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Fragile X syndrome (FXS) is characterized by synaptic immaturity, cognitive impairment, and behavioral changes. The disorder is caused by transcriptional shutdown in neurons of thefragile X mental retardation 1gene product, fragile X mental retardation protein. Fragile X mental retardation protein is a repressor of dendritic mRNA translation and its silencing leads to dysregulation of synaptically driven protein synthesis and impairments of intellect, cognition, and behavior, and FXS is a disorder that currently has no effective therapeutics. Here, young fragile X mice were treated with chronic bryostatin-1, a relatively selective protein kinase Cεactivator, which induces synaptogenesis and synaptic maturation/repair. Chronic treatment with bryostatin-1 rescues young fragile X mice from the disorder phenotypes, including normalization of most FXS abnormalities in 1) hippocampal brain-derived neurotrophic factor expression, 2) postsynaptic density-95 levels, 3) transformation of immature dendritic spines to mature synapses, 4) densities of the presynaptic and postsynaptic membranes, and 5) spatial learning and memory. The therapeutic effects were achieved without downregulation of metabotropic glutamate receptor (mGluR) 5 in the hippocampus and are more dramatic than those of a late-onset treatment in adult fragile X mice. mGluR5 expression was in fact lower in fragile X mice and its expression was restored with the bryostatin-1 treatment. Our results show that synaptic and cognitive function of young FXS mice can be normalized through pharmacological treatment without downregulation of mGluR5 and that bryostatin-1-like agents may represent a novel class of drugs to treat fragile X mental retardation at a young age and in adults.

Astrocytes have critical functions throughout the central nervous system (CNS) and have emerged as regulators of synaptic development and function. With their highly complex morphologies, they are able to interact with thousands of synapses via peripheral astrocytic processes (PAPs), ensheathing neuronal axons and dendrites to form the tripartite synapse. In this way, astrocytes engage in crosstalk with neurons to mediate a variety of CNS processes including the regulation of extracellular matrix protein signaling, formation and maintenance of the blood-brain barrier (BBB), axon growth and guidance, homeostasis of the synaptic microenvironment, synaptogenesis, and the promotion of synaptic diversity. In this review, we discuss several key astrocyte signaling factors (thrombospondins, netrins, apolipoproteins, neuregulins, bone morphogenetic proteins, and neuroligins) in the maintenance and regulation of synapse formation. We also explore how these astrocyte signaling factors are impacted by and contribute to substance abuse, particularly alcohol and cocaine use.

Opioid-induced hyperalgesia and analgesic tolerance can lead to dose escalation and inadequate pain treatment with μ-opioid receptor agonists. Opioids cause tonic activation of glutamate NMDA receptors (NMDARs) at primary afferent terminals, increasing nociceptive input. However, the signaling mechanisms responsible for opioid-induced activation of pre-synaptic NMDARs in the spinal dorsal horn remain unclear. In this study, we determined the role of MAPK signaling in opioid-induced pre-synaptic NMDAR activation caused by chronic morphine administration. Whole-cell recordings of excitatory post-synaptic currents (EPSCs) were performed on dorsal horn neurons in rat spinal cord slices. Chronic morphine administration markedly increased the frequency of miniature EPSCs, increased the amplitude of monosynaptic EPSCs evoked from the dorsal root, and reduced the paired-pulse ratio of evoked EPSCs. These changes were fully reversed by an NMDAR antagonist and normalized by inhibiting extracellular signal-regulated kinase 1/2 (ERK1/2), p38, or c-Jun N-terminal kinase (JNK). Furthermore, intrathecal injection of a selective ERK1/2, p38, or JNK inhibitor blocked pain hypersensitivity induced by chronic morphine treatment. These inhibitors also similarly attenuated a reduction in morphine's analgesic effect in rats. In addition, co-immunoprecipitation assays revealed that NMDARs formed a protein complex with ERK1/2, p38, and JNK in the spinal cord and that chronic morphine treatment increased physical interactions of NMDARs with these three MAPKs. Our findings suggest that opioid-induced hyperalgesia and analgesic tolerance are mediated by tonic activation of pre-synaptic NMDARs via three functionally interrelated MAPKs at the spinal cord level. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

GABA synapses in the brain undergo depolarization-induced suppression of inhibition (DSI) that requires activation of presynaptic cannabinoid type 1 receptors (CB1Rs). The brevity of DSI, lasting ∼1 min in most brain regions, has been ascribed to the transient production of 2-arachidonoylglycerol (2-AG). Here, we propose that the duration of DSI is controlled by heterologous interactions between presynaptic mGluRs and CB1Rs. By examining GABA synapses on parvocellular corticotropin-releasing hormone-expressing neurons in the paraventricular nucleus of the hypothalamus (PVN) of male and female mice, we show that DSI decays quickly in experimental conditions in which both GABA and glutamate are released from adjacent nerve terminals. Pharmacological inhibition of group I mGluRs prolongs DSI, whereas prior activation of mGluRs inhibits DSI, collectively suggesting that group I mGluRs quench presynaptic CB1R signaling. When photostimulation of genetically identified terminals is used to release only GABA, CB1R-dependent DSI persists for many minutes. Under the same conditions, activation of group I mGluRs reestablishes classical, transient DSI. The long-lasting DSI observed when GABA synapses are independently recruited functionally uncouples inhibitory input to PVN neurons. These observations suggest that heterologous interactions between mGluRs and CB1Rs control the temporal window of DSI at GABA synapses, providing evidence for a powerful new way to affect functional circuit connectivity in the brain.SIGNIFICANCE STATEMENTPostsynaptic depolarization liberates endocannabinoids, resulting in a rapid and transient decrease in release probability at GABA synapses. We discovered that mGluRs control the duration of depolarization-induced suppression of inhibition (DSI), most likely through heterologous desensitization of cannabinoid type 1 receptors by presynaptic mGluR5. By shortening the duration of DSI, mGluRs control the temporal window for retrograde signaling at GABA synapses. Physiological or pathological changes that affect glutamate spillover may profoundly affect network excitability by shifting the duration of cannabinoid inhibition at GABA synapses.

In the classical view, postsynaptic NMDA receptors (NMDARs) trigger Hebbian plasticity via Ca2+ influx. However, unconventional presynaptic NMDARs (preNMDARs) which regulate both long-term and short-term plasticity at several synapse types have also been found. A lack of sufficiently specific experimental manipulations and a poor understanding of how preNMDARs signal have contributed to long-standing controversy surrounding these receptors. Although several prior studies linked preNMDARs to neocortical timing-dependent long-term depression (tLTD), a recent study argues that the NMDARs are actually postsynaptic and signal metabotropically, that is, without Ca2+. Other recent work indicates that, whereas ionotropic preNMDARs signaling controls evoked release, spontaneous release is regulated by metabotropic NMDAR signaling. We argue that elucidating unconventional NMDAR signaling modes-both presynaptically and metabotropically-is key to resolving the preNMDAR debate.

Inflammation-induced activation of the hypothalamic-pituitary-adrenal (HPA) axis and the ensuing release of anti-inflammatory glucocorticoids are critical for the fine-tuning of the inflammatory response. This immune-induced neuroendocrine response is in large part mediated by prostaglandin E

Glucagon-like peptide-1 receptors (GLP-1Rs) have been shown to mediate cognitive-enhancing and neuroprotective effects in the central nervous system. However, little is known about their physiological roles on central neurotransmission, especially at the presynaptic level. Using purified synaptosomal preparations and immunofluorescence techniques, here we show for the first time that GLP-1Rs are localized on mouse cortical and hippocampal synaptic boutons, in particular on glutamatergic and GABAergic nerve terminals. Their activation by the selective agonist exendin-4 (1-100 nM) was able to increase the release of either [

NMDA receptors (NMDARs) are glutamate-gated ion channels widely expressed in the central nervous system (CNS) and endowed with unique biophysical, pharmacological and signaling properties. These receptors are best known for their critical roles in synaptic plasticity and their implications in a variety of neurological and psychiatric disorders. Since their discovery three decades ago, NMDARs have been thoroughly studied as components of postsynaptic excitatory potentials. Early on, however, both anatomical and physiological evidence pointed out to the existence of NMDARs away from the postsynaptic density. Some were found to be extrasynaptic, while others seemed to be specifically present at presynaptic (i.e. axonal) elements. Although presynaptic NMDARs (preNMDARs) were at first thought to be exceptional, there is now strong evidence that these receptors are relatively widespread in the CNS and regulate synaptic strength in specific sets of synapses. In this review, we compile our current knowledge on preNMDARs, presenting their anatomical distribution, developmental regulation, subunit composition, activation mechanisms as well as their downstream effects on synapse function. Contentious issues that animate the field are also discussed. Finally, particular emphasis is put on the molecular and cellular diversity of preNMDARs which translates into a variety of effects, both short- and long-term, on synaptic efficacy. Overshadowed by their postsynaptic counterparts, preNMDARs are progressively emerging as important regulators of neuronal signaling.

