Synaptic Plasticity and Hebbian Cell Assemblies

Synaptic dynamics are critical to the function of neuronal circuits on multiple timescales. In the first part of this dissertation, I tested the roles of action potential timing and NMDA receptor composition in long-term modifications to synaptic efficacy. In a computational model I showed that the dynamics of the postsynaptic [Ca2+] time course can be used to map the timing of pre- and postsynaptic action potentials onto experimentally observed changes in synaptic strength. Using dual patch-clamp recordings from cultured hippocampal neurons, I found that NMDAR subtypes can map combinations of pre- and postsynaptic action potentials onto either long-term potentiation (LTP) or depression (LTD). LTP and LTD could even be evoked by the same stimuli, and in such cases the plasticity outcome was determined by the availability of NMDAR subtypes. The expression of LTD was increasingly presynaptic as synaptic connections became more developed. Finally, I found that spike-timing-dependent potentiability is history-dependent, with a non-linear relationship to the number of pre- and postsynaptic action potentials. After LTP induction, subsequent potentiability recovered on a timescale of minutes, and was dependent on the duration of the previous induction. While activity-dependent plasticity is putatively involved in circuit development, I found that it was not required to produce small networks capable of exhibiting rhythmic persistent activity patterns called reverberations. However, positive synaptic scaling produced by network inactivity yielded increased quantal synaptic amplitudes, connectivity, and potentiability, all favoring reverberation. These data suggest that chronic inactivity upregulates synaptic efficacy by both quantal amplification and by the addition of silent synapses, the latter of which are rapidly activated by reverberation. Reverberation in previously inactivated networks also resulted in activity-dependent outbreaks of spontaneous network activity. Applying a model of short-term synaptic dynamics to the network level, I argue that these experimental observations can be explained by the interaction between presynaptic calcium dynamics and short-term synaptic depression on multiple timescales. Together, the experiments and modeling indicate that ongoing activity, synaptic scaling and metaplasticity are required to endow networks with a level of synaptic connectivity and potentiability that supports stimulus-evoked persistent activity patterns but avoids spontaneous activity

Time-coded neurotransmitter release at excitatory and inhibitory synapses.

Communication between neurons at chemical synapses is regulated by hundreds of different proteins that control the release of neurotransmitter that is packaged in vesicles, transported to an active zone, and released when an input spike occurs. Neurotransmitter can also be released asynchronously, that is, after a delay following the spike, or spontaneously in the absence of a stimulus. The mechanisms underlying asynchronous and spontaneous neurotransmitter release remain elusive. Here, we describe a model of the exocytotic cycle of vesicles at excitatory and inhibitory synapses that accounts for all modes of vesicle release as well as short-term synaptic plasticity (STSP). For asynchronous release, the model predicts a delayed inertial protein unbinding associated with the SNARE complex assembly immediately after vesicle priming. Experiments are proposed to test the model's molecular predictions for differential exocytosis. The simplicity of the model will also facilitate large-scale simulations of neural circuits

