Extrasynaptic neurotransmission in the modulation of brain function. Focus on the striatal neuronal glial networks

Extrasynaptic neurotransmission is an important short distance form of volume transmission (VT) and describes the extracellular diffusion of transmitters and modulators after synaptic spillover or extrasynaptic release in the local circuit regions binding to and activating mainly extrasynaptic neuronal and glial receptors in the neuroglial networks of the brain. Receptor-receptor interactions in G protein-coupled receptor (GPCR) heteromers play a major role, on dendritic spines and nerve terminals including glutamate synapses, in the integrative processes of the extrasynaptic signaling. Heteromeric complexes between GPCR and ion-channel receptors play a special role in the integration of the synaptic and extrasynaptic signals. Changes in extracellular concentrations of the classical synaptic neurotransmitters glutamate and GABA found with microdialysis is likely an expression of the activity of the neuron-astrocyte unit of the brain and can be used as an index of VT-mediated actions of these two neurotransmitters in the brain. Thus, the activity of neurons may be functionally linked to the activity of astrocytes, which may release glutamate and GABA to the extracellular space where extrasynaptic glutamate and GABA receptors do exist. Wiring transmission (WT) and VT are fundamental properties of all neurons of the CNS but the balance between WT and VT varies from one nerve cell population to the other. The focus is on the striatal cellular networks, and the WT and VT and their integration via receptor heteromers are described in the GABA projection neurons, the glutamate, dopamine, 5-hydroxytryptamine (5-HT) and histamine striatal afferents, the cholinergic interneurons, and different types of GABA interneurons. In addition, the role in these networks of VT signaling of the energy-dependent modulator adenosine and of endocannabinoids mainly formed in the striatal projection neurons will be underlined to understand the communication in the striatal cellular networks

Linking supply to demand: the neuronal monocarboxylate transporter MCT2 and the alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid receptor GluR2/3 subunit are associated in a common trafficking process.

MCT2 is the major neuronal monocarboxylate transporter (MCT) that allows the supply of alternative energy substrates such as lactate to neurons. Recent evidence obtained by electron microscopy has demonstrated that MCT2, like alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid (AMPA) receptors, is localized in dendritic spines of glutamatergic synapses. Using immunofluorescence, we show in this study that MCT2 colocalizes extensively with GluR2/3 subunits of AMPA receptors in neurons from various mouse brain regions as well as in cultured neurons. It also colocalizes with GluR2/3-interacting proteins, such as C-kinase-interacting protein 1, glutamate receptor-interacting protein 1 and clathrin adaptor protein. Coimmunoprecipitation of MCT2 with GluR2/3 and C-kinase-interacting protein 1 suggests their close interaction within spines. Parallel changes in the localization of both MCT2 and GluR2/3 subunits at and beneath the plasma membrane upon various stimulation paradigms were unraveled using an original immunocytochemical and transfection approach combined with three-dimensional image reconstruction. Cell culture incubation with AMPA or insulin triggered a marked intracellular accumulation of both MCT2 and GluR2/3, whereas both tumor necrosis factor alpha and glycine (with glutamate) increased their cell surface immunolabeling. Similar results were obtained using Western blots performed on membrane or cytoplasm-enriched cell fractions. Finally, an enhanced lactate flux into neurons was demonstrated after MCT2 translocation on the cell surface. These observations provide unequivocal evidence that MCT2 is linked to AMPA receptor GluR2/3 subunits and undergoes a similar translocation process in neurons upon activation. MCT2 emerges as a novel component of the synaptic machinery putatively linking neuroenergetics to synaptic transmission.

Storage and uptake of D-serine into astrocytic synaptic-like vesicles specify gliotransmission.