303 Objectives 90Y microspheres are increasingly used for the treatment of inoperable hepatocellular carcinoma (HCC). Currently, the confirmation of 90Y biodistribution after treatment is assessed indirectly by Bremsstrahlung-SPECT, which is known to have poor statistical counting and inaccurate quantification of actual delivered dose to the tumor (TD) and nontumorous liver (NTD). TD and NTD are important prognostic and radiobiological factors in HCC patients; therefore, we aim to evaluate the efficacy of 90Y PET/CT post radioembolization for dosimetry measurement and for quantitative optimization of injected activity (IA) of 90Y glass microspheres individualized to HCC patients. Methods From April 2014 to Oct 2015, 34 patients (M: 28, F: 6; age range: 34~82y, mean=60.1±13.9y; HBV: 24, HCV: 1) treated by 90Y glass microspheres for intermediate or advanced stage HCC were recruited. All patients underwent pretreatment 99mTc-MAA planar and SPECT/CT to estimate the lung shunting (S:% of IA) and tumor-to-nontumorous liver ratio (TNR), respectively. The dose to the injected liver (ILD) was determined by adjustment of the fixed radiation dose (120Gy recommended by pharmaceutical company) according to patient’s tumor extent, liver size and liver function. The IA was then calculated by a simplified MIRD equation: IA(GBq)= ILD(Gy)/[(1-S)×49.67/W(kg)] (W=ILvolume×1.03, where 1.03kg/L is the liver density). Regional 90Y PET/CT (Biograph mCT) was performed 17~22 hours post radioembolization with 15-min acquisition/bed and “time-of-flight” reconstruction (2 iterations, 21 subsets, 10-mm at FWHM, matrix size 200×200). For each patient, right & left nontumorous liver (NL), lung and all HCC lesions were contoured by automatic algorithms on PET/CT with mean activity concentration (Bq/ml) and volume measured for each VOI. They are then corrected for the 90Y positron branching ratio (3.186×10−5) and time decay factors to obtain the actual PET-measured activities (PA) in tumor, NL and lung. The TD, NTD and lung dose were calculated by D(Gy)=PA(GBq)×49.67/W(kg). Partial volume correction was not considered due to voluminous lesions (>6cm) in all patients. The accuracy of 90Y PET/CT for dosimetry measurement was checked by comparing the summation of PA in tumor, NL and lung with the injected activity. Results 33/34 patients received reduced IA with ILD ranging from 40 to 100Gy (median: 60Gy). The IA was 2.45±1.11GBq. 90Y PET/CT post radioembolization showed high TD (>120Gy) in 30/34 patients (mean: 215±72Gy, range: 139~363Gy) and 73~117Gy in the other 4 patients. The NTD in all patients was less than the limiting dose of 70Gy recommended by the literature (mean: 37±11Gy, range: 16~55Gy). The lung shunting on PET/CT was minimal in 33/34 patients (mean: 1.8±1.2%, range: 0.4~4.6%) and mild in 1 patient (14.6%), lower than that on 99mTc-MAA planar (mean: 6.8±3.8%, range: 2.9~15.4%) in all patients. The summation of PA in tumor, NL and lung was comparable to the IA (91.2~99.3% with a median of 96.4% of IA), indicating that quantification by 90Y PET/CT for dosimetry measurement is feasible and accurate. The TNR on pretreatmet 99mTc-MAA SPECT/CT strongly correlated with 90Y PET/CT post radioembolization (mean=5.4±3.0 vs 5.6±3.6, linear curve: TNRY90=1.044×TNRMAA-0.033, P Conclusions PET/CT post 90Y-radioembolization could provide accurate dosimetry measurement and confirmation of biodistribution in HCC patients, thus obviating the conventional use of Bremsstrahlung-SPECT. The TNR predicted by 99mTc-MAA SPECT/CT was accurate but lung shunting in 90Y treatment was overestimated by 99mTc-MAA planar imaging, therefore, the threshold limit might possibly be given greater allowance. Quantitative optimization of IA of 90Y glass microspheres could be individualized and guided by target TD or limiting NTD instead of a routinely fixed ILD.

Corticosteroid and endocannabinoid actions converge on prefrontocortical circuits associated with neuropsychiatric illnesses. Corticosteroids can also modulate forebrain synapses by using endocannabinoid effector systems. Here, we determined whether corticosteroids can modulate transmitter release directly in the frontal cortex and, in doing so, whether they affect presynaptic CB1 cannabinoid receptor- (CB1R) mediated neuromodulation. By Western blotting of purified subcellular fractions of the rat frontal cortex, we found glucocorticoid receptors (GcRs) and CB1Rs enriched in isolated frontocortical nerve terminals (synaptosomes). CB1Rs were predominantly presynaptically located while GcRs showed preference for the post-synaptic fraction. Additional confocal microscopy analysis of cortical and hippocampal regions revealed vesicular GABA transporter-positive and vesicular glutamate transporter 1-positive nerve terminals endowed with CB1R immunoreactivity, apposing GcR-positive post-synaptic compartments. In functional transmitter release assay, corticosteroids, corticosterone (0.1–10 microM) and dexamethasone (0.1–10 microM) did not significantly affect the evoked release of [3H]GABA and [14C]glutamate in superfused synaptosomes, isolated from both rats and mice. In contrast, the synthetic cannabinoid, WIN55212-2 (1 microM) diminished the release of both [3H]GABA and [14C]glutamate, evoked with various depolarization paradigms. This effect of WIN55212-2 was abolished by the CB1R neutral antagonist, O-2050 (1 microM), and was absent in the CB1R KO mice. CB2R-selective agonists did not affect the release of either neurotransmitter. The lack of robust presynaptic neuromodulation by corticosteroids was unchanged upon either CB1R activation or genetic inactivation. Altogether, corticosteroids are unlikely to exert direct non-genomic presynaptic neuromodulation in the frontal cortex, but they may do so indirectly, via the stimulation of trans-synaptic endocannabinoid signaling.

Aims This study was performed to examine whether hypertension affects the sympathetic transmission to resistance vessels of pithed rats via inhibitory presynaptic cannabinoid CB1 receptors and whether endocannabinoids are involved in this response. Materials and methods We compared uninephrectomised rats rendered hypertensive by high salt diet and deoxycorticosterone acetate (DOCA) injections with normotensive animals (uninephrectomy only). Experiments were performed on vagotomised and pithed animals. Increases in diastolic blood pressure (DBP) were induced four times (S1–S4) by electrical stimulation or phenylephrine injection. Key findings Electrical stimulation (0.75 Hz, 1 ms, 50 V, 5 impulses) of the preganglionic sympathetic nerve fibres innervating the blood vessels more strongly increased DBP in normotensive than in DOCA–salt rats. Phenylephrine (0.01 μmol/kg) induced similar increases in DBP in both groups. The cannabinoid receptor agonist CP55940 (0.01–1 μmol/kg) did not modify the rises in DBP induced by phenylephrine. However, it inhibited the electrically stimulated increases in DBP, more strongly in DOCA–salt than in normotensive animals (maximally by 50 and 30%, respectively). The effect of CP55940 was attenuated by the CB1 antagonist AM251 (3 μmol/kg). AM251 enhanced the neurogenic vasopressor response during S4 by itself in hypertensive rats only. URB597 (3 μmol/kg), which inhibits degradation of the endocannabinoid anandamide, did not modify the electrically stimulated increases in DBP. Significance The function of inhibitory presynaptic CB1 receptors on sympathetic nerves is enhanced in DOCA–salt hypertensive rats. Thus, the CB1 receptor-mediated inhibition of noradrenaline release from the sympathetic nerve fibres innervating the resistance vessels might play a protective role in hypertension.

We investigated the anti-nociceptive effects of GABA-C receptors in the central nervous system. Intracisternal injection of CACA, a GABA-C receptor agonist or isoguvacine, a GABA-A receptor agonist, significantly increased the tail-withdrawal latency. TPMPA, a GABA-C receptor antagonist blocked the effects of CACA but not isoguvacine indicating that GABA-C receptors are involved in regulating pain. Further, double-labelled immunofluorescence studies revealed that GABA-Cρ2 receptors are expressed presynaptically in the spinal dorsal horn, especially, substantia gelatinosa, a region that has been previously implicated in analgesia by regulating nociceptive inflow. These data provide a provenance for future work looking at presynaptic spinal GABA-C receptors in the control of nociception.

