticide.

Short-Term Epileptiform Activity Potentiates Excitatory Synapses but Does Not Affect Intrinsic Membrane Properties of Pyramidal Neurons in the Rat Hippocampus In Vitro.

Even brief epileptic seizures can lead to activity-dependent structural remodeling of neural circuitry. Animal models show that the functional plasticity of synapses and changes in the intrinsic excitability of neurons can be crucial for epileptogenesis. However, the exact mechanisms underlying epileptogenesis remain unclear. We induced epileptiform activity in rat hippocampal slices for 15 min using a 4-aminopyridine (4-AP) in vitro model and observed hippocampal hyperexcitability for at least 1 h. We tested several possible mechanisms of this hyperexcitability, including changes in intrinsic membrane properties of neurons and presynaptic and postsynaptic alterations. Neither input resistance nor other essential biophysical properties of hippocampal CA1 pyramidal neurons were affected by epileptiform activity. The glutamate release probability also remained unchanged, as the frequency of miniature EPSCs and the paired amplitude ratio of evoked responses did not change after epileptiform activity. However, we found an increase in the AMPA/NMDA ratio, suggesting alterations in the properties of postsynaptic glutamatergic receptors. Thus, the increase in excitability of hippocampal neural networks is realized through postsynaptic mechanisms. In contrast, the intrinsic membrane properties of neurons and the probability of glutamate release from presynaptic terminals are not affected in a 4-AP model.

Long-term depression links amyloid-β to the pathological hyperphosphorylation of tau.

SummaryIn Alzheimer’s disease, soluble oligomers of the amyloid-β peptide (Aβo) trigger a cascade of events that includes abnormal hyperphosphorylation of the protein tau, which is essential for pathogenesis. However, the mechanistic link between these two key pathological proteins remains unclear. Using hippocampal slices, we show here that an Aβo-mediated increase in glutamate release probability causes enhancement of synaptically evoked N-methyl-d-aspartate subtype glutamate receptor (NMDAR)-dependent long-term depression (LTD). We also find that elevated glutamate release probability is required for Aβo-induced pathological hyperphosphorylation of tau, which is likewise NMDAR dependent. Finally, we show that chronic, repeated chemical or optogenetic induction of NMDAR-dependent LTD alone is sufficient to cause tau hyperphosphorylation without Aβo. Together, these results support a possible causal chain in which Aβo increases glutamate release probability, thus leading to enhanced LTD induction, which in turn drives hyperphosphorylation of tau. Our data identify a mechanistic pathway linking the two critical pathogenic proteins of AD.