Glial cells are increasingly recognized as active players that profoundly influence neuronal synaptic transmission by specialized signaling pathways. In particular, astrocytes have been shown recently to release small molecules, such as the amino acids l-glutamate and d-serine as "gliotransmitters," which directly control the efficacy of adjacent synapses. However, it is still controversial whether gliotransmitters are released from a cytosolic pool or by Ca(2+)-dependent exocytosis from secretory vesicles, i.e., by a mechanism similar to the release of synaptic vesicles in synapses. Here we report that rat cortical astrocytes contain storage vesicles that display morphological and biochemical features similar to neuronal synaptic vesicles. These vesicles share some, but not all, membrane proteins with synaptic vesicles, including the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) synaptobrevin 2, and contain both l-glutamate and d-serine. Furthermore, they show uptake of l-glutamate and d-serine that is driven by a proton electrochemical gradient. d-Serine uptake is associated with vesicle acidification and is dependent on chloride. Whereas l-serine is not transported, serine racemase, the synthesizing enzyme for d-serine, is anchored to the membrane of the vesicles, allowing local generation of d-serine. Finally, we reveal a previously unexpected mutual vesicular synergy between d-serine and l-glutamate filling in glia vesicles. We conclude that astrocytes contain vesicles capable of storing and releasing d-serine, l-glutamate, and most likely other neuromodulators in an activity-dependent manner.

Intraneuronal angiotensinergic system in rat and human dorsal root ganglia.

To elucidate the local formation of angiotensin II (Ang II) in the neurons of sensory dorsal root ganglia (DRG), we studied the expression of angiotensinogen (Ang-N)-, renin-, angiotensin converting enzyme (ACE)- and cathepsin D-mRNA, and the presence of protein renin, Ang II, Substance P and calcitonin gene-related peptide (CGRP) in the rat and human thoracic DRG. Quantitative real time PCR (qRT-PCR) studies revealed that rat DRG expressed substantial amounts of Ang-N- and ACE mRNA, while renin mRNA as well as the protein renin were untraceable. Cathepsin D-mRNA and cathepsin D-protein were detected in the rat DRG indicating the possibility of existence of pathways alternative to renin for Ang I formation. Angiotensin peptides were successfully detected with high performance liquid chromatography and radioimmunoassay in human DRG extracts. In situ hybridization in rat DRG confirmed additionally expression of Ang-N mRNA in the cytoplasm of numerous neurons. Intracellular Ang II staining could be shown in number of neurons and their processes in both the rat and human DRG. Interestingly we observed neuronal processes with angiotensinergic synapses en passant, colocalized with synaptophysin, within the DRG. In the DRG, we also identified by qRT-PCR, expression of Ang II receptor AT(1A) and AT(2)-mRNA while AT(1B)-mRNA was not traceable. In some neurons Substance P and CGRP were found colocalized with Ang II. The intracellular localization and colocalization of Ang II with Substance P and CGRP in the DRG neurons may indicate a participation and function of Ang II in the regulation of nociception. In conclusion, these results suggest that Ang II may be produced locally in the neurons of rat and human DRG and act as a neurotransmitter.

Differential changes in synaptic proteins in the Alzheimer frontal cortex with marked increase in PSD-95 postsynaptic protein

We investigated how synaptic plasticity is related to the neurodegeneration process in the human dorsolateral prefrontal cortex. Pre- and postsynaptic proteins of Brodmann's area 9 from patients with Alzheimer's disease (AD) and age-matched controls were quantified by immunohistochemical methods and Western blots. The main finding was a significant increase in the expression of postsynaptic density protein PSD-95 in AD brains, revealed on both sections and immunoblots, while the expression of spinophilin, associated to spines, remained quantitatively unchanged despite qualitative changes with age and disease. Presynaptic protein alpha-synuclein indicated an increased immunohistochemical level, while synaptophysin remained unchanged. MAP2, a somatodendritic microtubule protein, as well as AD markers such as amyloid-beta protein and phosphorylated protein tau showed an increased expression on immunosections in AD. Altogether these changes suggest neuritic and synaptic reorganization in the process of AD. In particular, the significant increase in PSD-95 expression suggests a change in NMDA receptors trafficking and may represent a novel marker of functional significance for the disease.

Neuroscience: Astrocytes as aide-mémoires.

Memory formation is known to occur at the level of synaptic contacts between neurons. It therefore comes as a surprise that another type of brain cell, the astrocyte, is also involved in establishing memory.

Stable masking by H-2Dd cis ligand limits Ly49A relocalization to the site of NK cell/target cell contact.