Immunoelectron microscopy revealed that peripheral afferent nerve fibers innervating taste buds contain calcitonin gene-related peptide (CGRP), which may be as an efferent transmitter released from peripheral axon terminals. In this report, we determined the targets of CGRP within taste buds and studied what effect CGRP exerts on taste bud function. We isolated mouse taste buds and taste cells, conducted functional imaging using Fura-2, and used cellular biosensors to monitor taste-evoked transmitter release. The findings showed that a subset of Presynaptic (Type III) taste cells (53%) responded to 0.1 μmCGRP with an increase in intracellular Ca2+. In contrast, Receptor (Type II) taste cells rarely (4%) responded to 0.1 μmCGRP. Using pharmacological tools, the actions of CGRP were probed and elucidated by the CGRP receptor antagonist CGRP8-37. We demonstrated that this effect of CGRP was dependent on phospholipase C activation and was prevented by the inhibitor U73122. Moreover, applying CGRP caused taste buds to secrete serotonin (5-HT), a Presynaptic (Type III) cell transmitter, but not ATP, a Receptor (Type II) cell transmitter. Further, our previous studies showed that 5-HT released from Presynaptic (Type III) cells provides negative paracrine feedback onto Receptor (Type II) cells by activating 5-HT1Areceptors, and reducing ATP secretion. Our data showed that CGRP-evoked 5-HT release reduced taste-evoked ATP secretion. The findings are consistent with a role for CGRP as an inhibitory transmitter that shapes peripheral taste signals via serotonergic signaling during processing gustatory information in taste buds.SIGNIFICANCE STATEMENTThe taste sensation is initiated with a highly complex set of interactions between a variety of cells located within the taste buds before signal propagation to the brain. Afferent signals from the oral cavity are carried to the brain in chemosensory fibers that contribute to chemesthesis, the general chemical sensitivity of the mucus membranes in the oronasal cavities and being perceived as pungency, irritation, or heat. This is a study of a fundamental question in neurobiology: how are signals processed in sensory end organs, taste buds? More specifically, taste-modifying interactions, via transmitters, between gustatory and chemosensory afferents inside taste buds will help explain how a coherent output is formed before being transmitted to the brain.

Mon1 is an evolutionarily conserved protein involved in the conversion of Rab5 positive early endosomes to late endosomes through the recruitment of Rab7. We have identified a role for Drosophila Mon1 in regulating glutamate receptor levels at the larval neuromuscular junction. We generated mutants in Dmon1 through P-element excision. These mutants are short-lived with strong motor defects. At the synapse, the mutants show altered bouton morphology with several small supernumerary or satellite boutons surrounding a mature bouton; a significant increase in expression of GluRIIA and reduced expression of Bruchpilot. Neuronal knockdown of Dmon1 is sufficient to increase GluRIIA levels, suggesting its involvement in a presynaptic mechanism that regulates postsynaptic receptor levels. Ultrastructural analysis of mutant synapses reveals significantly smaller synaptic vesicles. Overexpression of vglut suppresses the defects in synaptic morphology and also downregulates GluRIIA levels in Dmon1 mutants, suggesting that homeostatic mechanisms are not affected in these mutants. We propose that DMon1 is part of a presynaptically regulated transsynaptic mechanism that regulates GluRIIA levels at the larval neuromuscular junction.

Herbal products containing synthetic cannabinoids-initially sold as legal alternatives to marijuana-have become major drugs of abuse. Among the synthetic cannabinoids, [1-(5-fluoropentyl)-1H-indol-3-yl](4-methyl-1-naphthalenyl)-methanone (MAM-2201) has been recently detected in herbal products and has psychoactive and intoxicating effects in humans, suggesting that MAM-2201 alters brain function. Nevertheless, the pharmacological actions of MAM-2201 on cannabinoid receptor type 1 (CB1R) and neuronal functions have not been elucidated. We found that MAM-2201 acted as an agonist of human CB1Rs expressed in AtT-20 cells. In whole-cell patch-clamp recordings made from Purkinje cells (PCs) in slice preparations of the mouse cerebellum, we also found that MAM-2201 inhibited glutamate release at parallel fiber-PC synapses via activation of presynaptic CB1Rs. MAM-2201 inhibited neurotransmitter release with an inhibitory concentration 50% of 0.36 μM. MAM-2201 caused greater inhibition of neurotransmitter release than Δ(9)-tetrahydrocannabinol within the range of 0.1-30 μM and JWH-018, one of the most popular and potent synthetic cannabinoids detected in the herbal products, within the range of 0.03-3 μM. MAM-2201 caused a concentration-dependent suppression of GABA release onto PCs. Furthermore, MAM-2201 induced suppression of glutamate release at climbing fiber-PC synapses, leading to reduced dendritic Ca(2+) transients in PCs. These results suggest that MAM-2201 is likely to suppress neurotransmitter release at CB1R-expressing synapses in humans. The reduction of neurotransmitter release from CB1R-containing synapses could contribute to some of the symptoms of synthetic cannabinoid intoxication including impairments in cerebellum-dependent motor coordination and motor learning.

Endomorphin-2 (EM2) and Substance P (SP) exert suppressive and facilitative influences upon nociception, respectively. Although EM2 and SP were often co-expressed in single neurons in dorsal root ganglion (DRG), it is still unknown if and how the nociception-suppressive influences of EM2 might be exerted upon nociception-facilitative effects of SP in the DRG neurons. We examined these issues in the inflammatory pain model rats produced by subcutaneous injection of the complete Freund's adjuvant into the hind paw. The paw withdrawal threshold for mechanical allodynia was measured. Changes of EM2 and SP release were estimated by measuring intrathecal levels of EM2 and SP through in vivo microdialysis analysis of cerebrospinal fluid. The mechanical allodynia was dose-dependently attenuated by intrathecal injection of EM2 or a neurokinin-1 receptor antagonist, and facilitated by intrathecal injection of SP or a mu-opioid receptor (MOR) antagonist. Importantly, intrathecal level of SP was found to be lowered by intrathecal injection of EM2. Morphologically, colocalization of EM2-, MOR- and SP-immunoreactivity in single DRG neurons was observed by immunofluorescent histochemistry, and co-expression of EM2 and SP in large, dense-cored presynaptic vesicles in primary afferents, as well as localization of MOR on pre- and postsynaptic membrane in spinal dorsal horn, was also confirmed electron miscroscopically. Thus, the results indicated that analgesic influences of EM2 upon inflammatory pain might be exerted through suppression of SP release, supporting the assumptions that binding of EM2 to presynaptic MOR might induce such effects.

Parallel fiber synapses in the cerebellum express a wide range of presynaptic receptors. However, presynaptic receptor expression at individual parallel fiber synapses is quite heterogeneous, suggesting physiological mechanisms regulate presynaptic receptor expression. We investigated changes in presynaptic GABAB receptors at parallel fiber-stellate cell synapses in acute cerebellar slices from juvenile mice. GABAB receptor-mediated inhibition of excitatory postsynaptic currents (EPSCs) is remarkably diverse at these synapses, with transmitter release at some synapses inhibited by >50% and little or no inhibition at others. GABAB receptor-mediated inhibition was significantly reduced following 4 Hz parallel fiber stimulation but not after stimulation at other frequencies. The reduction in GABAB receptor-mediated inhibition was replicated by bath application of forskolin and blocked by application of a PKA inhibitor, suggesting activation of adenylyl cyclase and PKA are required. Immunolabeling for an extracellular domain of the GABAB2 subunit revealed reduced surface expression in the molecular layer after exposure to forskolin. GABAB receptor-mediated inhibition of action potential evoked calcium transients in parallel fiber varicosities was also reduced following bath application of forskolin, confirming presynaptic receptors are responsible for the reduced EPSC inhibition. These data demonstrate that presynaptic GABAB receptor expression can be a plastic property of synapses, which may compliment other forms of synaptic plasticity. This opens the door to novel forms of receptor plasticity previously confined primarily to postsynaptic receptors.

