Short-term plasticity of Bergmann glial cell extrasynaptic currents during parallel fiber stimulation in rat cerebellum

Bergmann glial cells (BGC) enclose the synapses of Purkinje neurons (PN) and interneurons in the molecular layer of the cerebellar cortex. During synaptic transmission, glutamate evokes inward currents in the glia by activation of Ca2+-permeable aminohydroxymethylisoxazole propionic acid receptors (AMPAR) and electrogenic transporters. We describe the plasticity of BGC currents during paired-pulse and repetitive stimulation of parallel fibers in cerebellar slices. Paired-pulse facilitation (PPF) of BGC AMPAR currents was 4-fold, twice that of PN PPF. Experiments with a low-affinity AMPAR antagonist showed an increase in extrasynaptic glutamate concentration during the second pulse of the pair. PPF of glial transporter currents was 1.8-fold, similar to synaptic PPF. Tetanic stimulation revealed that facilitation of BGC AMPAR currents is not sustained during high-frequency stimulation, and substantial depression is observed after a few pulses. Consequently, Ca2+ influx through glial AMPARs would initially be facilitated but subsequently depressed, generating a transient Ca2+ influx in response to a sustained tetanus. This pattern of plasticity may be important in enabling Bergmann glial cell processes to detect and support synapses with high-frequency input. Finally, a new current was observed in BGC during repetitive stimulation. It was blocked by NBQX and intracellular GDP-beta-S, increased by glutamate uptake inhibition, had PPF similar to synaptic PPF, and was unaffected by an inhibitor of fast glial AMPAR currents. The evidence suggests that activation of neuronal AMPARs causes the release of a paracrine messenger to activate a G-protein coupled receptor in the BGC.     

Long-term depression of neuron to glial signalling in rat cerebellar cortex

Bergmann glial cells enclose synapses throughout the molecular layer of the cerebellum and express extrasynaptic AMPA receptors and glutamate transporters. Accordingly, stimulation of parallel fibres leads to the generation of inward currents in the glia due to AMPA receptor activation and electrogenic uptake of glutamate. Elimination of AMPA receptor Ca(2+) permeability leads to the withdrawal of glial processes and synaptic dysfunction, suggesting that AMPA receptor-mediated Ca(2+) signalling is essential for glial support of the neuronal network. Here we show that glial extrasynaptic currents (ESCs) exhibit activity-dependent plasticity, specifically, long-term depression during repetitive stimulation of parallel fibres at low frequencies (0.033-1 Hz) -- conditions in which Purkinje neuron excitatory postsynaptic currents (EPSCs) remain stable. Both the rate of onset and the magnitude of ESC depression increased with stimulation frequency. Depression was reversible following brief periods of stimulation, but became increasingly persistent as the duration of repetitive stimulation increased. All glial currents -- AMPA receptors, glutamate transporter and a recently discovered slow 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulphonamide (NBQX)-sensitive current -- were depressed. Increasing presynaptic release probability by doubling external Ca(2+) concentration did not affect the time course of depression, suggesting that neither decreased release probability nor fatigue of release sites contribute to depression. Inhibition of glutamate uptake caused a dramatic enhancement of the rate of depression, implicating glutamate in the underlying mechanism. The strength of neuron to glial signalling in the cerebellum is therefore dynamically regulated, independently of adjacent synapses, by the frequency of parallel fibre activity.     

Adenosine released by astrocytes contributes to hypoxia-induced modulation of synaptic transmission