Synaptotagmin in asynchronous neurotransmitter release and synaptic disease

2018 Summer.Includes bibliographical references.The majority of cell-to-cell communication relies on the stimulated release of neurotransmitter. Two forms of Ca2+-dependent stimulated release, synchronous and asynchronous, have been identified. Synchronous release is the initial release that occurs within milliseconds of stimulation. Critical for efficient synaptic communication, synchronous release is the dominant form of release at most synapses. Alternatively, asynchronous release occurs over longer time periods, with implications in synaptic plasticity and development. However, its mechanisms are poorly understood. Both synchronous and asynchronous release rely on Ca2+ sensors to confer their distinct characteristics. Synaptotagmin 1 is widely accepted as the Ca2+ sensor for fast, synchronous release, but its role in asynchronous release is unclear. Previous studies have led to the hypothesis that synaptotagmin 1, particularly Ca2+ binding by its C2A domain, is needed to inhibit aberrant asynchronous fusion events. However, recent studies have raised questions regarding the interpretation of the results that led to this conclusion. In chapter 2, I have directly tested the effect of Ca2+ binding by synaptotagmin 1's C2A domain on asynchronous release utilizing an alternant Ca2+-binding mutant. This novel mutation was designed to block Ca2+ binding without introducing the artifacts of the original Ca2+-binding mutation. By investigating asynchronous events in vivo at the Drosophila neuromuscular junction, I found no significant effect on asynchronous release when C2A Ca2+ binding was blocked. Thus, I conclude that Ca2+ binding by synaptotagmin's C2A domain is not needed for regulation of asynchronous release, in contrast to the previous study that inadvertently introduced an artifact described below. To prevent Ca2+ binding, the original aspartate to asparagine mutations (sytD-N) removed some of the negatively-charged residues that coordinate Ca2+. This simultaneously introduced aberrant fusion events, because it also interrupted the electrostatic repulsion between synaptotagmin's negatively-charged C2A Ca2+-binding pocket and the negatively-charged presynaptic membrane which is required to clamp constitutive SNARE-mediated fusion. Previous Reist lab results demonstrate that the sytD-N mutations in the C2A domain are likely behaving as ostensibly constitutively bound Ca2+. Indeed, I report that the sytD-N mutation displays slower release kinetics. To directly test if this mutation is the cause of the increase in asynchronous events, I generated additional mutations that prevent interactions with the presynaptic membrane coupled to the originally published sytD-N mutations. In chapter 3 of this dissertation, I investigated these novel mutations at the Drosophila neuromuscular junction. I reported no increase in asynchronous release relative to control, providing evidence that the increased asynchronous events in sytD-N mutants are a result of the original mutation acting as an asynchronous sensor. Together, my results contradict the current hypothesis in the field and provide the likely mechanism for the increased asynchronous release observed in the original study. This dissertation also investigated the relatively new role for synaptotagmin mutations in the etiology of neuromuscular disease. With increased availability of high-throughput sequencing, over 20 candidate genes have been implicated in different forms of congential myasthenic syndromes. These inherited disorders are caused by mutations in genes needed for effective neuromuscular signaling. Two families, presenting with similar myasthenic syndromes, carry point mutations in the C2B Ca2+ binding pocket of synaptotagmin, expressed as an autosomal dominant disorder. One of theses families contains a proline to leucine substitution (sytP-L) a residue that had not been previously investigated for synaptotagmin function. In chapter 4, I investigated the functional importance of this mutation and created a disease model for this familial condition by driving the expression of a homolous proline-leucine synaptotagmin substitution in the central nervous system of Drosophila. I demonstrated that the proline residue plays a functional role in efficient transmitter release by testing its function in an otherwise synaptotagmin null genetic background. Additionally, this mutation displayed characteristics similar to the human disorder when expressed in a heterozygous synaptotagmin background, similar to the familial expression. Namely, the sytP-L mutants exhibited a decreased release probability, which resulted in decreased evoked responses that facilitate upon high frequency stimulation, a rightward shift in Ca2+ sensitivity, and behavioral deficits, including decreased motor output and increased fatigability. Thus, these studies establish the causative nature of the sytP-L mutation in this rare form of congenital myasthenic syndrome and highlight the utility of the Drosophila system for disease modeling

Impact of Second Messenger Modulation on Activity-Dependent and Basal Properties of Excitatory Synapses

Cognitive processing in the central nervous system relies on accurate information propagation; neurotransmission is the fundamental mechanism underlying network information flow. Because network information is coded by the timing and the strength of neuronal activity, synaptic properties that translate neuronal activity into synaptic output profoundly determine the precision of information transfer. Synaptic properties are in turn shaped by changes in network activity to ensure appropriate synaptic output. Activity-dependent adjustment of synaptic properties is often initiated by second messenger signals. Understanding how second messengers sculpt synaptic properties and produce changes in synaptic output is key for elucidating the interplay between network activity and synaptic properties. We studied the effect of second messenger modification on activity-dependent and static properties of rat hippocampal excitatory synapses using electrophysiological and optical approaches. We focused on two second-messenger pathways that potentiate transmission: cAMP and diacyl glycerol: DAG) signals. In parallel, we also compared the effects of manipulating calcium influx, which is known to potentiate synaptic transmission through increasing release probability: Pr). During high frequency stimulation, we found that both cAMP and DAG signals potentiated phasic transmission, as previously characterized. In parallel with increasing phasic transmission, the modulators also enhanced high-frequency associated asynchronous transmission, which emerges late during stimulus trains and is relatively long-lasting. However, such parallel potentiation of phasic and asynchronous transmission was not seen in elevated calcium; high calcium preferentially promoted asynchronous transmission. With low frequency stimulation, we found that cAMP and high calcium enhanced synaptic output by potentiating synapses with basally high Pr. Conversely, DAG signals recruited neurotransmission from both high Pr and low Pr terminals, which include presynaptically quiescent synapses. Taken together, these results suggest that second messenger modulation of synapses differentially shapes the static properties of the synapses; second messengers also fine-tune activity-dependent synaptic responses differently from manipulating calcium influx. These results likely have physiological relevance to second messenger-dependent sculpting of temporal and spatial synaptic properties