Ly49A is an inhibitory receptor, which counteracts natural killer (NK) cell activation on the engagement with H-2D(d) (D(d)) MHC class I molecules (MHC-I) on target cells. In addition to binding D(d) on apposed membranes, Ly49A interacts with D(d) ligand expressed in the plane of the NK cells' membrane. Indeed, multivalent, soluble MHC-I ligand binds inefficiently to Ly49A unless the NK cells' D(d) complexes are destroyed. However, it is not known whether masked Ly49A remains constitutively associated with cis D(d) also during target cell interaction. Alternatively, it is possible that Ly49A has to be unmasked to significantly interact with its ligand on target cells. These two scenarios suggest distinct roles of Ly49A/D(d) cis interaction for NK cell function. Here, we show that Ly49A contributes to target cell adhesion and efficiently accumulates at synapses with D(d)-expressing target cells when NK cells themselves lack D(d). When NK cells express D(d), Ly49A no longer contributes to adhesion, and ligand-driven recruitment to the cellular contact site is strongly reduced. The destruction of D(d) complexes on NK cells, which unmasks Ly49A, is necessary and sufficient to restore Ly49A adhesive function and recruitment to the synapse. Thus, cis D(d) continuously sequesters a considerable fraction of Ly49A receptors, preventing efficient Ly49A recruitment to the synapse with D(d)+ target cells. The reduced number of Ly49A receptors that can functionally interact with D(d) on target cells explains the modest inhibitory capacity of Ly49A in D(d) NK cells. This property renders Ly49A NK cells more sensitive to react to diseased host cells.

Neurophysiological effects of vasopressin and oxytocin in the central amygdala of the rat : a potential mechanism for the opposite modulation of anxiety and fear behavior