Astrocytes play a critical role in brain homeostasis controlling the local environment in normal as well as in pathological conditions, such as during hypoxic/ischemic insult. Since astrocytes have recently been identified as a source for a wide variety of gliotransmitters that modulate synaptic activity, we investigated whether the hypoxia-induced excitatory synaptic depression might be mediated by adenosine release from astrocytes. We used electrophysiological and Ca2+ imaging techniques in hippocampal slices and transgenic mice, in which ATP released from astrocytes is specifically impaired, as well as chemiluminescent and fluorescence photometric Ca2+ techniques in purified cultured astrocytes. In hippocampal slices, hypoxia induced a transient depression of excitatory synaptic transmission mediated by activation of presynaptic A1 adenosine receptors. The glia-specific metabolic inhibitor fluorocitrate (FC) was as effective as the A1 adenosine receptor antagonist CPT in preventing the hypoxia-induced excitatory synaptic transmission reduction. Furthermore, FC abolished the extracellular adenosine concentration increase during hypoxia in astrocyte cultures. Several lines of evidence suggest that the increase of extracellular adenosine levels during hypoxia does not result from extracellular ATP or cAMP catabolism, and that astrocytes directly release adenosine in response to hypoxia. Adenosine release is negatively modulated by external or internal Ca2+ concentrations. Moreover, adenosine transport inhibitors did not modify the hypoxia-induced effects, suggesting that adenosine was not released by facilitated transport. We conclude that during hypoxia, astrocytes contribute to regulate the excitatory synaptic transmission through the release of adenosine, which acting on A1 adenosine receptors reduces presynaptic transmitter release. Therefore, adenosine release from astrocytes serves as a protective mechanism by down regulating the synaptic activity level during demanding conditions such as transient hypoxia.     

The neuropeptide corticotropin-releasing factor regulates excitatory transmission and plasticity at the climbing fibre-Purkinje cell synapse

The climbing fibre (CF) input controls cerebellar Purkinje cell (PC) activity as well as synaptic plasticity at parallel fibre (PF)-PC synapses. Under high activity conditions, CFs release not only glutamate, but also the neuropeptide corticotropin-releasing factor (CRF). Brief periods of such high CF activity can lead to the induction of long-term depression (LTD) at CF-PC synapses. Thus, we have examined for the first time the role of CRF in regulating excitatory postsynaptic currents (EPSCs) and long-term plasticity at this synapse. Exogenous application of CRF alone transiently mimicked three aspects of CF-LTD, causing reductions in the CF-evoked excitatory postsynaptic current, complex spike second component and complex spike afterhyperpolarization. The complex spike first component is unaffected by CF-LTD induction and was similarly unaffected by CRF. Application of a CRF receptor antagonist reduced the expression amplitude and induction probability of CF-LTD monitored at the EPSC level. Collectively, these results suggest that under particular sensorimotor conditions, co-release of CRF from climbing fibres could down-regulate excitatory transmission and facilitate LTD induction at CF-PC synapses. Inhibition of either protein kinase C (PKC) or protein kinase A (PKA) attenuated the effects of CRF upon CF-EPSCs. We have previously shown that CF-LTD induction is PKC-dependent, and here demonstrate PKA-dependence as well. These results suggest that both the acute effects of CRF on CF-EPSCs as well as the facilitating effect of CRF on CF-LTD induction can be explained by a CRF-mediated recruitment of PKC and PKA.     

Effects of FAK ablation on cerebellar foliation, Bergmann glia positioning and climbing fiber territory on Purkinje cells

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that is widely expressed in the brain, and plays key roles in various cellular processes in response to both extracellular and intracellular stimuli. Here, we explored the role of FAK in cerebellar development. In the mouse cerebellum, FAK was found to be distributed as tiny cytoplasmic aggregates in various neuronal and glial elements, including Purkinje cells (PCs), Bergmann glia (BG), parallel fiber (PF)-terminals and climbing fiber (CF)-terminals. The neuron/glia-specific ablation of FAK impaired cerebellar foliation, such as variable decreases in foliation sizes and the lack of intercrural and precentral fissures. Some of the BG cells became situated ectopically in the molecular layer. Furthermore, the FAK ablation altered the innervation territories of CFs and PFs on PCs. CF innervation regressed to the basal portion of proximal dendrites and somata, whereas ectopic spines protruded from proximal dendrites and PFs expanded their territory by innervating the ectopic spines. Furthermore, the persistence of surplus CFs innervating PC somata caused multiple innervation. When FAK was selectively ablated in PCs, diminished dendritic innervation and persistent somatic innervation by CFs were observed, whereas cerebellar foliation and cell positioning of BG were normally retained. These results suggest that FAK in various neuronal and glial elements is required for the formation of normal histoarchitecture and cytoarchitecture in the cerebellum, and for the construction of proper innervation territory and synaptic wiring in PCs.     