Synaptic mechanisms of Hebbian and homeostatic plasticity driven by intrinsic activity in the developing hippocampus

The formation of synaptic connections in the brain is guided by genetic and activity-dependent mechanisms. The initial hard-wiring of the circuitry is followed by a phase during which connections are refined. During this process the genetic factors are less important and refinement is guided by electrical activity. However, the mechanisms underlying the activity-driven synaptic fine-tuning are still poorly understood. The features of electrical activity and the mechanisms of synaptic transmission differ in the developing networks as compared to those of the adult. Electrical activity in the developing networks comprises of intermittent, highly synchronous bursts of action potentials interleaved by more silent, asynchronous neuronal firing. In the hippocampus this immature-type electrical patternity coincides temporally with the intense synaptic reorganization. Moreover, there is a parallel, developmentally-regulated expression GluA4 subunit of AMPA-type ionotropic glutamate receptors in the hippocampal neurons. Despite frequent speculation on the relative importance of synchronous vs. asynchronous neuronal activity on the synaptic development in the brain, there have been no direct experiments to study this issue. In this thesis we have, for the first time, been able to experimentally dissect the roles of asynchronous vs. synchronous activity on synaptic refinement in the hippocampus. Specifically, we show that spontaneous synchronous activity is essential for the stabilization and maturation of immature CA3-CA1 synapses, and that network desynchronization leads to weakening of glutametergic transmission in the CA1 area. Plasticity changes caused by different endogenous activity patterns were strongly dependent on the synapse type (glutamatergic vs. GABAergic), the anatomical area (CA1 vs. CA3) and maturational stage of the neurons. In addition, the GluA4 was shown to be critical for both the Hebbian type and homeostatic plasticity mechanisms in developing glutamatergic synapses. In the absence of GluA4, the homeostatic regulation of the immature glutamatergic networks in response to manipulation of endogenous activity patterns was perturbed. Finally, GluA4 was shown to be necessary and sufficient for protein kinase A dependent long-term potentiation (LTP), typical of immature CA3-CA1 synapses. These data demonstrate the instrumental role of spontaneous synchronous activity and GluA4 AMPAR subunit expression in the formation and refinement of hippocampal synaptic networks.Aivojen hermosolujen välinen tiedonsiirto perustuu niiden välisiin synaptisiin yhteyksiin. Yksittäinen hermosolu voi olla synapsiyhteydessä useisiin satoihin tai tuhansiin muihin hermosoluihin. Osa näistä yhteyksistä on hermosolun toimintaa edistäviä (eksitoivia) ja osa estäviä (inhiboivia). Yksi aivojen keskeisimmistä ominaisuuksista on plastisuus, eli kyky muuttaa hermosolujen välisten synapsien määrää ja vahvuutta. Plastisuusmekanismit luovat molekulaarisen pohjan mm. oppimisen ja muistin solutason mekanismeille. Voimakas hermosolujen samanaikainen sähköinen aktiivisuus tai korkeataajuinen ärsytys johtaa yleensä kyseisten solujen välisen synapsiyhteyden vahvistumiseen (LTP, long-term potentiation), kun taas matalalla