Дисертації з теми "GABAergic/glycinergic synaptic transmission"

La sclérose latérale amyotrophique (SLA) est une maladie neurodégénérative fatale de l’adulte caractérisée par la dégénérescence des motoneurones (MNs) et ayant une étiologie multifactorielle. La plupart des études sur la SLA se sont focalisées aux stades symptomatiques selon l’hypothèse que la pathogénicité apparaît lorsque la maladie devient symptomatique. Cependant, un nombre grandissant d’évidences indique que la pathogénicité se développerait bien avant les symptômes. Mon travail de thèse de Doctorat a été basé sur l’hypothèse selon laquelle la SLA – familiale et sporadique – découlerait de déficits présents dès le développement précoce. La première partie de ma thèse a consisté à analyser les courants post-synaptiques GABA/glycine (IPSCs) au niveau des MNs embryonnaire (E) E17,5, localisés dans la colonne motrice ventro-laterale, chez la souris SOD1G93A (SOD) modèle de la SLA, en parallèle à l’analyse de l’homéostasie chlorure. Nos résultats ont montré que les IPSCs sont moins fréquents chez les animaux SOD en accord avec une réduction des terminaisons synaptiques VIAAT autour des MNs. Les MNs SODs avaient un ECI 10 mV plus positif que les MNs sauvages (WT) de la même portée. Ce déficit était lié à une réduction du co-transporteur chlorure KCC2. Les IPSCs évoqués et spontanés présentaient une relaxation plus longue chez les MNs SOD, en corrélation à une [Cl-]i plus élevée. La modélisation a montré que cet excès de relaxation permettait de compenser la moindre efficacité de l’inhibition GABA/glycine liée au ECI dépolarisé. De manière intéressante, les simulations ont révélé la nature excitatrice des potentiels dépolarisants post-synaptiques GABA/glycine (dGPSPs) survenant à basse fréquence (<50Hz) sur les MNs SOD mais pas sur les MNs WT. A plus haute fréquence, les dGPSPs basculaient vers une inhibition du MN liée à une sommation de composantes « shuntantes ». La seconde partie de ma thèse a donc focalisé sur les effets de dGPSPs évoqués électriquement at différentes fréquences (7,5 - 100 Hz) sur de vrais MNs E17,5 au niveau desquels un ECl dépolarisant (sous le seuil du PA) était imposé. Le but était d’examiner si l’effet excitateur pouvait être lié aux changements morphologiques des MNs E17,5 décrits précédemment. Les résultats ont montré que certains MNs étaient bien excités par les dGPSPs basse fréquence et inhibés à plus forte fréquence (MNs bi-effet) alors que d’autres MNs étaient inhibés quelles que soient les fréquences (MNs inhibés). L’effet double était plus souvent détecté au niveau des MNs SOD. Les MNs WT ont été classés en deux groupes en fonction de leur résistance d’entrée (Rin), les MNs bi-effet ayant une Rin élevée et les MNs inhibés une Rin basse. Les données morphométriques ont mis en avant un arbre dendritique réduit pour les MNs WT bi-effet (Rin élevée) et un arbre dendritique étendu pour les MNs inhibés (Rin basse). Ce n’était pas le cas des MNs SOD excités ou inhibés indépendamment de leur morphologie. En accord avec les simulations montrant qu’une baisse de la densité des courants inhibiteurs sur le soma du MN favorise l’excitation des dGPSPs, nous avons trouvé moins de terminaisons synaptiques VIAAT sur le soma et dendrites proximales des MNs SOD, et une fréquence réduite des dGPSPs spontanés. Dans leur ensemble, les données de ma thèse soulignent une altération précoce de l’homéostasie chlorure et de l’innervation GABA/glycine des MNs SOD1G93A. Avant la naissance, une population dominante de MNs avec Rin basse émerge chez les animaux WT. Ces MNs qui sont inhibés par les dGPSPs pourraient correspondre aux futures MNs vulnérables (rapides, FF). Ces MNs ne sont pas inhibés chez les animaux SOD. Le dysfonctionnement de l’inhibition pourrait être attribué à deux facteurs distincts : la morphologie et la densité des synapses inhibitrices péri-somatiques. Parmi ces facteurs, le deuxième joue un rôle majeur en contrôlant la capacité des neurones GABA/glycine à façonner la sortie motrice spinale