Sodium signals in cerebellar Purkinje neurons and Bergmann glial cells evoked by glutamatergic synaptic transmission

Glial cells express specific high-affinity transporters for glutamate that play a central role in glutamate clearance at excitatory synapses in the brain. These transporters are electrogenic and are mainly energized by the electrochemical gradient for sodium. In the present study, we combined somatic whole-cell patch-clamp recordings with quantitative Na+ imaging in fine cellular branches of cerebellar Bergmann glial cells and in dendrites of Purkinje neurons to analyze intracellular Na+ signals close to activated synapses. We demonstrate that pressure application of glutamate and glutamate agonists causes local Na+ signals in the mM range. Furthermore, we analyzed the pharmacological profile, as well as the time course and spatial distribution of Na+ signals following short synaptic burst stimulation of parallel or climbing fibers. While parallel fibers stimulation resulted in local sodium transients that were largest in processes close to the stimulation pipette, climbing fibers stimulation elicited global sodium transients throughout the entire cell. Glial sodium signals amounted to several mM, were mainly caused by sodium influx following inward transport of glutamate and persisted for tens of seconds. Sodium transients in dendrites of Purkinje neurons, in contrast, were mainly caused by activation of AMPA receptors and had much faster kinetics. By reducing the driving force for sodium-dependent glutamate uptake, intracellular sodium accumulation in glial cells upon repetitive activity might provide a negative feedback mechanism, promoting the diffusion of glutamate and the activation of extrasynaptic glutamate receptors at active synapses in the cerebellum.     

Afferent-target interactions during olivocerebellar development: transcommissural reinnervation indicates interdependence of Purkinje cell maturation and climbing fibre synapse elimination

We have used a model of postlesional reinnervation to observe the interactions between synaptic partners during neosynaptogenesis to determine how the developmental states of the pre- and postsynaptic cells influence circuit maturation. After unilateral transection of the neonatal rat olivocerebellar pathway (pedunculotomy), axons from the remaining ipsilateral inferior olive grow into the denervated hemicerebellum and develop climbing fibre (CF) terminal arbors on Purkinje cells (PCs) at a later stage of development than normal. However, the significance of delayed CF-PC interactions on subsequent circuit maturation remains poorly defined. To examine this question, we recorded CF-induced currents in PCs and analysed PC morphology during the first two postnatal weeks in control animals and following left unilateral inferior cerebellar pedunculotomy on postnatal day (P)3. Our results show that transcommissural olivary axons multiply-reinnervate PCs in the denervated hemisphere over 4 days following pedunculotomy. Each PC received fewer CFs than did age-matched controls and the maximal multi-reinnervation was reached on P7, 2 days later than in controls. Consequently, the onset of CF synapse elimination in reinnervated PCs was delayed, but then proceeded in parallel with controls so that all PCs were monoinnervated by P15. Furthermore, reinnervated PCs had delayed dendritic maturation and subsequent dendritic abnormalities consistent with the role of CF innervation in PC dendritic growth. Thus, within the olivocerebellar system, our data suggest that target neurons depend upon sufficient afferent investment arriving at the correct time for their normal development, and maturation of the target neuron regulates afferent selection and therefore circuit maturation.     