taajuudella toistuva ärsytys heikentää kyseisten solujen välisen kontaktin vahvuutta (LTD, long-term deprssion). Tätä ominaisuutta kutsutaan hebbiläiseksi plastisuudeksi. Yksittäisten synapsiyhteyksien liiallinen vahvistuminen tai heikkeneminen voi kuitenkin johtaa hermosolujen yliaktiivisuuteen tai totaaliseen hiljaisuuteen. Näitä ääripäitä välttääkseen aivot käyttävät ns. tasapainottavia eli homeostaattisia plastisuusmekanismeja, jotka muuttavat hermosolujen yhteyksiä niin, että yksittäisten synapsien vahvuuserot ja erojen sisältämä informaatio säilyvät. Sekä hebbiläiset että homeostaattiset plastisuusmekanismit ovat tärkeitä jo varhaiskehityksen aikana ensimmäisten hermosolujen yhteyksien muodostuessa. Aluksi synapsien muodostus on runsasta ja ensimmäisten yhteyksien muodostumista seuraa niiden testaus ja hienosäätö, jonka aikana tarpeelliset synapsit vahvistuvat ja tarpeettomat poistetaan. Tätä hienosäätöä ja siihen tarvittavia plastisuusmekanismeja ohjaa hermosolujen sähköinen aktiivisuus. Kaikkien nisäkäsaivojen varhaiskehitykselle on ominaista spontaani eli sisäsyntyinen sähköinen aktiivisuus. Tälle aktiivisuudelle on tyypillistä hermosolujen samanaikaisen (synkronisen) aktiivisuuden muodostamat hermoverkkoryöpyt, joilla on uskottu olevan tärkeä rooli synapsiyhteyksien hienosäädössä ja aivojen kehitykselle sopivan sähköisen aktiivisuustason ylläpidossa. Synaptiseen plastisuuteen liittyvien solutason mekanismien sekä hermoverkkoryöppyjen merkitys synaptisten kontaktien synnyssä varhaiskehityksen aikana on kuitenkin ollut tähän asti epäselvää. Tärkein aivojen viestien välitystä edistävä synapsissa vaikuttava välittäjäaine on glutamaatti. Tässä väitöskirjatyössä on ensimmäistä kertaa osoitettu, että sisäsyntyiset hermoverkkoryöpyt ohjaavat aivojen viestinvälitystä edistävien glutamaattivälitteisten synapsien kehitystä hippokampuksessa. Ilman synkronista aktiivisuutta glutamaattivälitteinen aktiivisuus heikkenee ja toimimattomien ns. hiljaisten synapsien määrä kasvaa. Glutamaatin vapautumisen aikaansaama viestinvälitys synapsissa perustuu sen vastaanottajasolun synapsin solukalvolla sijaitsevien reseptorimolekyylien aktivaatioon. Väitöskirjassa osoitettiin myös, että synapsien kehityksen solutason mekanismit riippuvat tietyn glutamaattireseptorin, 1-amino-3-hydroksi-5-metyyli-iso-oksatsoli-4-propionaatti (AMPA)-reseptorin, alayksikön, GluA4, ilmentymisestä. Tämän alayksikön ilmentyminen hippokampuksessa katoaa samaan aikaan sisäsyntyisen aktiivisuuden kanssa ja se korvataan muilla alayksiköillä aikuisissa aivoissa. GluA4:n ohimenevän ilmentymisen fysiologista merkitystä ei ole aikaisemmin tiedetty. Nykykäsityksen mukaan alttius monille hermostoperäisille sairauksille saattaa juontaa juurensa jo keskushermoston varhaiskehityksen aikaisista häiriöistä. Tässä väitöskirjassa tutkittua sisäsyntyistä spontaania aktiivisuutta havaitaan ihmissikiöillä viimeisen raskauskolmanneksen aikana. Tulosten perusteella voidaan olettaa, että jo pienet häiriöt aivojen spontaanissa aktiivisuudessa voivat aiheuttaa merkittäviä muutoksia hermosolujen synapsiyhteyksien muodostumisessa. Häiriö voi olla esimerkiksi alkoholin tai lääkeaineiden aiheuttama. Väitöskirjassa löydetyt synapsiyhteyksien kehitysmekanismit ja niiden muutokset auttavat ymmärtämään tiettyjen kehitysperäisten keskushermostosairauksien syntymekanismeja