Amyotrophic lateral sclerosis (ALS) is a fatal and adult-onset neurodegenerative disease characterized by a progressive degeneration of motoneurons (MNs) with complex multifactorial aetiology. Most ALS studies have focused on symptomatic stages based on the hypothesis that ALS pathogenesis occurs when the disease becomes symptomatic. However, growing evidence indicates that ALS pathogenesis might start long before symptom onset. My PhD thesis work was based on the hypothesis that ALS - familial and sporadic - stems from deficits taking place during early development. With the aim of identifying early changes underpinning ALS neurodegeneration, the first part of my thesis analysed the GABAergic/glycinergic inhibitory postsynaptic currents (IPSCs) to embryonic (E) E17.5 MNs located in the ventro-lateral motor column from SOD1G93A (SOD) mice, in parallel with the analyse of chloride homeostasis. Our results showed that IPSCs are less frequent in SOD animals in accordance with a reduction of synaptic VIAAT-positive terminals in the close proximity of MN somata. SOD MNs exhibited an ECI 10 mV more depolarized than wild type (WT) MNs. This deficit in GABA/glycine inhibition was due to a reduction of the neuronal chloride transporter KCC2. SOD spontaneous IPSCs and evoked GABAAR-currents exhibited a slower decay correlated to elevated [Cl-]i. Using computer modelling approach, we revealed that the slower relaxation of synaptic inhibitory events acts as a compensatory mechanism to strengthen or increase the efficacy of GABA/glycine inhibition when ECI is more depolarized. Interestingly, simulations revealed an excitatory effect of low frequency (<50Hz) depolarizing GABA/glycine post-synaptic potentials (dGPSPs) in SOD-like MNs but not in WT-like littermates. At high frequency, dGPSPs switched to inhibitory effect resulting from the summation of the shunting components. The second part of my PhD thesis focussed on the effect of electrically evoked-dGPSPs, at different frequencies (7.5 to 100 Hz), on real lumbar E17.5 MNs in which a depolarized ECI (below spike threshold) was imposed. The aim was to examine whether the excitatory effect could be linked to morphological changes previously described in E17.5 SOD MNs. Results showed that some MNs were excited by low frequency dGPSPs and inhibited by high frequency dGPSPs (Dual MNs) and others were inhibited at all frequencies (Inhibited MNs). Dual effect was more often detected in SOD MNs. WT MNs were classified into two clusters according to their input resistance (Rin), Dual MNs being specific to high Rin and Inhibited MNs to low Rin. Morphometric data pointed out a reduced dendritic tree in high Rin WT Dual MNs and a large dendritic tree in low Rin Inhibited MNs. This was not the case in SOD MNs that were excited or inhibited whatever their morphology and Rin. In agreement with simulation showing that a less density of inhibitory current on MNs soma favours excitatory dGPSPs, we found less synaptic VIAAT terminals on the soma and proximal dendrites of SOD MNs, compared to littermate WT MNs, as well as a lower frequency of spontaneous dGPSPs. Altogether, my thesis data emphasize a prenatal defect in the CI- homeostasis and GABA/glycine innervation in the SOD1G93A ALS MNs. Before birth, a dominant population of MNs with low Rin emerges in WT animals. These MNs that are inhibited by dGPSPs could represent future ALS vulnerable fast MNs (putative FF). Interestingly, those MNs are not inhibited in SOD animals. The inhibitory dysfunction could be attributed to two distinct factors: morphology and perisomatic inhibitory synapse density. Of these two factors, the latter plays a major role by controlling capability of GABAergic/glycinergic neurons for shaping spinal motor output

The correct functioning of the central nervous system relies on the rapid and efficient communication between neurons. This occurs at highly specialized functional points of contact called synapses. Synapses are extremely plastic in structure and function, strongly influenced by their own histories of impulse traffic and by signals from nearby cells. Synaptic contacts are fundamental to the development, homeostasis and remodeling of neural circuits. All these events are achieved through different mechanisms operating at both pre- and postsynaptic sites. At the level of the post synaptic density (PSD) compartment, scaffolding molecules and trans-membrane proteins are known to orchestrate proper synapses formation, maturation and rearrangement required to sustain plasticity processes. Protein phosphorylation represents one of the most important mechanisms engaged in affecting the molecular composition of the post-synaptic device. Most studies have focused on the impact of phosphorylation on the gating properties, surface mobility and trafficking of neurotransmitter receptors while much less is known about the effect of post-translational