Abstract The amygdala is a group of nuclei in the temporal lobe of the brain that plays a crucial role in anxiety and fear behavior. Sensory information converges in the basolateral and lateral nuclei of the amygdala, which have been the first regions in the brain where the acquisition of new (fear) memories has been associated with long term changes in synaptic transmission. These nuclei, in turn, project to the central nucleus of the amygdala. The central amygdala, through its extensive projections to numerous nuclei in the midbrain and brainstem, plays a pivotal role in the orchestration of the rapid autonomic and endocrine fear responses. In the central amygdala a large number of neuropeptides and receptors is expressed, among which high levels of vasopressin and oxytocin receptors. Local injections of these peptides into the amygdala modulate several aspects of the autonomic fear reaction. Interestingly, their effects are opposing: vasopressin tends to enhance the fear reactions, whereas oxytocin has anxiolytic effects. In order to investigate the neurophysiological mechanisms that could underlie this opposing modulation of the fear behavior, we studied the effects of vasopressin and oxytocin on the neuronal activity in an acute brain slice preparation of the rat central amygdala. We first assessed the effects of vasopressin and oxytocin on the spontaneous activity of central amygdala neurons. Extracellular single unit recordings revealed two major populations of neurons: a majority of neurons was excited by vasopressin and inhibited by oxytocin, whereas other neurons were only excited by oxytocin receptor activation. The inhibitory effect of oxytocin could be reduced by the block of GABAergic transmission, whereas the excitatory effects of vasopressin and oxytocin were not affected. In a second step we identified the cellular mechanisms for the excitatory effects of both peptides as well as the morphological and biochemical mechanisms underlying the opposing effects, by using sharp electrode recordings together with intracellular labelings. We revealed that oxytocin-excited neurons are localized in the lateral part (CeL) whereas vasopressin excited cells are found in the medial part of the central amygdala (CeM). The tracing of the neuronal morphology showed that the axon collaterals of the oxytocin-excited neurons project from the CeL, far into the CeM. Combined immunohistochemical stainings indicated that these projections are GABAergic. In the third set of experiments we investigated the synaptic interactions between the two identified cell populations. Whole-cell patch-clamp recordings in the CeM revealed that the inhibitory effect of oxytocin was caused by the massive increase of inhibitory GABAergic currents, which was induced by the activation of CeL neurons. Finally, the effects of vasopressin and oxytocin on evoked activity were investigated. We found on the one hand, that the probability of evoking action potentials in the CeM by stimulating the basolateral amygdala afferents was enhanced under vasopressin, whereas it decreased under oxytocin. On the other hand, the impact of cortical afferents stimulation on the CeL neurons was enhanced by oxytocin application. Taken together, these findings have allowed us to develop a model, in which the opposing behavioral effects of vasopressin and oxytocin are caused by a selective activation of two distinct populations of neurons in the GABAergic network of the central amygdala. Our model could help to develop new anxiolytic treatments, which modulate simultaneously both receptor systems. By acting on a GABAergic network, such treatments can further be tuned by combinations with classical benzodiazepines. Résumé: L'amygdale est un groupe de noyaux cérébraux localisés dans le lobe temporal. Elle joue un rôle essentiel dans les comportements liés à la peur et l'anxiété. L'information issue des aires sensorielles converge vers les noyaux amygdaliens latéraux et basolatéraux, qui sont les projections vers différents noyaux du tronc cérébral et de l'hypothalamus, joue un rôle clef premières régions dans lesquelles il a été démontré que l'acquisition d'une nouvelle mémoire (de peur) était associée à des changements à long terme de la transmission synaptique. Ces noyaux envoient leurs projections sur l'amygdale centrale, qui à travers ses propres dans l'orchestration des réponses autonomes et endocrines de peur. Le contrôle de l'activité neuronale dans l'amygdale centrale module fortement la réaction de peur. Ainsi, un grand nombre de neuropeptides sont spécifiquement exprimés dans l'amygdale centrale et un bon nombre d'entre eux interfère dans la réaction de peur et d'anxiété. Chez les rats, une forte concentration de récepteurs à l'ocytocine et à la vasopressine est exprimée dans le noyau central, et l'injection de ces peptides dans l'amygdale influence différents aspects de la réaction viscérale associée à la peur. Il est intéressant de constater que ces peptides exercent des effets opposés. Ainsi, la vasopressine augmente la réaction de peur alors que l'ocytocine a un effet anxiolytique. Afin d'investiguer les mécanismes neurophysiologiques responsables de ces effets opposés, nous avons étudié l'effet de la vasopressine et de l'ocytocine sur l'activité neuronale de préparations de tranches de cerveau de rats contenant entre autres de l'amygdale centrale. Tout d'abord, notre intérêt s'est porté sur les effets de ces deux neuropeptides sur l'activité spontanée dans l'amygdale centrale. Des enregistrements extracellulaires ont révélé différentes populations de neurones ; une majorité était excitée par la vasopressine et inhibée par l'ocytocine ; d'autres étaient seulement excités par l'activation du récepteur à l'ocytocine. L'effet inhibiteur de l'ocytocine a pu être réduit par l'inhibition de la transmission GABAergique, alors que ses effets excitateurs n'étaient pas affectés. Dans un deuxième temps, nous avons identifié les mécanismes cellulaires responsables de l'effet excitateur de ces deux peptides et analysé les caractéristiques morphologiques et biochimiques des neurones affectés. Des enregistrements intracellulaires ont permis de localiser les neurones excités par l'ocytocine dans la partie latérale de l'amygdale centrale (CeL), et ceux excités par la vasopressine dans sa partie médiale (CeM). Le traçage morphologique des neurones a révélé que les collatérales axonales des cellules excitées par l'ocytocine projetaient du CeL loin dans le CeM. De plus, des colorations immuno-histochimiques ont révélé que ces projections étaient GABAergiques. Dans un troisième temps, nous avons étudié les interactions synaptiques entre ces deux populations de cellules. Les enregistrements en whole-cell patch-clamp dans le CeM ont démontré que les effets inhibiteurs de l'ocytocine résultaient de l'augmentation massive des courants GABAergique résultant de l'activation des neurones dans le CeL. Finalement, les effets de l'ocytocine et de la vasopressine sur l'activité évoquée ont été étudiés. Nous avons pu montrer que la probabilité d'évoquer un potentiel d'action dans le CeM, par stimulation de l'amygdale basolatérale, était augmentée sous l'effet de la vasopressine et diminuée sous l'action de l'ocytocine. Par contre, l'impact de la stimulation des afférences corticales sur les neurones du CeL était augmenté par l'application de l'ocytocine. L'ensemble de ces résultats nous a permis de développer un modèle dans lequel les effets comportementaux opposés de la vasopressine et de l'ocytocine sont causés par une activation sélective des deux différentes populations de neurones dans un réseau GABAergique. Un tel modèle pourrait mener au développement de nouveaux traitements anxiolytiques en modulant l'activité des deux récepteurs simultanément. En agissant sur un réseau GABAergique, les effets d'un tel traitement pourraient être rendus encore plus sélectifs en association avec des benzodiazépines classiques.