A REST/NRSF-dependent transcriptional remodeling governs GABAergic synaptic upscaling induced by chronic hyperactivity

REST/NRFS (RE1-silencing transcription factor) has been initially identified as a negative transcription factor. Its target genes encode postsynaptic receptors, ion channels and transporters, neuropeptides and synaptic proteins. Evidences show that in mature neurons, REST can be upregulated by neuronal hyperactivity and works as a master epigenetic modulator, acting mostly as transcriptional repressor and, occasionally, as a transcriptional activator (Perera et al., 2015; Kallunki et al., 1998). We have previously demonstrated that REST is critical for the downscaling of intrinsic excitability in excitatory neurons subjected to prolonged elevation of electrical activity (Pozzi et al., 2013) and that it participates to the synaptic homeostasis of glutamatergic synapses by reducing their strength at the presynaptic level (Pecoraro-Bisogni et al., 2017). The aim of our work is to verify if REST plays a role in the synaptic homeostasis of GABAergic transmission evoked by hyperexcitability. Here we show that neuronal hyperactivity, obtained by treating for two days primary hippocampal neurons with 4-aminopyridine (4AP), induces a REST-dependent potentiation of the strength and number of somatic GABAergic synapses onto excitatory neurons, while the effect was lacking when the postsynaptic target cell was another inhibitory neuron. Our data suggest that the postsynaptic target specificity depends on a REST-dependent induction of a downstream transcription factor, NPAS4, known for its role in the development of inhibitory synapses, thanks to its capability of activating BDNF release from excitatory neurons upon hyperactivity (Lin et al. 2008). BDNF is synthetized only by excitatory neurons (Hofer et al., 1990). Thus, the retrograde action of BDNF, released from the soma of excitatory neurons onto the somatic GABAergic presynaptic contacts, could explain the observed postsynaptic target specificity of REST-action

Rôle de deux groupes de vésicules dans la transmission synaptique

Les synapses formées par les fibres moussues (FM) sur les cellules principales de la région CA3 (FM-CA3) jouent un rôle crucial pour la formation de la mémoire spatiale dans l’hippocampe. Une caractéristique des FM est la grande quantité de zinc localisée avec le glutamate dans les vésicules synaptiques recyclées par la voie d’endocytose dépendante de l’AP3. En combinant l’imagerie calcique et l’électrophysiologie, nous avons étudié le rôle des vésicules contenant le zinc dans la neurotransmission aux synapses FM-CA3. Contrairement aux études précédentes, nous n’avons pas observé de rôle pour le zinc dans l’induction des vagues calciques. Nos expériences ont révélé que les vagues calciques sont dépendantes de l’activation des récepteurs métabotropiques et ionotropiques du glutamate. D’autre part, nos données indiquent que les vésicules dérivées de la voie dépendante de l’AP3 forment un groupe de vésicules possédant des propriétés spécifiques. Elles contribuent principalement au relâchement asynchrone du glutamate. Ainsi, les cellules principales du CA3 de souris n’exprimant pas la protéine AP3 avaient une probabilité inférieure de décharge et une réduction de la synchronie des potentiels d’action lors de la stimulation à fréquences physiologiques. Cette diminution de la synchronie n’était pas associée avec un changement des paramètres quantiques ou de la taille des groupes de vésicules. Ces résultats supportent l’hypothèse que deux groupes de vésicules sont présents dans le même bouton synaptique. Le premier groupe est composé de vésicules recyclées par la voie d’endocytose utilisant la clathrine et participe au relâchement synchrone du glutamate. Le second groupe est constitué de vésicules ayant été recyclées par la voie d’endocytose dépendante de l’AP3 et contribue au relâchement asynchrone du glutamate. Ces deux groupes de vésicules sont nécessaires pour l’encodage de l’information et pourraient être importants pour la formation de la mémoire. Ainsi, les décharges de courte durée à haute fréquence observées lorsque les animaux pénètrent dans les places fields pourraient causer le relâchement asynchrone de glutamate. Finalement, les résultats de mon projet de doctorat valident l’existence et l’importance de deux groupes de vésicules dans les MF qui sont recyclées par des voies d’endocytoses distinctes et relâchées durant différents types d’activités.Mossy fiber-CA3 pyramidal cell synapses play a crucial role in the hippocampal formation of spatial memories. These synaptic connections possess a number of unique features substantial for its role in the information processing and coding. One of these features is presence of zinc co-localized with glutamate within a subpopulation of synaptic vesicles recycling through AP3-dependent bulk endocytosis. Using Ca2+ imaging and electrophysiological recordings we investigated role of these zinc containing vesicles in the neurotransmission. In contrast to previous reports, we did not observe any significant role of vesicular zinc in the induction of large postsynaptic Ca2+ waves triggered by burst stimulation. Moreover, our experiments revealed that Ca2+ waves mediated by Ca2+ release from internal stores are dependent not only on the activation of metabotropic, but also ionotropic glutamate receptors. Nevertheless, subsequent experiments unveiled that the vesicles derived via AP3-dependent endocytosis primary contribute to the asynchronous, but not synchronous mode of glutamate release. Futhermore, knockout mice lacking adaptor protein AP3 had a reduced synchronization of postsynaptic action potentials and impaired information transfer; this was not associated with any changes in the synchronous release quantal parameters and vesicle pool size. These findings strongly support the idea that within a single presynaptic bouton two heterogeneous pools of releasable vesicles are present. One pool of readily releasable vesicles forms via clathrin mediated endocytosis and mainly participates in the synchronous release; a second pool forms through bulk endocytosis and primarily supplies asynchronous release. The existence of two specialized pools is essential for the information coding and transfer within hippocampus. It also might be important for hippocampal memory formation. In contrast to low firing rates at rest, dentate gyrus granule cells tend to fire high frequency bursts once an animal enters a place field. These burst activities, embedded in the lower gamma frequency, should be especially efficient in the triggering of substantial asynchronous glutamate release. Therefore, the results of my PhD project for the first time provide strong evidence for the presence and physiological importance of two vesicle pools with heterogeneous release and recycling properties via separate endocytic pathways within the same mossy fiber bouton