modifications on scaffolding and cell adhesion molecules functionally linked to neurotransmitter receptors. At GABAergic synapses specific phosphorylation events of the scaffolding molecule gephyrin were shown to alter its multimerization properties, thus producing parallel changes in the number of receptors trapped by the scaffold leading to alterations of synaptic strength. Most of these phosphorylation events occur at serine or threonine residues preceding a proline, underlying a potential role of proline-directed phosphorylation as modulator of synaptic strength. The key player of such signalling cascade is represented by a small enzyme called peptidyl-prolyl isomerase Pin1 (protein interacting with NIMA 1). Pin1, upon recruitment by its substrates in a phosphorylation-dependent manner, catalyzes the cis/tran isomerization of phospho‐Ser/Thr‐Pro motifs leading to changes in target protein conformation and biological activity. Pin1 is highly expressed in neurons suggesting that it can exert a crucial role in synaptic transmission and plasticity processes at both inhibitory and excitatory synapses. In the first part of my PhD thesis I focused on the impact of Pin1-dependent signalling on GABAergic transmission. I found that the cell adhesion molecule of the neuroligin family enriched at GABAergic synapses, Neuroligin 2 (NL2), undergoes post-phosphorylation prolyl-isomerization modulation of its activity. Using biochemical approaches I found that the unique Pin1 consensus motif present within the cytoplasmic tail of NL2, Serine 714-proline, is indeed phosphorylated in vivo. Proline-directed phosphorylation at Serine 714 of NL2 strongly impacts on NL2 ability to complex with gephyrin. In particular, at this site, post-phosphorylation prolyl-isomerization negatively regulates the ability of NL2 to interact with gephyrin. In line with biochemical results, immunocytochemical analysis reveal that, in the absence of Pin1 expression, NL2/gephyrin complexes are enriched at GABAergic post-synaptic sites and this enrichment is accompanied by an enhanced synaptic recruitment of GABAA receptors (GABAAR). This effect was associated with a concomitant increase in the amplitude, but not in frequency, of spontaneous inhibitory post-synaptic currents (IPSCs). These findings unveil the existence of a new signalling pathway operating at inhibitory GABAergic synapses able to alter the efficacy of GABAergic transmission by modulating NL2/gephyrin interaction. Given the high abundance of Pin1 at excitatory synaptic contacts, in the second part of my PhD thesis I focused on the impact of Pin1-dependent signalling on excitatory glutamatergic transmission. In particular, I started to investigate whether the scaffolding molecule PSD-95, a member of the Disc-Large (DGL)-Membrane-associated guanylate kinase, could be a target of Pin1-dependent signalling cascade. I observed that Pin1, known to reside in post-synaptic structures, is recruited by PSD-95 at specific Serine-Threonine/Proline consensus motifs localized in the linker region connecting PDZ2 to PDZ3 domains. These sites are represented by Treonine287-Proline, Serine290-Proline and Serine295-Proline, and deletion of all of them almost completely abolished Pin1 interaction with PSD-95. Pin1 exerts a negative control on PSD-95 ability to complex with N-Methyl-D-Aspartate receptors (NMDARs). Indeed an enhanced PSD-95/NMDAR complex formation was detected in brain extracts derived from Pin1-/- mice. In electrophysiological experiments, larger NMDA-mediated synaptic currents were detected in CA1 principal cells in hippocampal slices obtained from Pin1-/- mice as compared to controls, an effect that was associated with an enhancement in spine density and size. These data indicate that Pin1 controls the synaptic content of NMDARs via PSD-95 prolyl-isomerization and the expression of dendritic spines, both required for the maintenance of long-term potentiation. Overall, this study highlights the crucial role of Pin1-dependent signalling in the functional organization of both inhibitory and excitatory synapses.

Medium Spiny projection neurons, the sole output neurons of the striatum, are connected by GABAergic synapses. Such connections are classically assumed to be inhibitory, thus it has been suggested that one of the main functions of the striatum is to detect and classify cortical representations of sensory events to trigger appropriate motor responses through a winner-take-all network dynamic.