Functional Microdomains Control Glutamate Exocytosis from Astrocytes

The discovery that astrocytes possess a non-electrical form of excitability (Ca21-excitability) that leads to the release of chemical transmitters, an activity called ''gliotransmission'', indicates that these cells may have additional important roles in brain function. Elucidating the stimulus-secretion coupling leading to the exocytic release of chemical transmitters (such as glutamate, Bezzi et al., Nature Neurosci, 2004) may therefore clarify i) whether astrocytes represent in full a new class of secretory cells in the brain and ii) whether they can participate to the fast brain signaling in the brain. We have recently discovered the existence in astrocytes of functional sub-membrane microdomains of Ca21 release from the internal stores in response to mGluR5 activation (Marchaland et al., J of Neurosci., 2008). Such Ca21 microdomains control exocytosis of astrocytic glutamate signalling to neurons. Homer proteins are scaffold proteins controlling Ca21 signalling in different cellular microdomains, including dendritic spines in neurons (Sala et al., J of Neurosci., 2005). Thus, similarly to dendritic pines, Homer1 could be implicated in the coupling between astrocytic mGluR5 and IP3Rs on the ER. Here, by using a recently developed approach for studying vesicle recycling dynamics at synapses (Voglmaier et al., Neuron, 2006; Balaji and Ryan, PNAS, 2007) combined with epifluorescence and total internal reflection fluorescence (TIRF) imaging, we have investigated the involvement of Homer1 proteins in the Ca21-dependent stimulus-secretion coupling leading glutamate exocytosis of synaptic-like microvesicles (SLMVs) in astrocytes.