Mesoscale Systems, Finite Size Effects, and Balanced Neural Networks

Cortical populations are typically in an asynchronous state, sporadically interrupted by brief epochs of coordinated population activity. Current cortical models are at a loss to explain this combination of states. At one extreme are network models where recurrent in- hibition dynamically stabilizes an asynchronous low activity state. While these networks are widely used they cannot produce the coherent population-wide activity that is reported in a variety of datasets. At the other extreme are models where short term synaptic depression between excitatory neurons can generate the epochs of population-wide activity. However, in these networks inhibition plays only a perfunctory role in network stability, which is at odds with many reports across cortex. In this study we analyze spontaneously active in vitro preparations of primary auditory cortex that show dynamics that are emblematic of this mix- ture of states. To capture this complex population activity we consider models where large excitation is balanced by recurrent inhibition yet we include short term synaptic depression dynamics of the excitatory connections. This model gives very rich nonlinear behavior that mimics the core features of the in vitro data, including the possibility of low frequency (2- 12 Hz) rhythmic dynamics within population events. Our study extends balanced network models to account for nonlinear, population-wide correlated activity, thereby providing a critical step in a mechanistic theory of realistic cortical activity. We further investigate an extension of this model that l exhibits clearly non-Arrhenius behavior, whereby lower noise systems may exhibit faster escape from a stable state. We show that this behavior is due to the system size dependent vector field, intrinsically linking noise and dynamics

Effect of GABAA Receptor Clustering on Phasic and Tonic Inhibition in the Hippocampus

Inhibitory transmission plays a major role in information processing in the brain since it integrates excitatory signals and defines the gain between neural input and output. \u3b3-Amino butyric acid (GABA) is the main inhibitory neurotransmitter in the adult mammalian brain. By activating GABAA and GABAB receptors this neurotransmitter inhibits neuronal firing and stabilizes the membrane potential near the resting value. In particular GABAA receptors are permeable to chloride ions and are responsible for phasic and tonic hyperpolarizing responses. GABA-mediated currents are the result of rapid, sequential events including transmitter release from the presynaptic terminal, transmitter diffusion within and outside the cleft and post-synaptic receptors gating. The kinetics of each of these processes is crucial in determining the shape of post-synaptic currents. Therefore the modulation of any of these events leads to the heterogeneity of GABAergic responses and to changes in the potency of inhibition. In this thesis I have studied the sources of such variability at presynaptic/cleft and postsynaptic level. At presynaptic/cleft level I have focused on the influence of the agonist concentration profile in the synaptic cleft on GABA-mediated synaptic currents. Fast-off competitive antagonists and computer simulations allowed estimating the range of variability of the peak concentration and the speed of GABA clearance form the synaptic cleft. At postsynaptic level particular attention has been attributed to the impact of GABAA receptors clustering on both phasic and tonic GABAA-mediated inhibition. With ultrafast applications of GABA and computer simulations it was possible to describe the modulation of GABAA receptor gating induced by clustering

Trans-synaptic signaling at GABAergic connections: possible dysfunction in some forms of Autism Spectrum Disorders