Phosphorylation of cysteine string protein in the brain: developmental, regional and synaptic specificity

Protein phosphorylation modulates regulated exocytosis in most cells, including neurons. Cysteine string protein (CSP) has been implicated in this process because its phosphorylation on Ser10 alters its interactions with syntaxin and synaptotagmin, and because the effect of CSP overexpression on exocytosis kinetics in chromaffin cells requires phosphorylatable Ser10. To characterize CSP phosphorylation in the brain, we raised phosphospecific antibodies to Ser10. Western blotting revealed that the proportion of phosphorylated CSP (P-CSP) varies between distinct brain regions and also exhibits developmental regulation, with P-CSP highest early in development. Immunohistochemical analysis of the cerebellar cortex revealed a novel pool of P-CSP that did not colocalize with synaptic vesicle markers during early development. Strikingly, in the adult cerebellar granular layer P-CSP was highly enriched in a subset of glutamatergic synapses but undetectable in neighbouring GABA-ergic synapses. In view of the functional consequences of CSP phosphorylation, such differences could contribute to the synapse-specific regulation of neurotransmitter release.     

Pressure-induced depression of synaptic transmission in the cerebellar parallel fibre synapse involves suppression of presynaptic N-type Ca2+ channels

High pressure induces CNS hyperexcitability while markedly depressing synaptic transmitter release. We studied the effect of pressure (up to 10.1 MPa) on the parallel fibre (PF) synaptic response in biplanar cerebellar slices of adult guinea pigs. Pressure mildly reduced the PF volley amplitude and to a greater extent depressed the excitatory field postsynaptic potential (fPSP). The depression of the PF volley was noted even at supramaximal stimulus intensities, indicating an effect of pressure on the amplitude of the action potential in each axon. Low concentrations of TTX mimicked the effects of pressure on the PF volley without affecting the fPSP. Application omega-conotoxin GVIA (omega-CgTx) reduced the synaptic efficacy by 34.3+/-2.7%. However, in the presence of omega-CgTx the synaptic depression at pressure was significantly reduced. Reduced Ca2+ entry by application of Cd2+ or low [Ca2+]o did not have a similar influence on the effects of pressure. Application of omega-AGA IVA, omega-AGA TK and Funnel-web spider toxin did not affect the synaptic response in concentrations that usually block P-type Ca2+ channels, whilst the N/P/Q-type blocker omega-conotoxin MVIIC reduced the response to 52.7+/-5.0% indicating the involvement of Q-type channels and R-type channels in the non-N-type fraction of Ca2+ entry. The results demonstrate that N-type Ca2+ channels play a crucial role in the induction of PF synaptic depression at pressure. This finding suggests a coherent mechanism for the induction of CNS hyperexcitability at pressure.     

Cell-type specific GABA synaptic transmission and activity-dependent plasticity in rat hippocampal stratum radiatum interneurons

Abstract In hippocampal pyramidal cells, the efficacy of synaptic transmission at gamma-aminobutyric acid (GABA)ergic synapses, is modulated by activity. However, whether such plasticity occurs at inhibitory synapses on interneurons remains largely unknown. Using whole-cell voltage-clamp recordings of inhibitory postsynaptic currents (IPSCs) in Sprague-Dawley rat hippocampal slices, we examined whether GABA synapses of stratum radiatum interneurons were affected by stimulation protocols known to alter efficacy at inhibitory synapses of CA1 pyramidal cells. Monosynaptically evoked IPSCs (eIPSCs) exhibited different properties with significantly faster kinetics, higher coefficients of variation, a current-voltage (I-V) relationship shifted to depolarized values and a smaller paired-pulse depression, in interneurons than in pyramidal cells. GABA synapses on interneurons also showed a different capacity for plasticity. Indeed, theta-burst stimulation induced a long-term potentiation of eIPSCs in both cell types, but the induction mechanisms differed in interneurons, as it was not affected by antagonists of GABAB receptors and group I/II metabotropic glutamate receptors (mGluRs). Furthermore, 100-Hz tetanization selectively elicited a short-term depression of eIPSCs in pyramidal cells. A postsynaptic depolarization produced a transient suppression of eIPSCs (depolarization-induced suppression of inhibition) in pyramidal cells but not in interneurons. Spontaneous IPSCs were similarly reduced following depolarization in pyramidal cells, but not in interneurons. These results indicate that GABA synapses of stratum radiatum interneurons exhibit different properties and capacity for activity-dependent plasticity than those of pyramidal cells. This cell-type specific mode of transmission and adaptive regulation of GABA synapses may contribute to hippocampal plasticity and functions.     