Ultrastructural analysis of neuronal plasticity in the adult mouse

ABSTRACT Adult neuronal plasticity is a term that corresponds to a set of biological mechanisms allowing a neuronal circuit to respond and adapt to modifications of the received inputs. Mystacial whiskers of the mouse are the starting point of a major sensory pathway that provides the animal with information from its immediate environment. Through whisking, information is gathered that allows the animal to orientate itself and to recognize objects. This sensory system is crucial for nocturnal behaviour during which vision is not of much use. Sensory information of the whiskers are sent via brainstem and thalamus to the primary somatosensory area (S1) of the cerebral cortex in a strictly topological manner. Cell bodies in the layer N of S 1 are arranged in ring forming structures called barrels. As such, each barrel corresponds to the cortical representation in layer IV of a single whisker follicle. This histological feature allows to identify with uttermost precision the part of the cortex devoted to a given whisker and to study modifications induced by different experimental conditions. The condition used in the studies of my thesis is the passive stimulation of one whisker in the adult mouse for a period of 24 hours. It is performed by glueing a piece of metal on one whisker and placing the awake animal in a cage surrounded by an electromagnetic coil that generates magnetic field burst inducing whisker movement at a given frequency during 24 hours. I analysed the ultrastructure of the barrel corresponding the stimulated whisker using serial sections electron microscopy and computer-based three-dimensional reconstructions; analysis of neighbouring, unstimulated barrels as well as those from unstimulated mice served as control. The following elements were structurally analyzed: the spiny dendrites, the axons of excitatory as well as inhibitory cells, their connections via synapses and the astrocytic processes. The density of synapses and spines is upregulated in a barrel corresponding to a stimulated whisker. This upregulation is absent in the BDNF heterozygote mice, indicating that a certain level of activity-dependent released BDNF is required for synaptogenesis in the adult cerebral cortex. Synpaptogenesis is correlated with a modification of the astrocytes that place themselves in closer vicinity of the excitatory synapses on spines. Biochemical analysis revealed that the astrocytes upregulate the expression of transporters by which they internalise glutamate, the neurotransmitter responsible for the excitatory response of cortical neurons. In the final part of my thesis, I show that synaptogenesis in the stimulated barrel is due to the increase in the size of excitatory axonal boutons that become more frequently multisynaptic, whereas the inhibitory axons do not change their morphology but form more synapses with spines apposed to them. Taken together, my thesis demonstrates that all the cellular elements present in the neuronal tissue of the adult brain contribute to activity-dependent cortical plasticity and form part of a mechanism by which the animal responds to a modified sensory experience. Throughout life, the neuronal circuit keeps the faculty to adapt its function. These adaptations are partially transitory but some aspects remain and could be the structural basis of a memory trace in the cortical circuit. RESUME La plasticité neuronale chez l'adulte désigne un ensemble de mécanismes biologiques qui permettent aux circuits neuronaux de répondre et de s'adapter aux modifications des stimulations reçues. Les vibrisses des souris sont un système crucial fournissant des informations sensorielles au sujet de l'environnement de l'animal. L'information sensorielle collectée par les vibrisses est envoyée via le tronc cérébral et le thalamus à l'aire sensorielle primaire (S 1) du cortex cérébral en respectant strictement la somatotopie. Les corps cellulaires dans la couche IV de S 1 sont organisés en anneaux délimitant des structures nommées tonneaux. Chaque tonneau reçoit l'information d'une seule vibrisse et l'arrangement des tonneaux dans le cortex correspond à l'arrangement des vibrisses sur le museau de la souris. Cette particularité histologique permet de sélectionner avec certitude la partie du cortex dévolue à une vibrisse et de l'étudier dans diverses conditions. Le paradigme expérimental utilisé dans cette thèse est la stimulation passive d'une seule vibrisse durant 24 heures. Pour ce faire, un petit morceau de métal est collé sur une vibrisse et la souris est placée dans une cage entourée d'une bobine électromagnétique générant un champ qui fait vibrer le morceau de métal durant 24 heures. Nous analysons l'ultrastructure du cortex cérébral à l'aide de la microscopie électronique et des coupes sériées permettant la reconstruction tridimensionnelle à l'aide de logiciels informatiques. Nous observons les modifications des structures présentes : les dendrites épineuses, les axones des cellules excitatrices et inhibitrices, leurs connections par des synapses et les astrocytes. Le nombre de synapses et d'épines est augmenté dans un tonneau correspondant à une vibrisse stimulée 24 heures. Basé sur cela, nous montrons dans ces travaux que cette réponse n'est pas observée dans des souris hétérozygotes BDNF+/-. Cette neurotrophine sécrétée en fonction de l'activité neuronale est donc nécessaire pour la synaptogenèse. La synaptogenèse est accompagnée d'une modification des astrocytes qui se rapprochent des synapses excitatrices au niveau des épines dendritiques. Ils expriment également plus de transporteurs chargés d'internaliser le glutamate, le neurotransmetteur responsable de la réponse excitatrice des neurones. Nous montrons aussi que les axones excitateurs deviennent plus larges et forment plus de boutons multi-synaptiques à la suite de la stimulation tandis que les axones inhibiteurs ne changent pas de morphologie mais forment plus de synapses avec des épines apposées à leur membrane. Tous les éléments analysés dans le cerveau adulte ont maintenu la capacité de réagir aux modifications de l'activité neuronale et répondent aux modifications de l'activité permettant une constante adaptation à de nouveaux environnements durant la vie. Les circuits neuronaux gardent la capacité de créer de nouvelles synapses. Ces adaptations peuvent être des réponses transitoires aux stimuli mais peuvent aussi laisser une trace mnésique dans les circuits.

An Ion Transport-Independent Role for the Cation-Chloride Cotransporter KCC2 in Dendritic Spinogenesis In Vivo.