Synapses are recognized as being highly plastic in structure and function, strongly influenced by their own histories of impulse traffic and by signals from nearby cells. Synaptic contacts are fundamental for the development, homeostasis and remodeling of complex neural circuits. Synapses are highly varied in their molecular composition. Understand this diversity is important because it sheds light on the way they function. In particular, this may be useful for understanding the mechanisms at the basis of synaptic dysfunctions associated with neurodevelopmental disorders, such as Autism Spectrum Disorders (ASD) in order to develop properly targeted therapeutic tools. During the first part of my Phd course I characterized the functional role of gephyrin at inhibitory synapses (paper N. 1). Gephyrin is a scaffold protein essential for stabilizing glycine and GABAA receptors at inhibitory synapses. Using recombinant intrabodies against gephyrin (scFv-gephyrin) I tested the hypothesis that this protein exerts a trans-synaptic action on GABA and glutamate release. Pair recordings from interconnected hippocampal cells in culture revealed a reduced probability of GABA release in scFv-gephyrintransfected neurons compared with controls. This effect was associated with a significant decrease in VGAT, the vesicular GABA transporter, and in neuroligin 2 (NL2), a protein that, interacting with the neurexins, ensures the cross-talk between the post- and presynaptic sites. I also found that, hampering gephyrin function produced a significant reduction in VGLUT, the vesicular glutamate transporter, an effect accompanied by a significant decrease in frequency of miniature excitatory postsynaptic currents. Over-expressing NLG2 in gephyrindeprived neurons rescued GABAergic but not glutamatergic innervation, suggesting that the observed changes in the latter were not due to a homeostatic compensatory mechanism. These results suggest a key role of gephyrin in regulating trans-synaptic signaling at both inhibitory and excitatory synapses. Several lines of evidence suggest that proteins involved in synaptic function are altered in ASDs. In particular, in a small percentage of cases, ASDs have been found to be associated with single mutations in genes encoding for cell adhesion molecules of the neuroligin-neurexin families. One of these involves the postsynaptic cell adhesion molecule neuroligin (NL) 3. In the second part of my PhD, I used transgenic mice carrying the human R451C mutation of Nlgn3, to study GABAergic and glutamatergic signaling in the hippocampus early in postnatal life (paper N. 2). I performed whole cell recordings from CA3 pyramidal neurons in hippocampal slices from NL3 R451C knock-in mice and I found an enhanced frequency of Giant Depolarizing Potentials, as compared to controls. This effect was probably dependent on an increased GABAergic drive to principal cells as demonstrated by the enhanced frequency of miniature GABAAmediated (GPSCs) postsynaptic currents, but not AMPA-mediated postsynaptic currents (EPSCs). The increase in frequency of mGPSCs suggest a presynaptic 9 type of action. This was further supported by the experiments with the fast-off GABAA receptor antagonist TPMPA that, as expected for an enhanced GABA transient in the cleft, showed a reduced blocking effect on miniature events. Although an increased number of available postsynaptic GABAA receptors, if these are not saturated by the content of a single GABA containing vesicle may account for these results, this was not the case since a similar number of receptor channels was revealed with peak-scaled non-stationary fluctuation analysis in both WT and NL3R451C knock-in mice, indicating that the observed effects were not postsynaptic in origin. Presynaptic changes in GABA release can be attributed to modifications in the probability of GABA release, in the number of release sites or in the content of GABA in single synaptic vesicles. Changes in probability of GABA release seem unlikely considering that we examined miniature events generated by the release of a single quantum. Our data do not allow distinguishing between the other two possibilities (changes in the number of release sites or in vesicle GABA content). However, in agreement with previous data from S\ufcdhof group showing an enhancement of the presynaptic GABAergic marker VGAT (but not VGlut1) in the hippocampus of NL3R451C KI mice (Tabuchi et al., 2007), it is likely that an increased GABAergic innervation may contribute to the enhancement of GABA release. In additional experiments I found that changes in frequency of miniature GABAergic events were associated with an acceleration of mGPSCs decay possibly of postsynaptic origin. The increased frequency of mEPSCs detected in adult, but not young NL3 R451C mice may represent a late form of compensatory homeostatic correction to counter the excessive GABAA-mediated inhibition. Therefore, it is reasonable to assume that alterations in the excitatory/inhibitory balance, crucial for the refinement of neuronal circuits early in postnatal development, accounts for the behavioral deficits observed in ASDs patients. Although also in the present case, a modification of gephyrin expression in R451C NL 3 knock-in mice was associated with changes in GABAergic innervations suggesting the involvement of a trans-synaptic signal, the role of NL3 mutation in this effect remains to be elucidated. Finally, I contribute in writing a review article (paper N. 3) that gives an up dated picture of alterations of GABAergic signaling present in different forms of Autism Spectrum Disorders