The ionotropic glutamate receptors (iGluRs) concertedly mediate neurotransmission to convey, process, and integrate acoustic information along the auditory pathway. In order to ensure these challenging tasks, the iGluRs are variously expressed in auditory neurons in an age- and site-dependent manner. The subunit compositions of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) and N-methyl-D-aspartate receptors (NMDARs) are altered with development, underlying the acceleration in kinetics of excitatory postsynaptic responses. AMPAR desensitization partly affects short-term synaptic plasticity upon repetitive stimuli in subsets of auditory neurons at a given period of maturation. NMDAR activation is required for long-term synaptic plasticity in a cerebellum-like microcircuit within the first auditory brainstem nucleus. Along with their postsynaptic functions, AMPARs and NMDARs fulfill essential roles in presynaptic modulation of auditory neurotransmission. Despite the expression of the kainate and delta receptors, their functions remain unknown. Here this review aims to discuss the diverse distribution and functions of pre- and postsynaptic iGluRs in the peripheral and central auditory systems.

Addiction is proposed to arise from alterations in synaptic strength via mechanisms of long-term potentiation (LTP) and depression (LTD). However, the causality between these synaptic processes and addictive behaviors is difficult to demonstrate. Here we report that LTP and LTD induction altered operant alcohol self-administration, a motivated drug-seeking behavior. We first induced LTP by pairing presynaptic glutamatergic stimulation with optogenetic postsynaptic depolarization in the dorsomedial striatum, a brain region known to control goal-directed behavior. Blockade of this LTP by NMDA-receptor inhibition unmasked an endocannabinoid-dependent LTD. In vivo application of the LTP-inducing protocol caused a long-lasting increase in alcohol-seeking behavior, while the LTD protocol decreased this behavior. We further identified that optogenetic LTP and LTD induction at cortical inputs onto striatal dopamine D1 receptor-expressing neurons controlled these behavioral changes. Our results demonstrate a causal link between synaptic plasticity and alcohol-seeking behavior and suggest that modulation of this plasticity may inspire a therapeutic strategy for addiction. Addiction-related behaviors are believed to result from drug-evoked synaptic changes, but their causality is unclear. The authors show that bidirectional optogenetic modifications of synaptic strength distinctly alter alcohol-seeking behavior.

Summary Homeostatic signaling systems are thought to interface with other forms of plasticity to ensure flexible yet stable levels of neurotransmission. The role of neurotransmitter receptors in this process, beyond mediating neurotransmission itself, is not known. Through a forward genetic screen, we have identified the Drosophila kainate-type ionotropic glutamate receptor subunit DKaiR1D to be required for the retrograde, homeostatic potentiation of synaptic strength. DKaiR1D is necessary in presynaptic motor neurons, localized near active zones, and confers robustness to the calcium sensitivity of baseline synaptic transmission. Acute pharmacological blockade of DKaiR1D disrupts homeostatic plasticity, indicating that this receptor is required for the expression of this process, distinct from developmental roles. Finally, we demonstrate that calcium permeability through DKaiR1D is necessary for baseline synaptic transmission, but not for homeostatic signaling. We propose that DKaiR1D is a glutamate autoreceptor that promotes robustness to synaptic strength and plasticity with active zone specificity.

Precise coordination of synaptic connections ensures proper information flow within circuits. The activity of presynaptic organizing molecules signaling to downstream pathways is essential for such coordination, though such entities remain incompletely known. We show that LRP4, a conserved transmembrane protein known for its postsynaptic roles, functions presynaptically as an organizing molecule. In the Drosophila brain, LRP4 localizes to the nerve terminals at or near active zones. Loss of presynaptic LRP4 reduces excitatory (not inhibitory) synapse number, impairs active zone architecture, and abolishes olfactory attraction - the latter of which can be suppressed by reducing presynaptic GABAB receptors. LRP4 overexpression increases synapse number in excitatory and inhibitory neurons, suggesting an instructive role and a common downstream synapse addition pathway. Mechanistically, LRP4 functions via the conserved kinase SRPK79D to ensure normal synapse number and behavior. This highlights a presynaptic function for LRP4, enabling deeper understanding of how synapse organization is coordinated. DOI: http://dx.doi.org/10.7554/eLife.27347.001, eLife digest The connections between nerve cells, called synapses, often malfunction in disease, injury and during aging, and to understand how this happens we first need to know how they work normally. At a synapse, one nerve cell sends a signal to the other. The signal is a chemical substance, which binds to specialized proteins called receptors on the receiving nerve cell. At excitatory synapses, the chemical signal activates the receiver; at inhibitory synapses, it does the opposite. Communication at synapses typically only goes in one direction because the sender and receiver at a synapse are not interchangeable; they contain different molecules that support their distinct roles. To complicate matters, the same molecule may sometimes be present on both sides of a synapse with a different role in each. Moreover, not all synapses exist between two nerve cells; some synapses also form between nerve cells and muscle fibers to control the movement of the muscles. Mosca et al. set out to identify new players involved in forming synapses, and to identify differences in the formation of nerve cell-to-nerve cell versus nerve cell-to-muscle connections. Mosca et al. were interested in particular in a protein called LRP4. In mammals, LRP4 is largely present on the muscle side of nerve cell-to-muscle synapses, where it acts as a receptor for a chemical signal called Agrin. However, fruit flies — which lack Agrin – also possess the gene for LRP4, suggesting that it has other roles too. Mosca et al. now show that LRP4 is present in the nerve cell-to-nerve cell synapses found in the fruit fly’s brain. Further experiments reveal that fruit fly LRP4 plays an important role on the sender side of these synapses. Reducing the amount of LRP4 in the fruit fly brain reduces the number of excitatory, but not inhibitory, synapses. This suggests that fruit fly LRP4 may help regulate the formation of excitatory synapses. Understanding how synapses form, and the differences between excitatory and inhibitory connections, could provide new insights into disorders of impaired synapse formation such as schizophrenia. LRP4 has also been implicated in disorders, such as amyotrophic lateral sclerosis (ALS) and myasthenia gravis, in which impaired communication between nerves and muscles causes muscles to weaken. Improved understanding of how synapses work may lead to better drugs to treat these disorders. DOI: http://dx.doi.org/10.7554/eLife.27347.002

ObjectiveWe investigated the impact of cardiac presynaptic norepinephrine recycling in patients with long-QT syndrome (LQTS) using positron emission tomography (PET) with 11C-meta-hydroxyephedrine ([11C]mHED-PET).Methods[11C]mHED-PET was performed in 25 patients with LQTS (LQT1: n=14; LQT2: n=11) and 20 healthy controls and correlated with clinical parameters. [11C]mHED-PET images were analysed for global and regional retention indices (RI) and washout rates (WO) reflecting dynamic parameters of the tracer activity.ResultsGlobal and regional RI values were similar between patients with LQTS and controls. Although the global WO rates were similar between these groups, regional WO rates were on average higher in the lateral left ventricle (LV) wall in patients with LQTS (dose, mean ±SD; 0.08±0.14 vs 0.00%±0.09% min–1; p=0.033). In addition, patients with LQTS with a longer QTc interval showed a higher global WO rate. Clinical symptoms correlated with higher global WO rates. In the presence of normal global WO rates, asymptomatic LQTS patients showed higher global RI values.ConclusionThe increased regional WO rate of [11C]mHED in the lateral LV suggests an imbalance of presynaptic catecholamine reuptake and release, resulting in a higher synaptic catecholamine concentration, in particular in LQT1 patients. This might enhance β-adrenoceptor signalling and thereby aggravate inherited ion channel dysfunction and may facilitate occurrence of ventricular tachyarrhythmias. Detection of regional differences in LV sympathetic nervous function may modify disease expression and potentially serve as a non-invasive risk marker in congenital LQTS.Trial registration number2006-002767-41;Results.

Chemotherapeutic drugs, including bortezomib, often cause painful peripheral neuropathy, which is a severe dose-limiting adverse effect experienced by many cancer patients. The glutamate N-methyl-d-aspartate receptors (NMDARs) at the spinal cord level are critically involved in the synaptic plasticity associated with neuropathic pain. In this study, we determined whether treatment with bortezomib, a proteasome inhibitor, affects the NMDAR activity of spinal dorsal horn neurons. Systemic treatment with bortezomib in rats did not significantly affect postsynaptic NMDAR currents elicited by puff application of NMDA directly to dorsal horn neurons. Bortezomib treatment markedly increased the baseline frequency of miniature excitatory postsynaptic currents (EPSCs), which was completely normalized by the NMDAR antagonist 2-amino-5-phosphonopentanoic acid (AP5). AP5 also reduced the amplitude of monosynaptic EPSCs evoked by dorsal root stimulation in bortezomib-treated, but not vehicle-treated, rats. Furthermore, inhibition of protein kinase C (PKC) with chelerythrine fully reversed the increased frequency of miniature EPSCs and the amplitude of evoked EPSCs in bortezomib-treated rats. Intrathecal injection of AP5 and chelerythrine both profoundly attenuated mechanical allodynia and hyperalgesia induced by systemic treatment with bortezomib. In addition, treatment with bortezomib induced striking membrane translocation of PKC-βII, PKC-δ, and PKC-e in the dorsal root ganglion. Our findings indicate that bortezomib treatment potentiates nociceptive input from primary afferent nerves via PKC-mediated tonic activation of presynaptic NMDARs. Targeting presynaptic NMDARs and PKC at the spinal cord level may be an effective strategy for treating chemotherapy-induced neuropathic pain.