Les interneurones GABAergiques du néocortex, englobent un grand nombre de types cellulaires: certains innervent la région périsomatique des neurones pyramidaux (NP), d'autres ciblent leurs dendrites. Ici, nous avons étudié la sous-unité alpha5 du récepteur GABAA(GABAAR), qui contribuerait significativement à l’inhibition tonique. Nous avons constaté que, dans les NP de la couche 2/3 du cortex somatosensoriel chez la souris, alpha5 a une contribution négligeable à l'inhibition tonique. Inversement nous avons montré qu’alpha5 est spécifiquement exprimée aux synapses dendritiques entre les cellules de Martinotti (MC) et les NP, indiquant l’importance des alpha5-GABAARs dans l’inhibition dendritique synaptique. Nous avons aussi montré qu’alpha5 est exclusivement exprimé aux synapses MC-NP, en proposant les alpha5-GABAARs comme signature moléculaire spécifique de ces synapses dendritiques. En plus, des nombreuses maladies du cerveau sont le résultat du dysfonctionnement de circuits inhibiteurs distincts: par exemple, il a été montré que le traitement avec un agoniste inverse spécifique du récepteur alpha5-GABAA(alpha5IA) a permis la récupération des déficits cognitifs chez des modèles animaux de trisomie 21(DS) mais les mécanismes de cette récupération cognitive sont inconnus au niveau du circuit. Nos résultats préliminaires indiquent que les synapses GABAergiques dendritiques formées par les MCs sont spécifiquement modifiées chez les souris DS. Nous définirons si cette modification est spécifique à un circuit particulier. Nos expériences visent à comprendre les altérations spécifiques des circuits de la DS afin d’ouvrir de nouvelles pistes thérapeutiques
Neocortical GABAergic interneurons encompass a vast number of cell types: some innervate the perisomatic region of cortical pyramidal neurons (PNs), whereas others target PN dendrites. Here we studied the alpha5 subunit of the GABAAR, which is believed to contribute significantly to tonic inhibition. We found that, in L 2/3 PNs of mouse somatosensory cortex, alpha5 provides a negligible contribution to tonic inhibition. Conversely, we found that alpha5 is specifically expressed at synapses between the dendrite-targeting interneurons Martinotti cells (MCs) thus indicating that GABAergic transmission through 5-GABAAR subtypes is important for synaptic dendritic inhibition. We also show that the expression of alpha5 is always present only at synapses made by MCs onto PNs. These results suggest alpha5-GABAARs as a molecular signature of specific inhibitory dendritic synapses involving MCs. Importantly, many brain diseases originate from dysfunctions of distinct inhibitory circuits and, in particular, alpha5-KO mice show improved learning: it was shown that the treatment with a highly specific alpha5 inverse agonist rescued learning and memory deficits in Ts65Dn mice, an animal model for Down syndrome (DS). Yet, the actual mechanisms underlying this cognitive rescue at the synaptic and circuit levels are unknown. Our preliminary results indicate that GABAergic synapses from dendrite-targeting MCs are specifically altered in DS. We are defining whether this alteration is circuit-specific. Our results will provide a better understanding of specific circuit alterations in DS, and will likely open new therapeutic avenues to alleviate cognitive impairment of this disease

Les glucocorticoïdes (GC) sont des hormones stéroïdes synthétisées par les glandes surrénales. La production de ces GC est une des réponses de l’organisme pour rétablir l’homéostasie grâce à différentes actions comprenant des effets centraux sur le comportement et la douleur. C’est ce dernier qui a fait l’objet de mes travaux dans le cadre de cette thèse.Les afférences sensorielles primaires véhiculent les informations de la périphérie dans les cornes dorsales de la moelle épinière. Ces informations qui peuvent être nociceptives sont modulées par un réseau de neurones spinal avant d’être transmises et intégrées. Nous avons montré que les GC sont impliqués dans la maturation et le fonctionnement de la transmission inhibitrice faisant intervenir le neurotransmetteur GABA. Dans les cornes dorsales, cette inhibition est cruciale pour limiter les mécanismes de transmission de l’information nociceptive
Glucocorticoids (GC) are steroid hormones synthesized in adrenals following HPA axis activation. GC production is a response of the organism to alleviate homeostasis perturbations through different actions. One of them involves central neuronal modulation of behavior and pain perception.Primary afferents convey peripheral sensory information in the dorsal horns of the spinal cord. This information can be nociceptive and are modulated by a spinal neuronal network before being transmitted and integrated. We showed that GC are implied in the maturation and functioning of the inhibitory transmission involving GABA neurotransmitter. In the dorsal horns this inhibitory transmission is of major importance, limiting the processing of nociceptive information

Tese de mestrado em Bioquímica, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2012