EPO induces changes in synaptic transmission and plasticity in the dentate gyrus of rats

Erythropoietin has shown wide physiological effects on the central nervous system in animal models of disease, and in healthy animals. We have recently shown that systemic EPO administration 15 min, but not 5 h, after daily training in a water maze is able to induce the recovery of spatial memory in fimbria‐fornix chronic‐lesioned animals, suggesting that acute EPO triggers mechanisms which can modulate the active neural plasticity mechanism involved in spatial memory acquisition in lesioned animals. Additionally, this EPO effect is accompanied by the up‐regulation of plasticity‐related early genes. More remarkably, this time‐dependent effects on learning recovery could signify that EPO in nerve system modulate specific living‐cellular processes. In the present article, we focus on the question if EPO could modulate the induction of long‐term synaptic plasticity like LTP and LTD, which presumably could support our previous published data. Our results show that acute EPO peripheral administration 15 min before the induction of synaptic plasticity is able to increase the magnitude of the LTP (more prominent in PSA than fEPSP‐Slope) to facilitate the induction of LTD, and to protect LTP from depotentiation. These findings showing that EPO modulates in vivo synaptic plasticity sustain the assumption that EPO can act not only as a neuroprotective substance, but is also able to modulate transient neural plasticity mechanisms and therefore to promote the recovery of nerve function after an established chronic brain lesion. According to these results, EPO could be use as a molecular tool for neurorestaurative treatments. Synapse 70:240–252, 2016 . © 2016 Wiley Periodicals, Inc.     

Long-term enhancement of CA1 synaptic transmission is due to increased quantal size, not quantal content.

Quantal components of Schaffer collateral synaptic transmission recorded intracellularly from CA1 pyramidal cells were examined using 2 methods: simultaneous recordings of CA3-CA1 cell-pairs, and minimal electrical stimulation in stratum radiatum. Quantal parameters estimated by the method of failures and by a computer algorithm that optimized parameter estimates using deconvolution of background noise were highly correlated. EPSP-amplitude histograms of CA3-CA1 cell pairs (N = 10) and minimal electrical stimulation (N = 33) could be adequately described either by Poisson or binomial statistics, or by both, and exhibited similar estimates of unit quantal size (q) and mean quantal content (m). Paired-pulse stimulation with 50 msec between stimuli resulted in an expected facilitation in the EPSP amplitude and increase in m during the second response, as estimated by noise deconvolution, by the decrease in apparent failures, and by a decrease in the coefficient of variation of the EPSP. Tetanization of the Schaffer collaterals that induced long-term enhancement (LTE/LTP) of the population response was associated with an average increase in q for minimal-stimulation responses, with no significant change in any estimate of m. Taken together, these data indicate that, under the present experimental conditions, LTE is expressed as an increase in quantal size, rather than an increase in the number of quanta released per presynaptic impulse. Although this is not definitive evidence for a postsynaptic mechanism, these findings do further restrict the classes of possible presynaptic mechanisms that may be proposed to account for LTE expression.     

Differential regulation of spontaneous and evoked inhibitory synaptic transmission in somatosensory cortex by retinoic acid

Retinoic acid (RA), a developmental morphogen, has emerged in recent studies as a novel synaptic signaling molecule that acts in mature hippocampal neurons to modulate excitatory and inhibitory synaptic transmission in the context of homeostatic synaptic plasticity. However, it is unclear whether RA is capable of modulating neural circuits outside of the hippocampus, and if so, whether the mode of RA's action at synapses is similar to that within the hippocampal network. Here we explore for the first time RA's synaptic function outside the hippocampus and uncover a novel function of all‐trans retinoic acid at inhibitory synapses. Acute RA treatment increases spontaneous inhibitory synaptic transmission in L2/3 pyramidal neurons of the somatosensory cortex, and this effect requires expression of RA's receptor RARα both pre‐ and post‐synaptically. Intriguingly, RA does not seem to affect evoked inhibitory transmission assayed with either extracellular stimulation or direct activation of action potentials in presynaptic interneurons at connected pairs of interneurons and pyramidal neurons. Taken together, these results suggest that RA's action at synapses is not monotonous, but is diverse depending on the type of synaptic connection (excitatory versus inhibitory) and circuit (hippocampal versus cortical). Thus, synaptic signaling of RA may mediate multi‐faceted regulation of synaptic plasticity. In addition to its classic roles in brain development, retinoic acid (RA) has recently been shown to regulate excitatory and inhibitory transmission in the adult brain. Here, the authors show that in layer 2/3 (L2/3) of the somatosensory cortex (S1), acute RA induces increases in spontaneous but not action‐potential evoked transmission, and that this requires retinoic acid receptor (RARα) both in presynaptic PV‐positive interneurons and postsynaptic pyramidal (PN) neurons.     