Different doses of an adenosine A2Areceptor antagonist MSX-3 [3,7-dihydro-8-[(1E)-2-(3-ethoxyphenyl)ethenyl]-7 methyl-3-[3-(phosphooxy)propyl-1-(2 propynil)-1H-purine-2,6-dione] were found previously to either decrease or increase self-administration of cannabinoids delta-9-tetrahydrocannabinol (THC) or anandamide in squirrel monkeys. It was hypothesized that the decrease observed with a relatively low dose of MSX-3 was related to blockade of striatal presynaptic A2Areceptors that modulate glutamatergic neurotransmission, whereas the increase observed with a higher dose was related to blockade of postsynaptic A2Areceptors localized in striatopallidal neurons. This hypothesis was confirmed in the present study by testing the effects of the preferential presynaptic and postsynaptic A2Areceptor antagonists SCH-442416 [2-(2-furanyl)-7-[3-(4-methoxyphenyl)propyl]-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine] and KW-6002 [(E)-1, 3-diethyl-8-(3,4-dimethoxystyryl)-7-methyl-3,7-dihydro-1H-purine-2,6-dione], respectively, in squirrel monkeys trained to intravenously self-administer THC. SCH-442416 produced a significant shift to the right of the THC self-administration dose–response curves, consistent with antagonism of the reinforcing effects of THC. Conversely, KW-6002 produced a significant shift to the left, consistent with potentiation of the reinforcing effects of THC. These results show that selectively blocking presynaptic A2Areceptors could provide a new pharmacological approach to the treatment of marijuana dependence and underscore corticostriatal glutamatergic neurotransmission as a possible main mechanism involved in the rewarding effects of THC.

Most neuronal communication relies upon the synchronous release of neurotransmitters, which occurs through synaptic vesicle exocytosis triggered by action potential invasion of a presynaptic bouton. However, neurotransmitters are also released asynchronously with a longer, variable delay following an action potential or spontaneously in the absence of action potentials. A compelling body of research has identified roles and mechanisms for synchronous release, but asynchronous release and spontaneous release are less well understood. In this review, we analyze how the mechanisms of the three release modes overlap and what molecular pathways underlie asynchronous and spontaneous release. We conclude that the modes of release have key fusion processes in common but may differ in the source of and necessity for Ca2+ to trigger release and in the identity of the Ca2+ sensor for release.

Neuromuscular transmission is clinically monitored using the train-of-four ratio (TOFratio), which is the quotient between twitch tension produced by the fourth (T4) and by the first (T1) stimulus within a train-of-four stimulation at 2Hz. Neostigmine causes a paradoxical depression of the TOFratio (TOFfade) that is amplified by intra-arterial atropine in cats. This led us to question the usefulness of the TOFratio as a sole testing element to control neostigmine-induced reversal of neuromuscular transmission block caused by non-depolarizing agents. We hypothesized that the inhibition of cholinesterase activity might increase acetylcholine bioavailability and consequently cholinoceptor activation, leading to concomitant adenosine release from nerve endings and skeletal muscle fibers. The involvement of presynaptic muscarinic and adenosine receptors in neostigmine-induced TOFfade in the rat phrenic nerve diaphragm was investigated. Blockade of adenosine A2A receptors with ZM241385 and of muscarinic M2 receptors with methoctramine or atropine amplified neostigmine-induced TOFfade. Notwithstanding TOFfade amplification, the blockade of M2 or A2A receptors increased both T1 and T4 twitch tensions above control during the first 3min of neostigmine application. Beyond that period, the T1 twitch tension returned to baseline, whereas T4 decreased further until the control value with neostigmine alone. Blockade of M1 receptors by pirenzepine did not change neostigmine-induced TOFfade, unless A2A receptors were concurrently blocked with ZM241385. Data indicate that the paradoxical neostigmine-induced fade involves presynaptic mechanisms that regulate transmitter release and synaptic adenosine accumulation, including the activation of adenosine A2A and muscarinic M2 receptors.

The cognitive impairment associated with schizophrenia is highly prevalent and affects the overall functioning of subjects. The stimulation of the serotonin 1A receptor is a primary characteristic of some atypical antipsychotic drugs. We measured the levels of cognitive impairment using the Morris water maze test and protein kinase A activity in hippocampal neurons on presynaptic and postsynaptic serotonin 1A receptors to investigate the effect of dizocilpine-induced cognitive impairment associated with atypical antipsychotic drugs in rats treated by quetiapine alone or combined with WAY100635/tandospirone. The results of the Morris water maze test presented evidence that quetiapine alone alleviated the cognitive impairment associated with atypical antipsychotic drugs induced by dizocilpine. However, quetiapine plus WAY100635 induced no improvement of cognitive impairment associated with atypical antipsychotic drugs. The results of protein kinase A assay suggested that neither quetiapine alone nor in combination with tandospirone, but not quetiapine plus WAY100635, raised protein kinase A activity in hippocampus neurons. The present study demonstrated the key role of presynaptic serotonin 1A receptors on the therapeutic effect of quetiapine on cognitive impairment associated with atypical antipsychotic drugs. Moreover, that protein kinase A activity in hippocampal cells is involved in the mechanism of quetiapine's effect on cognitive impairment associated with atypical antipsychotic drugs.

We studied the effects of selective blockade of various subtypes of α2-adrenoceptors on cardiac chronotropy in newborn rats. This period in rats is characterized by the absence of adrenergic regulation of heart function. Blockade of α2A/D- and α2B-adrenoceptors in 1-weekold rats provoked tachycardia. In contrast, blockade of α2C-adrenoceptors in newborn rats had no effect on heart rate.

In the classic model of the primary visual cortex, upper-layer complex cells are driven by feedforward inputs from layer 4 simple cells. Based on spike cross-correlation, previousin vivowork has suggested that this connection is strong and dense, with a high probability of connection (50%) and significant strength in connected pairs. A much sparser projection has been found in brain slices, however, with the probability of layer 4 cells connecting to layer 2/3 cells being relatively low (10%). Here, we explore this connectionin vivoin the cat primary visual cortex by recording simultaneously spikes of layer 4 simple cells and the membrane potential (Vm) of layer 2/3 complex cells. By triggering the average of the complex cell'sVmon the spikes of the simple cell (Vm-STA), we found functional coupling to be very common during visual stimulation: the simple cell's spikes tended to occur near the troughs of the complex cell'sVmfluctuations and were, on average, followed by a significant (∼1 mV) fast-rising (10 ms) depolarization in the complex cell. In the absence of visual stimulation, however, when single simple cells were activated electrically through the recording electrode, no significant depolarization, or at most a very weak input (0.1–0.2 mV), was detected in the complex cell. We suggest that the functional coupling observed during visual stimulation arises from coordinated or nearly synchronous activity among a large population of simple cells, only a small fraction of which are presynaptic to the recorded complex cell.

The amyloid precursor protein (APP) and its mammalian homologs, APLP1, APLP2, have been allocated to an organellar pool residing in the Golgi apparatus and in endosomal compartments, and in its mature form to a cell surface-localized pool. In the brain, all APPs are restricted to neurons; however, their precise localization at the plasma membrane remained enigmatic. Employing a variety of subcellular fractionation steps, we isolated two synaptic vesicle (SV) pools from rat and mouse brain, a pool consisting of synaptic vesicles only and a pool comprising SV docked to the presynaptic plasma membrane. Immunopurification of these two pools using a monoclonal antibody directed against the 12 membrane span synaptic vesicle protein2 (SV2) demonstrated unambiguously that APP, APLP1 and APLP2 are constituents of the active zone of murine brain but essentially absent from free synaptic vesicles. The specificity of immunodetection was confirmed by analyzing the respective knock-out animals. The fractionation experiments further revealed that APP is accumulated in the fraction containing docked synaptic vesicles. These data present novel insights into the subsynaptic localization of APPs and are a prerequisite for unraveling the physiological role of all mature APP proteins in synaptic physiology. We deciphered the precise subcellular localization of APP at the nerve terminal. We demonstrate that APP and its family members, APLP1 and APLP2, are constituents of the presynaptic active zone, albeit virtually absent in synaptic vesicles (SV). Our findings open new avenues for understanding the physiological role of the mature APP proteins at synaptic contacts, implying a function in the physiology of neurotransmitter release.