A transmissão inibitória desempenha um papel importante na regulação e estabilização da actividade neuronal e é essencial para diversas funções cerebrais como a cognição, percepção, movimento e emoção. As sinapses inibitórias, GABAérgica e glicinérgica, e a sua distribuição, apresentam diferenças no sistema nervoso central dos mamíferos (CNS). A maioria das sinapses inibitórias no cérebro são GABAérgicas, e as glicinérgicas, predominantes na espinal medula e tronco cerebral, tem sido bastante negligenciadas no cérebro. A glicina exerce a sua função através do receptor ionotrópico da glicina (GlyR), um canal pentamérico composto por dois tipos de subunidades (α e β) permeável a iões cloreto e localizado na membrana do terminal pós-sináptico. Os transportadores da glicina 2 (GlyT2) pertencem à família de transportadores dependentes de Na+/Cl-. Estão presentes na membrana dos terminais pré-sinápticos glicinérgicos, assegurando a remoção da glicina da fenda sináptica e permitindo a inserção do neurotransmissor em vesículas sinápticas. O presente estudo tem como principal objectivo investigar quais os principais intervenientes na aquisição do fenótipo glicinérgico. Para isso, efectuou-se uma abordagem farmacológica, em culturas primárias de neurónios, com o propósito de avaliar o fenótipo glicinérgico mediante o comprometimento da transmissão GABAérgica. Os resultados obtidos por western blot e por PCR quantitativo (qPCR) revelaram que a expressão de GlyR e de GlyT2 aumentava significativamente, após tratamento das células com antagonistas do receptor ionotrópico de GABA GABAA (GABAAR) ou do transportador de GABA GAT-1, gabazina e SKF89976a, respectivamente. Em sinaptossomas obtidos de cérebro, a dupla detecção por imunofluorescência, de GlyT2 (marcador de neurónios glicinérgicos) e GAD (marcador de neurónios GABAérgicos) revelou igualmente que, na presença de SKF89976a, a razão entre terminais GABAérgicos e glicinérgicos se apresentava alterada. O comprometimento do sistema GABAérgico resultou no aumento de terminais glicinérgicos puros e mistos, com a consequente diminuição de terminais GABAérgicos. Neste trabalho, a interacção entre o transportador vesicular de aminoácidos inibitórios (VIAAT) e o GlyT2 foi igualmente explorada por ensaios de imunoprecipitação. Os resultados obtidos nesta tese evidenciam, pela primeira vez, que o comprometimento da neurotransmissão GABAérgica induz um aumento dos marcadores da transmissão mediada pela glicina, nomeadamente GlyR e GlyT2, sugerindo assim um mecanismo de compensação entre os dois sistemas inibitórios no cérebro.
The inhibitory transmission plays an important role in the regulation and stabilization of brain network activity and is essential for a number of brain functions such as cognition, perception, movement and emotion. GABAergic and glycinergic inhibitory synapses, and their distribution, are very different in the mammalian central nervous system (CNS). Most inhibitory synapses in the brain are GABAergic, and glycinergic ones, predominant in the most caudal regions of the CNS, have been largely disregarded in the brain. Glycine exerts its action through glycine receptors (GlyR), which belong to the superfamily of ligand-gated ion channels, are localized in the postsynaptic membrane and form pentameric channels composed of two different subunits (α and β) permeable to chloride ions. Glycine transporters 2 (GlyT2) belong to the family of Na+/Cl--dependent transporter proteins. They are located in the membrane of glycinergic neurons and are responsible for terminating glycine-mediated neurotransmission by uptaking glycine into glycinergic nerve terminals, allowing for neurotransmitter reloading of synaptic vesicles. The present study aims to investigate which are the principal mediators for the acquisition of a glycinergic phenotype. A pharmacological approach, in primary neuronal cultures, was pursued in order to evaluate the glycinergic phenotype upon a GABAergic transmission impairment. Western blot analysis and quantitative real-time PCR (qPCR) revealed that GlyR and GlyT2 expression increased significantly after treating the cultures with blockers for either GABAA receptor or GABA transporter GAT-1, gabazine and SKF89976a, respectively. In brain synaptosomes, double immunofluorescence of GlyT2 (marker of glycinergic neurons) and GAD (marker of GABAergic neurons) also revealed that, in the presence of SKF89976a, the ratio of GABAergic vs glycinergic terminals changed. GABAergic impairment caused an increase in mixed (GABA and glycine-containing) and pure glycinergic terminals, with a concomitant decrease in GABA-containing boutons. Furthermore, a physical interaction was assessed between Vesicular Inhibitory Amino Acid Transporter (VIAAT) and GlyT2 by immunoprecipitation assays. These results obtained in this thesis have elucidated, for the first time, that impairment in GABA-mediated neurotransmission induces an increase in glycine- mediated transmission components, namely GlyR and GlyT2, and suggest a compensatory mechanism between the two inhibitory systems in the brain.