The sorting receptor SorCS3 is a stronger regulator of glutamate receptor functions compared to GABAergic mechanisms in the hippocampus

Correct function of glutamate receptors in the postsynaptic density is crucial to synaptic function and plasticity. SorCS3 (sortilin‐related receptor CNS expressed 3) is a sorting receptor which previously has been shown to interact with the key postsynaptic proteins; PSD‐95 and PICK1. In this study, we employed electrophysiological analyses of acute brain slices combined with immunohistochemistry to define the role of SorCS3 in hippocampal synapses in CA1 and the dentate gyrus. We analyzed a juvenile (P17‐21) and a young adult (P55‐65) group of animals from a Sorcs3 knockout mouse model. We show that the basal synaptic transmission is severely affected in SorCS3‐deficient neurons in CA1, while only slightly reduced in the dentate gyrus. Specifically, input/output curves of CA1 synapses revealed a 20% reduction of fEPSP (field excitatory postsynaptic potential) slopes at the highest stimulation intensity in knockouts of the juvenile group, which developed to a 33% decrease in young adult animals. These impairments may be a result of changes in the postsynaptic AMPA receptors. Interestingly, repetitive afferent stimulation demonstrated that SorCS3‐deficient slices respond with an enhanced synaptic facilitation and reduced synaptic depression. These changes also developed with age. A molecular mechanism underlying this relative increase during repetitive stimulations is compatible with enhanced mobility of postsynaptic AMPA receptors resulting in faster exchange of desensitized receptors in the postsynaptic density. The altered response during repetitive stimulation was characteristic for CA1 but not the dentate gyrus. Immunohistochemical analyses of parvalbumin positive neurons combined with paired‐pulse tests of network inhibition and patch‐clamp recordings only showed minute inhibitory changes in SorCS3‐deficient slices. Our results suggest that SorCS3 serves an important role in the postsynaptic protein network, which is more pronounced in CA1 compared to the dentate gyrus. These data support a role for SorCS3 in controlling proper positioning and mobility of glutamate receptors in the postsynaptic density. © 2016 Wiley Periodicals, Inc.     

Synaptic shunting by a baseline of synaptic conductances modulates responses to inhibitory input volleys in cerebellar Purkinje cells

When processing synaptic input in vivo, large neurons in the brain must cope with thousands of events each second. Much work has focused on the specific processing of synchronous excitatory input volleys, both in cerebellar and cerebral cortical research. Here we pursue the question of how a continuous background of ongoing 'noise' inputs interacts with the processing of synchronous inhibitory input volleys. Specifically we examine the processing of inhibitory input transients in cerebellar Purkinje cells, which by inducing pauses in Purkinje cell spike activity may lead to a disinhibition of the deep cerebellar nuclei and thus to cerebellar motor command signals. We use the technique of dynamic clamping in vitro to simulate controlled patterns of in vivo like background inputs. We use electrical stimulation of inhibitory interneurons in the deep or upper molecular layer to create inhibitory input transients that lead to spike pauses in Purkinje cell activity. These pauses were much longer in the absence than in the presence of background inputs applied with dynamic clamping. We found that a significant amount of the synaptic current elicited by electrical stimulation was shunted by the background inputs. The overall amount of background conductance as well as the pattern of background inputs modulated spike pause duration in a specific manner. This modulation by shunting may be employed in vivo to evaluate the salience of specific sensory input received by cerebellar cortex.     