Spreading depression (SD) is a slowly propagating neuronal depolarization that underlies certain neurologic conditions. The wave-like pattern of its propagation suggests that SD arises from an unusual form of neuronal communication. We used enzyme-based glutamate electrodes to show that during SD induced by transiently raising extracellular K+ concentrations ([K+]o) in rat brain slices, there was a rapid increase in the extracellular glutamate concentration that required vesicular exocytosis but unlike fast synaptic transmission, still occurred when voltage-gated sodium and calcium channels (VGSC and VGCC) were blocked. Instead, presynaptic N-methyl-D-aspartate (NMDA) receptors (NMDARs) were activated during SD and could generate substantial glutamate release to support regenerative glutamate release and propagating waves when VGSCs and VGCCs were blocked. In calcium-free solutions, high [K+]o still triggered SD-like waves and glutamate efflux. Under such a condition, glutamate release was blocked by mitochondrial Na+/Ca2+ exchanger inhibitors that likely blocked calcium release from mitochondria secondary to NMDA-induced Na+ influx. Therefore presynaptic NMDA receptor activation is sufficient for triggering vesicular glutamate release during SD via both calcium entry and release from mitochondria by mitochondrial Na+/Ca2+ exchanger. Our observations suggest that presynaptic NMDARs contribute to a cycle of glutamate-induced glutamate release that mediate high [K+]o-triggered SD.

We have investigated the mechanisms underlying the facilitatory modulation mediated by kainate receptor (KAR) activation in the cortex, using isolated nerve terminals (synaptosomes) and slice preparations. In cortical nerve terminals, kainate (KA, 100 μM) produced an increase in 4-aminopyridine (4-AP)-evoked glutamate release. In thalamocortical slices, KA (1 μM) produced an increase in the amplitude of evoked excitatory post-synaptic currents (eEPSCs) at synapses established between thalamic axon terminals from the ventrobasal nucleus onto stellate neurons of L4 of the somatosensory cortex. In both, synaptosomes and slices, the effect of KA was antagonized by 6-cyano-7-nitroquinoxaline-2,3-dione, and persisted after pre-treatment with a cocktail of antagonists of other receptors whose activation could potentially have produced facilitation of release indirectly. Mechanistically, the observed effects of KA appear to be congruent in synaptosomal and slice preparations. Thus, the facilitation by KA of synaptosomal glutamate release and thalamocortical synaptic transmission were suppressed by the inhibition of protein kinase A and occluded by the stimulation of adenylyl cyclase. Dissecting this G-protein-independent regulation further in thalamocortical slices, the KAR-mediated facilitation of synaptic transmission was found to be sensitive to the block of Ca(2+) permeant KARs by philanthotoxin. Intriguingly, the synaptic facilitation was abrogated by depletion of intracellular Ca(2+) stores by thapsigargin, or inhibition of Ca(2+) -induced Ca(2+) -release by ryanodine. Thus, the KA-mediated modulation was contingent on both Ca(2+) entry through Ca(2+) -permeable KARs and liberation of intracellular Ca(2+) stores. Finally, sensitivity to W-7 indicated that the increased cytosolic [Ca(2+) ] underpinning KAR-mediated regulation of synaptic transmission at thalamocortical synapses, requires downstream activation of calmodulin. We conclude that neocortical pre-synaptic KARs mediate the facilitation of glutamate release and synaptic transmission by a Ca(2+) -calmodulin dependent activation of an adenylyl cyclase/cAMP/protein kinase A signalling cascade, independent of G-protein involvement.

Because of the progressive loss of nigro-striatal dopaminergic terminals in Parkinson's disease (PD), in vivo quantitative imaging of dopamine (DA) containing neurons in animal models of PD is of critical importance in the preclinical evaluation of highly awaited disease-modifying therapies. Among existing methods, the high sensitivity of positron emission tomography (PET) is attractive to achieve that goal. The aim of this study was to perform a quantitative comparison of brain images obtained in 6-hydroxydopamine (6-OHDA) lesioned rats using two dopaminergic PET radiotracers, namely [

Extraclassical surround suppression is a prominent receptive field property of neurons in the lateral geniculate nucleus (LGN) of the dorsal thalamus, influencing stimulus size tuning, response gain control, and temporal features of visual responses. Despite evidence for the involvement of both retinal and nonretinal circuits in the generation of extraclassical suppression, we lack an understanding of the relative roles played by these pathways and how they interact during visual stimulation. To determine the contribution of retinal and nonretinal mechanisms to extraclassical suppression in the feline, we made simultaneous single-unit recordings from synaptically connected retinal ganglion cells and LGN neurons and measured the influence of stimulus size on the spiking activity of presynaptic and postsynaptic neurons. Results show that extraclassical suppression is significantly stronger for LGN neurons than for their retinal inputs, indicating a role for extraretinal mechanisms. Further analysis revealed that the enhanced suppression can be accounted for by mechanisms that suppress the effectiveness of retinal inputs in evoking LGN spikes. Finally, an examination of the time course for the onset of extraclassical suppression in the LGN and the size-dependent modulation of retinal spike efficacy suggests the early phase of augmented suppression involves local thalamic circuits. Together, these results demonstrate that the LGN is much more than a simple relay for retinal signals to cortex; it also filters retinal spikes dynamically on the basis of stimulus statistics to adjust the gain of visual signals delivered to cortex.SIGNIFICANCE STATEMENTThe lateral geniculate nucleus (LGN) is the gateway through which retinal information reaches the cerebral cortex. Within the LGN, neuronal responses are often suppressed by stimuli that extend beyond the classical receptive field. This form of suppression, called extraclassical suppression, serves to adjust the size tuning, response gain, and temporal response properties of neurons. Given the important influence of extraclassical suppression on visual signals delivered to cortex, we performed experiments to determine the circuit mechanisms that contribute to extraclassical suppression in the LGN. Results show that suppression is augmented beyond that provided by direct retinal inputs and delayed, consistent with polysynaptic inhibition. Importantly, these mechanisms influence the effectiveness of incoming retinal signals, thereby filtering the signals ultimately conveyed to cortex.

Excitatory amino acid transporters (EAATs) are abundantly expressed by astrocytes, rapidly remove glutamate from the extracellular environment, and restrict the temporal and spatial extent of glutamate signaling. Studies probing EAAT function suggest that their capacity to remove glutamate is large and does not saturate, even with substantial glutamate challenges. In contrast, we report that neuronal activity rapidly and reversibly modulates EAAT-dependent glutamate transport. To date, no physiological manipulation has shown changes in functional glutamate uptake in a nonpathological state. Using iGluSnFr-based glutamate imaging and electrophysiology in the adult mouse cortex, we show that glutamate uptake is slowed up to threefold following bursts of neuronal activity. The slowing of glutamate uptake depends on the frequency and duration of presynaptic neuronal activity but is independent of the amount of glutamate released. The modulation of glutamate uptake is brief, returning to normal within 50 ms after stimulation ceases. Interestingly, the slowing of glutamate uptake is specific to activated synapses, even within the domain of an individual astrocyte. Activity-induced slowing of glutamate uptake, and the increased persistence of glutamate in the extracellular space, is reflected by increased decay times of neuronal NR2A-mediated NMDA currents. These results show that astrocytic clearance of extracellular glutamate is slowed in a temporally and spatially specific manner following bursts of neuronal activity ≥30 Hz and that these changes affect the neuronal response to released glutamate. This suggests a previously unreported form of neuron–astrocyte interaction.SIGNIFICANCE STATEMENTWe report the first fast, physiological modulation of astrocyte glutamate clearance kinetics. We show that presynaptic activity in the cerebral cortex increases the persistence of glutamate in the extracellular space by slowing its clearance by astrocytes. Because of abundant EAAT expression, glutamate clearance from the extracellular space has been thought to have invariant kinetics. While multiple studies report experimental manipulations resulting in altered EAAT expression, our findings show that astrocytic glutamate uptake is dynamic on a fast time-scale. This shows rapid plasticity of glutamate clearance, which locally modulates synaptic signaling in the cortex. As astrocytic glutamate uptake is a fundamental and essential mechanism for neurotransmission, this work has implications for neurotransmission, extrasynaptic receptor activation, and synaptic plasticity.