In the cerebellum, the corticonuclear projection subserves as the major efferent pathway for the cerebellar cortical networks. This pathway, consists of a direct axonal projection from the Purkinje cells to the neurons of the deep cerebellar nuclei (DCN). It has been demonstrated both in vivo and in vitro that stimulation of the Purkinje cell axons exerts a powerful inhibitory influence on DCN neurons mediated by the neurotransmitter, ƴ-aminobutyric acid (GABA). However, despite the wealth of anatomical and biochemical information, few electrophysiological studies have been done to characterize GABAergic synaptic transmission in DCN neurons. For example, it is not clear whether synaptic release of GABA activates pre- or postsynaptic GABAB receptors despite the finding that GABAB binding sites are present in the DCN. Furthermore, although GABAergic transmission in the DCN exhibits paired-pulse, frequency-dependent, as well as long-term depressions, the mechanisms underlying these plasticity's are yet to be resolved. In the present study, both perforated and whole-cell patch clamp recording techniques were utilized to determine whether preand postsynaptic GABAB receptors are present in the DCN and to test if endogenous release of GABA can activate either of the receptors. In addition, the contribution of GABAB receptors to paired-pulse and frequency-dependent depression of the deep nuclear inhibitory postsynaptic current (IPSC) was also assessed. Finally, experiments were conducted to investigate the properties of a tetanic stimulation-induced deep nuclear long-term depression (LTD) of the IPSC and to examine the role of Ca^2+ and protein phosphatases as potential mediators of the sustained depression. The results of the studies indicated that postsynaptic GABAB receptors are present on the membrane of DCN neurons. Activation of these receptors produces a G-protein-dependent response similar to that observed in other central neurons. In addition, presynaptic GABAB receptors are also present in the DCN. Activation of these receptors produces a suppression of deep nuclear IPSCs. However, deep nuclear preand postsynaptic GABAB receptors were found not to be activated by endogenous release of GABA. Furthermore, these receptors appear not to be involved in pairedpulse and frequency-dependent depressions of the IPSC. In voltage-clamped DCN neurons, LTD of the IPSC was induced reliably if the LTD-inducing train was delivered under current-clamp conditions where the membrane potential was allowed to fluctuate. Using this protocol in subsequent experiments, it was found that currents elicited by iontophoretic applications of THIP, a GABAA agonist, also exhibited LTD following a tetanic stimulation of the input. It was also demonstrated that LTD can be induced heterosynaptically. Furthermore, activation of the IPSC during the train was not required for LTD to occur. However, postsynaptic Ca^2+ accumulations via influx though A/-methyl-D-aspartate receptor-gated channels and/or voltage-gated Ca^2+ channels appear to play an important role in the generation of LTD. Moreover, protein phosphatase activity appears to be necessary for the induction of the depression. It is concluded that postsynaptic mechanisms contribute to LTD of GABAergic transmission in neurons of the DCN. Bhagavatula R. Sastry, Ph.D., Research Supervisor.

Neuronal communication in the mammalian brain relies on the presynaptic release of neurotransmitters which bind to ligand-gated ion channels found on postsynaptic neurons to modulate neuronal excitability. One such neurotransmitter is acetylcholine (ACh), a small molecule that is the signalling messenger of the cholinergic system. The cholinergic system is involved in a variety of behavioural functions including motor activity, sensory function, and higher executive commands. Dopaminergic neurons in the substantia nigra pars compacta (SNc) and the basal ganglia in general have long been implicated in initiation and completion of voluntary movement. Studies have shown that cholinergic neurons from two brainstem nuclei, the laterodorsal tegmental nucleus and the pedunculopontine nucleus, project onto substantia nigra dopaminergic (DA) neurons in the midbrain and release ACh, GABA or both to modulate motor behaviours. However, with prior research primarily focused on demonstrating the phenomenon of co-transmission itself, the subcellular distribution and dynamics of ACh and GABA release onto SN DA neurons receiving co-transmitted inputs largely remains to be investigated. The present study investigates the spatial and physiological properties of ACh/GABA co- transmission from brainstem cholinergic axons synapsing onto medial SN DA neurons to understand its role in tuning the neuron’s excitatory-inhibitory balance. To that end, we developed a channelrhodopsin (ChR2)-based functional input mapping technique with high spatial resolution to probe the dendritic distribution of ACh and GABA synaptic inputs onto DA neurons in ChATcre::ChR2 mice. Using this technique, we discovered three different types of monosynaptic inputs from cholinergic axons onto DA cells: co-transmitted ACh/GABA, GABA only, and ACh only. Furthermore, we revealed a somatodendritic patterning of cholinergic input distribution onto DA cells with a predominant GABA conductance along the lateral dendrites and a soma-centered mix ACh/GABA transmission. Physiological findings were corroborated using immunolabeling against VGAT and VAChT, which showed many closely spatially clustered ACh and GABA- specific cholinergic terminals and few truly colocalized VAChT and VGAT terminals. This result revealed that true co-transmission represents a minority of the presynaptic mode of release from cholinergic axons onto medial SN DA neurons, and that the majority actually share closely spatially clustered ACh and GABA-specific cholinergic terminals. To investigate the dynamic properties of soma-centered ACh/GABA transmission, we restricted our stimulation field to the cell body to measure the contribution of nAChR and GABAR-mediated conductances without recruiting the lateralized population of primary GABA inputs. We then employed a deconvolution method to understand the relative plasticity of contributions of nAChRs and GABARs to ACh/GABA transmission onto DA cells. We confirmed an initial dominant GABAergic component of ACh/GABA transmission that was previously reported. However, we found that the GABAergic contribution had a greater decay compared to the ACh component with repeated stimulations. As such the predominant initial inhibition is followed by a subsequent equalization of excitatory and inhibitory conductances. Finally, we performed similar experiments to compare the short-term plasticity of the isolated GABA conductance during 15 Hz stimulation between the populations of mix ACh/GABA inputs proximally and the population of primary GABA inputs found on the lateral dendrites 160 μm from the cell body. Interestingly, the lateral GABA component was more sustained across repeated stimulations compared to the proximal GABA conductance, suggesting a differential contribution to excitation/inhibition balance by spatially distributed populations of ACh and GABA inputs from cholinergic axons onto the dendrites of medial SN DA neurons. To our knowledge, this is the first study to examine the distribution and dynamics of ACh/GABA transmission onto midbrain DA system using fine-scale ChR2-assisted subcellular input mapping and conductance deconvolution.