Resistance to ethanol sensitization is associated with a loss of synaptic plasticity in the hippocampus

Behavioral sensitization to repeated ethanol (EtOH) exposure induces an increase in locomotor activity in mice. However, not all animals express such sensitization. Although the literature indicated that the hippocampus may play a role in EtOH sensitization, it is not known whether behavioral sensitization to EtOH is associated with preferential changes in bidirectional synaptic plasticity, i.e., LTP and LTD, two markers of learning capabilities that have also been shown to be involved in addictive behavior. In the present study, we examined whether the vulnerability to develop and express behavioral sensitization to EtOH is associated with altered bidirectional synaptic plasticity in the CA1 area of the dorsal hippocampus. For this purpose, we analyzed both LTP and LTD in resistant and sensitized mice during the expression phase, i.e., 7 days after 10 days of repeated EtOH i.p. administration. We found that resistant mice showed a lack of LTD without changes in LTP. The lack of LTD was associated with an increase in GluN2A protein level and was not due to an altered level of neuronal activity, since no difference was observed between the number of c‐FOS positive neurons in sensitized and resistant mice. Given that both types of synaptic plasticity signals may have distinct roles in specific learning and behaviors, our results suggest that resistant mice could exhibit different phenotypes in terms of learning/memory and addictive behaviors compared to sensitized ones. Synapse 71:e21899, 2017 . © 2016 Wiley Periodicals, Inc.     

Membrane‐tethered AKT kinase regulates basal synaptic transmission and early phase LTP expression by modulation of post‐synaptic AMPA receptor level

The serine/threonine kinase AKT/PKB plays a fundamental role in a wide variety of neuronal functions, including neuronal cell development, axonal growth, and synaptic plasticity. Multiple evidence link AKT signaling pathways to regulation of late phase long‐term synaptic plasticity, synaptogenesis, and spinogenesis, as well as long‐term memory formation. Nevertheless, the downstream effectors mediating the effects of AKT on early phase long‐term potentiation (eLTP) are currently unknown. Here we report that using different regimes of pharmacological activation and inhibition of AKT activity in acute hippocampal slices, we found that AKT regulates the post‐synaptic expression of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptor (AMPA) receptors affecting solely the expression of eLTP, with no effect on its induction and maintenance. We further show that both maintenance of basal synaptic activity and expression of eLTP require plasma membrane tethering by activated AKT and that basal synaptic activity may be regulated via the direct effects of AKT1 on the expression level of post‐synaptic AMPA receptors bypassing the canonical AKT signaling. Finally, we establish that eLTP expression requires the involvement of both the canonical AKT signaling pathways and the direct effect of AKT1 on AMPA receptor activity/expression in the post‐synaptic membrane. © 2016 Wiley Periodicals, Inc.     

Characterization in vivo of the NMDA receptor-mediated component of dentate granule cell population synaptic responses to perforant path input.