Synaptic dysfunctions and altered neuronal activity play major role in the pathophysiology of Alzheimer's disease (AD), with underlying mechanisms largely unknown. We report that in the prefrontal cortex of amyloid precursor protein-presenilin 1 and APP23 AD mice, baseline activity of pyramidal cells is disrupted by episodes of paroxysmal hyperactivity. Induced by spontaneous EPSC bursts, these incidents are prevalent in neurons proximal to amyloid plaques and involve enhanced activity of glutamate with metabotropic effects. Abolition of EPSC bursts by tetrodotoxin and SERCA ATPase blockers thapsigargin or cyclopiasonic acid suggests their presynaptic origin and sensitized store-released calcium. Accordingly, the rate of EPSC bursts activated by single axon stimulation is enhanced. Aggravation of the hyperactivity by blockers of excitatory amino acid transporter (±)-HIP-A and DL-TBOA together with histochemical and ultrastructural evidence for enrichment of plaque-related dystrophies with synaptic vesicles and SNARE protein SNAP-25 infer the later as hot-spots for ectopic release of glutamate. Inhibition of EPSC bursts by I/II mGluR1 blocker MCPG or selective mGluR1 antagonist LY367385 implicate metabotropic glutamatergic effects in generation of paroxysmal bursts. These findings demonstrate for the first time that at amyloid plaques, enhanced activity of nonsynaptic glutamate can promote irregular EPSC bursts with hyperactivity of pyramidal cells via mGluR1 receptors.

The development of the nervous system involves the initial overproduction of synapses, which promotes connectivity. Hebbian competition between axons with different activities leads to the loss of roughly half of the overproduced elements and this refines connectivity. We used quantitative immunohistochemistry to investigate, in the postnatal day 7 (P7) to P9 neuromuscular junctions, the involvement of muscarinic receptors (muscarinic acetylcholine autoreceptors and the M1, M2, and M4 subtypes) and adenosine receptors (A1 and A2A subtypes) in the control of axonal elimination after the mouse levator auris longus muscle had been exposed to selective antagonists in vivo. In a previous study we analyzed the role of each of the individual receptors. Here we investigate the additive or occlusive effects of their inhibitors and thus the existence of synergistic activity between the receptors. The main results show that the A2A, M1, M4, and A1 receptors (in this order of ability) delayed axonal elimination at P7. M4 produces some occlusion of the M1 pathway and some addition to the A1 pathway, which suggests that they cooperate. M2 receptors may modulate (by allowing a permissive action) the other receptors, mainly M4 and A1. The continued action of these receptors (now including M2 but not M4) finally promotes axonal loss at P9. All 4 receptors (M2, M1, A1, and A2A, in this order of ability) are necessary. The M4 receptor (which in itself does not affect axon loss) seems to modulate the other receptors. We found a synergistic action between the M1, A1, and A2A receptors, which show an additive effect, whereas the potent M2 effect is largely independent of the other receptors (though can be modulated by M4). At P9, there is a full mutual dependence between the A1 and A2A receptors in regulating axon loss. In summary, postnatal axonal elimination is a regulated multireceptor mechanism that involves the cooperation of several muscarinic and adenosine receptor subtypes.

Depolarisation-induced suppression of inhibition (DSI) is a form of short-term synaptic plasticity at gamma-aminobutyric-acid-(GABA)ergic synapses between principal neurons and interneurons in both the cerebellum and the hippocampus. The induction of DSI involves an intracellular calcium-dependent release of a retrograde messenger from the postsynaptic principal neuron (Purkinje cell/pyramidal cell in cerebellum/hippocampus) onto presynaptic interneurons, where it is thought to bind to guanine nucleotide-binding protein (G protein)-coupled receptors and subsequently reducing GABA release from these interneurons onto the postsynaptic principal neuron. Pharmacological studies have indicated that glutamate might be a retrograde messenger in both cerebellum and hippocampus, where, in the former at least, it seems to activate type-II metabotropic glutamate receptors (mGluRs). Using LY-341495, a recently described, highly specific and potent antagonist of type-II mGluRs, to block these receptors reduced DSI slightly, but significantly, in spite of the fact that this antagonist completely suppressed the effects of stimulating type-II mGluRs with a specific agonist. Activation of type II mGluRs alone thus cannot account fully for DSI in cerebellum and hence other mechanisms are involved in its induction. Such mechanisms probably involve an additional retrograde signal.

Schizophrenia is a serious psychiatric illness which is experienced by about 1 % of individuals worldwide and has a debilitating impact on perception, cognition, and social function. Over the years, several models/hypotheses have been developed which link schizophrenia to dysregulations of the dopamine, glutamate, and serotonin receptor pathways. An important segment of these pathways that have been extensively studied for the pathophysiology of schizophrenia is the presynaptic neurotransmitter release mechanism. This set of molecular events is an evolutionarily well-conserved process that involves vesicle recruitment, docking, membrane fusion, and recycling, leading to efficient neurotransmitter delivery at the synapse. Accumulated evidence indicate dysregulation of this mechanism impacting postsynaptic signal transduction via different neurotransmitters in key brain regions implicated in schizophrenia. In recent years, after ground-breaking work that elucidated the operations of this mechanism, research efforts have focused on the alterations in the messenger RNA (mRNA) and protein expression of presynaptic neurotransmitter release molecules in schizophrenia and other neuropsychiatric conditions. In this review article, we present recent evidence from schizophrenia human postmortem studies that key proteins involved in the presynaptic release mechanism are dysregulated in the disorder. We also discuss the potential impact of dysfunctional presynaptic neurotransmitter release on the various neurotransmitter systems implicated in schizophrenia.

Abnormal Glu release occurs in the spinal cord of SOD1(G93A) mice, a transgenic animal model for human ALS. Here we studied the mechanisms underlying Glu release in spinal cord nerve terminals of SOD1(G93A) mice at a pre-symptomatic disease stage (30days) and found that the basal release of Glu was more elevated in SOD1(G93A) with respect to SOD1 mice, and that the surplus of release relies on synaptic vesicle exocytosis. Exposure to high KCl or ionomycin provoked Ca(2+)-dependent Glu release that was likewise augmented in SOD1(G93A) mice. Equally, the Ca(2+)-independent hypertonic sucrose-induced Glu release was abnormally elevated in SOD1(G93A) mice. Also in this case, the surplus of Glu release was exocytotic in nature. We could determine elevated cytosolic Ca(2+) levels, increased phosphorylation of Synapsin-I, which was causally related to the abnormal Glu release measured in spinal cord synaptosomes of pre-symptomatic SOD1(G93A) mice, and increased phosphorylation of glycogen synthase kinase-3 at the inhibitory sites, an event that favours SNARE protein assembly. Western blot experiments revealed an increased number of SNARE protein complexes at the nerve terminal membrane, with no changes of the three SNARE proteins and increased expression of synaptotagmin-1 and β-Actin, but not of an array of other release-related presynaptic proteins. These results indicate that the abnormal exocytotic Glu release in spinal cord of pre-symptomatic SOD1(G93A) mice is mainly based on the increased size of the readily releasable pool of vesicles and release facilitation, supported by plastic changes of specific presynaptic mechanisms.

Auditory nerve fibers in the adult ear are divided into functional subgroups according to spontaneous rate (SR) and threshold sensitivity. The high-threshold, low-SR fibers are morphologically and spatially distinct from the low-threshold high-SR fibers at their synaptic contacts with inner hair cells. This distinction between SR groups in the adult ear is visible in confocal microscopy as complementary size gradients of presynaptic ribbons and post-synaptic glutamate receptor patches across the modiolar-pillar and habenular-cuticular axes in the inner hair cell area. The aim of the present study was to track the post-natal development of this morphological gradient, in mouse, to determine the earliest age at which this important aspect of cochlear organization is fully mature. Here we show, using morphometric analysis of the organ of Corti immunostained for pre- and post-synaptic markers of efferent and afferent innervation, that this SR-based morphological gradient is not fully established until postnatal day 28, well after other features, such as synaptic counts and efferent innervation density in both the inner and outer hair cell areas, appear fully mature.

The nocioceptive information carried by neurons of the pontine parabrachial nucleus to neurons of the lateral division of the central amydala (CeA-L) is thought to contribute to the affective components of pain and is required for the formation of conditioned-fear memories. Importantly, excitatory transmission between parabrachial axon terminals and CeA-L neurons can be inhibited by a number of presynaptic receptors linked to Gi/o-type G-proteins, including α2-adrenoceptors and GABAB receptors. While the intracellular signalling pathway responsible for α2-adrenoceptor inhibition of synaptic transmission at this synapse is known, the mechanism by which GABAB receptors inhibits transmission has not been determined. The present study demonstrates that activation of presynaptic GABAB receptors reduces excitatory transmission between parabrachial axon terminals and CeA-L neurons by inhibiting N-type calcium channels. While the involvement of Gβγ subunits in mediating the inhibitory effects of GABAB receptors on N-type calcium channels is unclear, this inhibition does not involve Gβγ-independent activation of pp60C-src tyrosine kinase. The results of this study further enhance our understanding of the modulation of the excitatory input from parabrachial axon terminals to CeA-L neurons and indicate that presynaptic GABAB receptors at this synapse could be valuable therapeutic targets for the treatment of fear- and pain-related disorders.