The neuron-specific K-Cl cotransporter, KCC2, is highly expressed in the vicinity of excitatory synapses in pyramidal neurons, and recent in vitro data suggest that this protein plays a role in the development of dendritic spines. The in vivo relevance of these observations is, however, unknown. Using in utero electroporation combined with post hoc iontophoretic injection of Lucifer Yellow, we show that premature expression of KCC2 induces a highly significant and permanent increase in dendritic spine density of layer 2/3 pyramidal neurons in the somatosensory cortex. Whole-cell recordings revealed that this increased spine density is correlated with an enhanced spontaneous excitatory activity in KCC2-transfected neurons. Precocious expression of the N-terminal deleted form of KCC2, which lacks the chloride transporter function, also increased spine density. In contrast, no effect on spine density was observed following in utero electroporation of a point mutant of KCC2 (KCC2-C568A) where both the cotransporter function and the interaction with the cytoskeleton are disrupted. Transfection of the C-terminal domain of KCC2, a region involved in the interaction with the dendritic cytoskeleton, also increased spine density. Collectively, these results demonstrate a role for KCC2 in excitatory synaptogenesis in vivo through a mechanism that is independent of its ion transport function.

Gliotransmitters Travel in Time and Space.

The identification of the presence of active signaling between astrocytes and neurons in a process termed gliotransmission has caused a paradigm shift in our thinking about brain function. However, we are still in the early days of the conceptualization of how astrocytes influence synapses, neurons, networks, and ultimately behavior. In this Perspective, our goal is to identify emerging principles governing gliotransmission and consider the specific properties of this process that endow the astrocyte with unique functions in brain signal integration. We develop and present hypotheses aimed at reconciling confounding reports and define open questions to provide a conceptual framework for future studies. We propose that astrocytes mainly signal through high-affinity slowly desensitizing receptors to modulate neurons and perform integration in spatiotemporal domains complementary to those of neurons.

Derivation of traceable and transplantable photoreceptors from mouse embryonic stem cells.

Retinal degenerative diseases resulting in the loss of photoreceptors are one of the major causes of blindness. Photoreceptor replacement therapy is a promising treatment because the transplantation of retina-derived photoreceptors can be applied now to different murine retinopathies to restore visual function. To have an unlimited source of photoreceptors, we derived a transgenic embryonic stem cell (ESC) line in which the Crx-GFP transgene is expressed in photoreceptors and assessed the capacity of a 3D culture protocol to produce integration-competent photoreceptors. This culture system allows the production of a large number of photoreceptors recapitulating the in vivo development. After transplantation, integrated cells showed the typical morphology of mature rods bearing external segments and ribbon synapses. We conclude that a 3D protocol coupled with ESCs provides a safe and renewable source of photoreceptors displaying a development and transplantation competence comparable to photoreceptors from age-matched retinas.

The Munc13 proteins differentially regulate readily releasable pool dynamics and calcium-dependent recovery at a central synapse.

The Munc13 gene family encodes molecules located at the synaptic active zone that regulate the reliability of synapses to encode information over a wide range of frequencies in response to action potentials. In the CNS, proteins of the Munc13 family are critical in regulating neurotransmitter release and synaptic plasticity. Although Munc13-1 is essential for synaptic transmission, it is paradoxical that Munc13-2 and Munc13-3 are functionally dispensable at some synapses, although their loss in other synapses leads to increases in frequency-dependent facilitation. We addressed this issue at the calyx of Held synapse, a giant glutamatergic synapse that we found to express all these Munc13 isoforms. We studied their roles in the regulation of synaptic transmission and their impact on the reliability of information transfer. Through detailed electrophysiological analyses of Munc13-2, Munc13-3, and Munc13-2-3 knock-out and wild-type mice, we report that the combined loss of Munc13-2 and Munc13-3 led to an increase in the rate of calcium-dependent recovery and a change in kinetics of release of the readily releasable pool. Furthermore, viral-mediated overexpression of a dominant-negative form of Munc13-1 at the calyx demonstrated that these effects are Munc13-1 dependent. Quantitative immunohistochemistry using Munc13-fluorescent protein knock-in mice revealed that Munc13-1 is the most highly expressed Munc13 isoform at the calyx and the only one highly colocalized with Bassoon at the active zone. Based on these data, we conclude that Munc13-2 and Munc13-3 isoforms limit the ability of Munc13-1 to regulate calcium-dependent replenishment of readily releasable pool and slow pool to fast pool conversion in central synapses.