The NMDA receptor-mediated component of the hippocampal granule cell population excitatory postsynaptic potential response to low frequency (< 0.2 Hz) stimulation of the medial perforant path was characterized in vivo. Extracellular recordings were obtained from the dentate molecular layer in anesthetized rabbits, and glutamatergic and GABAergic antagonists were applied locally by pressure ejection. To measure the NMDA-mediated component, the NMDA receptor antagonist D-5-aminophosphonovalerate (APV) was applied during the constant ejection of physiological saline, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and/or bicuculline methiodide. In general agreement with the results of attempts by other investigators to identify NMDA responses in vivo, APV did not significantly reduce the response to a single stimulus impulse in the presence of saline. However, an NMDA-mediated response was revealed when alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprianate receptor-mediated current flow was eliminated by applying the non-NMDA receptor antagonist CNQX. The NMDA component was negative-going as predicted, but its duration was considerably less than indicated in other studies of the dentate in vitro. The relative magnitudes of the NMDA and non-NMDA components of the EPSP were found to vary as a function of stimulus intensity or frequency. The NMDA receptor-mediated component represented 12% of the control response and increased to over 25% in response to higher stimulus intensities. A brief, high-frequency burst of impulses evoked a larger NMDA component in the presence of CNQX and was able to evoke an NMDA component in the presence of saline. Surprisingly, short trains of stimulation at lower frequencies typically produced suppression of the NMDA component. In a final series of experiments, it was found that many characteristics of the NMDA component were substantially altered by GABAergic inhibition. In the presence of the GABAA antagonist bicuculline, the magnitude of NMDA receptor-mediated responses was increased and their duration was greatly extended. Additionally, in the presence of bicuculline, the NMDA component facilitated markedly in response to frequencies of stimulus input > 20 Hz. These results indicate in vivo that the initiation and duration of NMDA current flow depend strongly upon the intensity and frequency of perforant path stimulation. In addition, the NMDA response to a single impulse appears to be reduced and truncated by input from GABAA receptor-mediated feedback and/or feedforward inhibition, and this inhibition affects temporal summation of NMDA receptor-mediated responses over a wide range of input frequencies. It is suggested that such inhibition results from the activation of GABAA receptors located on granule cell dendritic shafts.(ABSTRACT TRUNCATED AT 400 WORDS)     

Selective synaptic distribution of AMPA and kainate receptor subunits in the outer plexiform layer of the carp retina.

The subunit composition of ionotropic glutamate receptors (GluRs) is extremely diverse and responsible for the diversity of postsynaptic responses to the release of glutamate, which is the major excitatory neurotransmitter in the retina. To understand the functional consequences of this diversity, it is necessary to reveal the synaptic localization and subunit composition of GluRs. We have used immuno light and electron microscopy to localize AMPA and kainate (GluR1, GluR2/3, GluR4, GluR5-7) subunits in identified carp retinal neurons contributing to the outer plexiform layer. GluR1 could not be detected within the outer plexiform layer. Rod and cone horizontal cells all express only GluR2/3 at the tips of their invaginating dendrites. These receptors are also inserted into the membrane of spinules, light-dependent protrusions of the horizontal cell dendrites, flanking the synaptic ribbon of the cone synapse. Bipolar cells express GluR2/3, GluR4, and GluR5-7 at their terminal dendrites invaginating cone pedicles and rod spherules. Colocalization data suggest that each subunit is expressed by a distinct bipolar cell type. The majority of bipolar cells expressing these receptors seem to be of the functional OFF-type; however, in a few instances, GluR2/3 could also be detected on dendrites of bipolar cells that, based on their localization within the cone synaptic complex, appeared to be of the functional ON-type. The spatial arrangement of the different subunits within the cavity of the cone pedicle appeared not to be random: GluR2/3 was found predominantly at the apex of the cavity, GluR4 at its base and GluR5-7 dispersed between the two.     

Effects of high-frequency synaptic stimulation on glumate receptor binding studied with a modified in vitro hippocampal slice preparation

Slices of the field CA1 minus the stratum moleculare were prepared from the rat hippocampus and maintained in an in vitro recording chamber. The physiological properties of these ‘minislices’ were similar to those reported for non-dissected hippocampal slices. Slices receiving various patterns of electrical stimulation through multiple electrodes were subsequently homogenized and crude membrane fractions prepared; the binding of [³H]glutamate was measured by a rapid filtration assay. Binding in membranes prepared from control slices exhibited kinetic properties and sensitivities to pharmacological and ionic manipulations which were comparable to those found in previous studies using conventional fractionation and assay techniques. Brief bursts of high-frequency stimulation increased [³H]glutamate binding compared to non-stimulated controls in 3 separate experiments. Stimulation at low frequency or at high frequency in low calcium medium did not produce this effect. In addition to suggesting that glutamate binding sites are regulated by patterns of afferent activity, these findings indicate that the minislice preparation should be of general utility in relating synaptic physiology to synaptic chemistry.