Graduate
2022-04-12

The number of newly detected autoantibodies (AB) targeting synaptic proteins in neurological disorders of the central nervous system (CNS) is steadily increasing. Direct interactions of AB with their target antigens have been shown in first studies but the exact pathomecha-nisms for most of the already discovered AB are still unclear. The present study investigates pathophysiological mechanisms of AB-fractions that are associated with the enigmatic CNS disease Stiff person syndrome (SPS) and target the synaptically located proteins amphiphysin or glutamate decarboxylase 65 (GAD65). In the first part of the project, effects of AB to the presynaptic endocytic protein amphiphysin were investigated. Ultrastructural investigations of spinal cord presynaptic boutons in an es-tablished in-vivo passive-transfer model after intrathecal application of human anti-amphiphysin AB showed a defect of endocytosis. This defect was apparent at high synaptic activity and was characterized by reduction of the synaptic vesicle pool, clathrin coated vesi-cles (CCVs), and endosome like structures (ELS) in comparison to controls. Molecular inves-tigation of presynaptic boutons in cultured murine hippocampal neurons with dSTORM microscopy after pretreatment with AB to amphiphysin revealed that marker proteins involved in vesicle exocytosis (synaptobrevin 2 and synaptobrevin 7) had an altered expression in GA-BAergic presynapses. Endophilin, a direct binding partner of amphiphysin also displayed a disturbed expression pattern. Together, these results point towards an anti-amphiphysin AB-induced defective organization in GABAergic synapses and a presumably compensatory rearrangement of proteins responsible for CME. In the second part, functional consequences of SPS patient derived IgG fractions containing AB to GAD65, the rate limiting enzyme for GABA synthesis, were investigated by patch clamp electrophysiology and immunohistology. GABAergic neurotransmission at low and high activity as well as short term plasticity appeared normal but miniature synaptic potentials showed an enhanced frequency with constant amplitudes. SPS patient IgG after preabsorption of GAD65-AB using recombinant GAD65 still showed specific synaptic binding to neu-rons and brain slices supporting the hypothesis that additional, not yet characterized AB are present in patient IgG responsible for the exclusive effect on frequency of miniature potentials. In conclusion, the present thesis uncovered basal pathophysiological mechanisms underlying paraneoplastic SPS induced by AB to amphiphysin leading to disturbed presynaptic architec-ture. In idiopathic SPS, the hypothesis of a direct pathophysiological role of AB to GAD65 was not supported and additional IgG AB are suspected to induce distinct synaptic malfunction
Die Anzahl neu charakterisierter Autoantikörper (AAK) gegen synaptische Proteine bei Er-krankungen des zentralen Nervensystems (ZNS) ist stetig wachsend. Direkte Interaktionen der AAK mit ihren Zielantigenen konnten in ersten Studien belegt werden, jedoch besteht weiterhin Unklarheit über die exakten zugrunde liegenden Pathomechanismen. In der vorliegenden Arbeit wurden pathophysiologische Mechanismen von AAK gegen die synaptisch lokalisierten Proteine Amphiphysin und Glutamatdecarboxylase 65 (GAD65) untersucht, die mit der ZNS Erkrankung Stiff Person Syndrom (SPS) assoziiert sind. Im ersten Projektteil wurden die Effekte von AAK gegen das Endozytoseprotein Amphiphysin analysiert: in einem etablierten in-vivo Tiermodell konnten nach intrathekalem passiven Transfer von AAK gegen Amphiphysin ultrastrukturelle Untersuchungen von präsynaptischen Terminalen im Rückenmark eine Störung der Endozytose aufzeigen. Dieser Defekt, der bei hoher synaptischer Aktivität eintrat, war durch eine Verminderung synaptischen Vesikelpools, Clathrin-ummantelter Vesikel und endosomähnlicher Strukturen charakterisiert. Molekulare Untersuchungen präsynaptischer Terminale kultivierter hippokampaler Zellkulturen mit dSTORM Mikroskopie zeigten, dass an der Exozytose beteiligte synaptische Vesikelproteine (Synaptobrevin 2 und