GABAB receptors are the G-protein-coupled receptors for GABA, the main inhibitory neurotransmitter in the central nervous system. Pharmacological activation of GABAB receptors regulates neurotransmission and neuronal excitability at pre- and postsynaptic sites. Electrophysiological activation of GABAB receptors in brain slices generally requires strong stimulus intensities. This raises the question as to whether behavioral stimuli are strong enough to activate GABAB receptors. Here we show that GABAB1a-/- mice, which constitutively lack presynaptic GABAB receptors at glutamatergic synapses, are impaired in their ability to acquire an operant learning task. In vivo recordings during the operant conditioning reveal a deficit in learning-dependent increases in synaptic strength at CA3-CA1 synapses. Moreover, GABAB1a-/- mice fail to synchronize neuronal activity in the CA1 area during the acquisition process. Our results support that activation of presynaptic hippocampal GABAB receptors is important for acquisition of a learning task and for learning-associated synaptic changes and network dynamics.

The nucleus accumbens (NAc) regulates motivated behavior by, in part, processing excitatory synaptic projections from several brain regions. Among these regions, the prefrontal cortex (PFC) and basolateral amygdala, convey executive control and affective states, respectively. Whereas glutamatergic synaptic transmission within the NAc has been recognized as a primary cellular target for cocaine and other drugs of abuse to induce addiction-related pathophysiological motivational states, the understanding has been thus far limited to drug-induced postsynaptic alterations. It remains elusive whether exposure to cocaine or other drugs of abuse influences presynaptic functions of these excitatory projections, and if so, in which projection pathways. Using optogenetic methods combined with biophysical assays, we demonstrate that the presynaptic release probability (Pr) of the PFC-to-NAc synapses was enhanced after short-term withdrawal (1 d) and long-term (45 d) withdrawal from either noncontingent (i.p. injection) or contingent (self-administration) exposure to cocaine. After long-term withdrawal of contingent drug exposure, the Pr was higher compared with i.p. injected rats. In contrast, within the basolateral amygdala afferents, presynaptic Pr was not significantly altered in any of these experimental conditions. Thus, cocaine-induced procedure- and pathway-specific presynaptic enhancement of excitatory synaptic transmission in the NAc. These results, together with previous findings of cocaine-induced postsynaptic enhancement, suggest an increased PFC-to-NAc shell glutamatergic synaptic transmission after withdrawal from exposure to cocaine. This presynaptic alteration may interact with other cocaine-induced cellular adaptations to shift the functional output of NAc neurons, contributing to the addictive emotional and motivational state.

Axonal organizations with specific patterns underlie the functioning of local intracortical circuitry, but their precise anatomy and development still remain elusive. Here, we selectively visualized layer 2/3 neurons using in utero electroporation and examined their axonal organization in the barrel cortex contralateral to the electroporated side. We found that callosal axons run preferentially in septal regions of layer 4 and showed a whisker-related pattern in the contralateral barrel cortex in rats and mice. In addition, presynaptic marker proteins were found in this whisker-related axonal organization. Although the whisker-related patterns were observed in both the ipsilateral and contralateral barrel cortex, we found a difference in their developmental processes. While the formation of the whisker-related pattern in the ipsilateral cortex consisted of two distinct steps, that in the contralateral cortex did not have the 1st step, in which the axons were diffusely distributed without preference to septal or barrel regions. We also found that these more diffuse axons ran close to radial glial fibers. Together, our results uncovered a whisker-related axonal pattern of callosal axons and two independent developmental processes involved in the formation of the axonal trajectories of layer 2/3 neurons.

Serotonin (5-HT) is a critical neurotransmitter in the control of autonomic functions. 5-HT3receptors participate in vagal afferent feedback to decrease food intake and regulate cardiovascular reflexes; however, the phenotype of the solitary tract nucleus (NTS) neurons involved is not known. A2/C2catecholamine (CA) neurons in the NTS are directly activated by visceral afferents and are important for the control of food intake and cardiovascular function, making them good candidates to respond to and mediate the effects of serotonin at the level of the NTS. This study examines serotonin's effects on NTS-CA neurons using patch-clamp techniques and transgenic mice expressing an enhanced green fluorescent protein driven by the tyrosine hydroxylase (TH) promoter (TH-EGFP) to identify catecholamine neurons. Serotonin increased the frequency of spontaneous glutamate excitatory postsynaptic currents (sEPSCs) in >90% of NTS-TH-EGFP neurons, an effect blocked by the 5-HT3receptor antagonist ondansetron and mimicked by the 5-HT3receptor agonists SR5227 and mCPBG. In contrast, 5-HT3receptor agonists increased sEPSCs on a minority (3receptor agonists increased the frequency, but not the amplitude, of mini-EPSCs, suggesting that their actions are presynaptic. 5-HT3receptor agonists increased the firing rate of TH-EGFP neurons, an effect dependent on the increased spontaneous glutamate inputs as it was blocked by the ionotropic glutamate antagonist NBQX, but independent of visceral afferent activation. These results demonstrate a cellular mechanism by which serotonin activates NTS-TH neurons and suggest a pathway by which it can increase catecholamine release in target regions to modulate food intake, motivation, stress, and cardiovascular function.

Elevated presynaptic striatal dopaminergic function is a robust feature of schizophrenia. However, the relationship between this dopamine abnormality and the response to dopamine-blocking antipsychotic treatments is unclear. The authors tested the hypothesis that in patients with schizophrenia the response to antipsychotic treatment would be related to the severity of presynaptic dopamine dysfunction, as indexed using [(18)F]-DOPA uptake positron emission tomography (PET).Twelve patients with treatment-resistant schizophrenia, twelve patients with schizophrenia who had responded to antipsychotics, and twelve healthy volunteers matched for gender, age, ethnicity, weight, and cigarette smoking underwent [(18)F]-DOPA PET scanning. [(18)F]-DOPA influx rate constants (K(i)(cer) values) were measured in the striatum and its functional subdivisions.Patients who had responded to antipsychotic treatment showed significantly higher K(i)(cer) striatal values than did patients with treatment-resistant illness (effect size=1.11) and healthy volunteers (effect size=1.12). The elevated [(18)F]-DOPA uptake was most marked in the associative (effect size=1.31) and the limbic (effect size=1.04) striatal subdivisions. There were no significant differences between patients with treatment-resistant illness and healthy volunteers in the whole striatum or any of its subdivisions.In some patients with schizophrenia, antipsychotic treatment may be ineffective because they do not exhibit the elevation in dopamine synthesis capacity that is classically associated with the disorder; this may reflect a different underlying pathophysiology or a differential effect of antipsychotic treatment.

Both the serotonergic and endocannabinoid systems modulate frontocortical glutamate release; thus they are well positioned to participate in the pathogenesis of psychiatric disorders. With the help of fluorescent and confocal microscopy, we localized the CB(1) cannabinoid receptor (CB(1)R) in VGLUT1- and 2- (i.e. glutamatergic) and serotonin transporter- (i.e. serotonergic) -positive fibers and nerve terminals in the mouse and rat frontal cortex. CB(1)R activation by the synthetic agonists, WIN55212-2 (1 μM) and R-methanandamide (1 μM) inhibited the simultaneously measured evoked Ca(2+)-dependent release of [(14)C]glutamate and [(3)H]serotonin from frontocortical nerve terminals of Wistar rats, in a fashion sensitive to the CB(1)R antagonists, O-2050 (1 μM) and LY320135 (5 μM). CB(1)R agonists also inhibited the evoked release of [(14)C]glutamate in C57BL/6J mice in a reversible fashion upon washout. Interestingly, the evoked release of [(14)C]glutamate and [(3)H]serotonin was significantly greater in the CB(1)R knockout CD-1 mice. Furthermore, CB(1)R binding experiments revealed similar frontocortical CB(1)R density in the rat and the CD-1 mouse. Still, the evoked release of [(3)H]serotonin was modulated by neither CB(1)R agonists nor antagonists in wild-type CD-1 or C57BL/6J mice. Altogether, this is the first study to demonstrate functional presynaptic CB(1)Rs in frontocortical glutamatergic and serotonergic terminals, revealing species differences.