Synaptobrevin 7) ein verändertes Expressionsmuster innerhalb GA-BAerger Synapsen aufweisen. Die Expression von Endophilin, einem direkten Bindungs-partner von Amphiphysin, war ebenso verändert. Zusammengefasst weisen diese Ergebnis-se auf einen Organisationsdefekt GABAerger Synapsen hin, die durch anti-Amphiphysin AAK induziert sind und eine kompensatorische Umverteilung von Endozytoseproteinen vermuten lassen. Im zweiten Teil der Arbeit wurden die funktionellen Effekte von SPS AAK gegen GAD65, dem geschwindigkeitsbestimmenden Enzym der GABA-Synthese, mittels Patch-Clamp Mes-sungen und Immunhistologie untersucht. Die GABAerge synaptische Übertragung bei niedri-ger als auch hoher synaptischer Aktivität sowie die synaptische Kurzzeitplastizität wurden durch die IgG Fraktionen mit GAD65-AAK nicht beeinträchtigt. Die Frequenz von GABAergen Miniaturpotentialen war jedoch bei ansonsten gleichbleibender Amplitude erhöht. SPS-Patienten-IgG zeigte allerdings auch nach Präabsorbtion von GAD65-AAK mit Hilfe von rekombinanten GAD65 eine spezifische Anfärbung neuronaler Synapsen, was die Hypothese von weiteren, funktionell wirksamen, aber noch nicht identifizierten AAK im Patienten-IgG unterstützt. Zusammenfassend konnten in der vorliegenden Arbeit grundlegende pathophysiologische Mechanismen aufgezeigt werden, wie pathogene Antikörper gegen Amphiphysin die Struktur präsynaptischer Boutons beeinträchtigen können. Im Falle des idiopathischen SPS konnte keine unterstützenden Befunde für die Hypothese einer direkten pathophysiologischen Rolle von GAD65 AAK erhoben werden. Nach den vorliegenden Ergebnissen wird das Vorhandensein weiterer, derzeit noch nicht beschriebener IgG AAK postuliert, die die synaptische Fehlfunktion erklären können

碩士
國立臺灣大學
生命科學系
105
It has been well demonstrated that the change of synaptic efficacy in neurocircuit of brain pain matrix is a cellular substrate for behavior hypersensitivity in animals with chronic pain. While most of previous studies focus on transmission at synapses between nociceptive inputs and principal neurons, the role of local GABAergic interneurons (IntNs) receives less attention. I address this issue by using the acid-induced muscle pain animal model (AIMP model) in mice and focusing on the rostral agranular insular cortex (RAIC). The RAIC is an important component of brain pain matrix as this cortical area is shown to tonically produce hyperalgesia signal and is a cortical area where nociceptive output originates. We propose that repeated acid saline injection may trigger a plastic change in synaptic efficacy of GABAergic IntNs onto pyramidal neurons (PNs) and cause an excitatory/inhibitory imbalance in neurocircuit in RAIC, which in turn alters cortical output of nociceptive signal in chronic pain. To test this possibility, dual-patch recording from a pair of IntN-PN in layer 5 was initially used to record unitary inhibitory postsynaptic current (IPSC) in previous experiments of our lab, and found that only 30% of all recorded IntN-PN pairs showed functional connectivity. To increase successful rate, here I employ optogenetic method to selectively active GABAergic IntNs. I injected a cre-dependent AAV that carries eYFP and channelrhodopsin2 sequences into RAIC in transgenic mice, in which the promoter of vesicular-GABA-transporter controls expression of cre recombinase. The animals were killed 2-3 weeks after AAV injection for brain slice preparation and whole-cell patch recording was made from PNs. Illuminating the slice with a single blue-light pulse (2 ms) evoked inhibitory postsynaptic current (IPSC) in PNs that was blocked by 20 uM bicuculline, a GABAA receptor antagonist. The paired-pulse ratio of the IPSC significantly reduced from 0.66 ± 0.10 (n = 13) in control mice to 0.37 ± 0.03 (n = 12) in muscle pain mice (P < 0.05; Mann-Whitney Test); the quantal size of the IPSC was significantly increased from 12.88 ± 1.22 pA (n = 13) in control mice to 18.82 ± 1.91 (n = 12) pA in muscle pain mice (P < 0.05; Mann-Whitney Test). These results show potential changes in synaptic function of GABAergic IntNs onto PNs in RAIC in chronic pain condition.