Know thy neighbour: A normative theory of synaptic depression

Synapses exhibit an extraordinary degree of short-term malleability, with release probabilities and effective synaptic strengths changing markedly over multiple timescales. From the perspective of a fixed computational operation in a network, this seems like a most unacceptable degree of added variability. We suggest an alternative theory according to which short-term synaptic plasticity plays a normatively-justifiable role. This theory starts from the commonplace observation that the spiking of a neuron is an incomplete, digital, report of the analog quantity that contains all the critical information, namely its membrane potential. We suggest that a synapse solves the inverse problem of estimating the pre-synaptic membrane potential from the spikes it receives, acting as a recursive filter. We show that the dynamics of short-term synaptic depression closely resemble those required for optimal filtering, and that they indeed support high quality estimation. Under this account, the local postsynaptic potential and the level of synaptic resources track the (scaled) mean and variance of the estimated presynaptic membrane potential. We make experimentally testable predictions for how the statistics of subthreshold membrane potential fluctuations and the form of spiking non-linearity should be related to the properties of short-term plasticity in any particular cell type.

Phosphorylation of synaptic vesicle proteins: modulation of the alpha SNAP interaction with the core complex.

We analyzed whether synaptic membrane trafficking proteins are substrates for casein kinase II, calcium/calmodulin-dependent protein kinase II, and cAMP-dependent protein kinase (PKA), three kinases implicated in the modulation of synaptic transmission. Each kinase phosphorylates a specific set of the vesicle proteins syntaxin 1A, N-ethylmaleimide-sensitive factor (NSF), vesicle-associated membrane protein (VAMP), synaptosome-associated 25-kDa protein (SNAP-25), n-sec1, alpha soluble NSF attachment protein (alpha SNAP), and synaptotagmin. VAMP is phosphorylated by calcium/calmodulin-dependent protein kinase II on serine 61. alpha SNAP is phosphorylated by PKA; however, the beta SNAP isoform is phosphorylated only 20% as efficiently. alpha SNAP phosphorylated by PKA binds to the core docking and fusion complex 10 times weaker than the dephosphorylated form. These studies provide a first glimpse at regulatory events that may be important in modulating neurotransmitter release during learning and memory.

Données nouvelles sur l’innervation à dopamine du striatum et son co-phénotype glutamatergique

Une sous-population des neurones à dopamine (DA) du mésencéphale ventral du rat et de la souris étant connue pour exprimer l'ARN messager du transporteur vésiculaire 2 du glutamate (VGLUT2), nous avons eu recours à l'immunocytochimie en microscopie électronique, après simple ou double marquage de l'enzyme de synthèse tyrosine hydroxylase (TH) et de VGLUT2, pour déterminer la présence de l'une et/ou l'autre protéine dans les terminaisons (varicosités) axonales de ces neurones et caractériser leur morphologie ultrastructurale dans diverses conditions expérimentales. Dans un premier temps, des rats jeunes (P15) ou adultes (P90), ainsi que des rats des deux âges soumis à l'administration intraventriculaire cérébrale de la cytotoxine 6-hydroxydopamine (6-OHDA) dans les jours suivant la naissance, ont été examinés, afin d'étayer l'hypothèse d'un rôle de VGLUT2 au sein des neurones DA, au cours du développement normal ou pathologique de ces neurones. Chez le jeune rat, ces études ont montré: i) la présence de VGLUT2 dans une fraction importante des varicosités axonales TH immunoréactives du coeur du noyau accumbens ainsi que du néostriatum; ii) une augmentation de la proportion de ces terminaisons doublement marquées dans le noyau accumbens par suite de la lésion 6-OHDA néonatale; iii) le double marquage fréquent des varicosités axonales appartenant à l'innervation DA aberrante (néoinnervation), qui se développe dans la substance noire, par suite de la lésion 6-OHDA néonatale. Des différences significatives ont aussi été notées quant à la dimension des terminaisons axonales marquées pour la TH seulement, VGLUT2 seulement ou TH et VGLUT2. Enfin, à cet âge (P15), toutes les terminaisons doublement marquées sont apparues dotées d'une spécialisation membranaire synaptique, contrairement aux terminaisons marquées pour la TH ou pour VGLUT2 seulement. Dans un deuxième temps, nous avons voulu déterminer le devenir du double phénotype chez le rat adulte (P90) soumis ou non à la lésion 6-OHDA néonatale. Contrairement aux observations recueillies chez le jeune rat, nous avons alors constaté: i) l'absence complète de terminaisons doublement marquées dans le coeur du noyau accumbens et le néostriatum d'animaux intacts, de même que dans les restes de la substance noire des animaux 6-OHDA lésés; ii) une très forte baisse de leurnombre dans le coeur du noyau accumbens des animaux 6-OHDA lésés. Ces observations, suggérant une régression du double phénotype TH/VGLUT2 avec l'âge, sont venues renforcer l'hypothèse d'un rôle particulier d'une co-libération de glutamate par les neurones mésencéphaliques DA au cours du développement. Dans ces conditions, il est apparu des plus intéressants d'examiner l'innervation DA méso-striatale chez deux lignées de souris dont le gène Vglut2 avait été sélectivement invalidé dans les neurones DA du cerveau, ainsi que leurs témoins et des souris sauvages. D'autant que malgré l'utilisation croissante de la souris en neurobiologie, cette innervation DA n'avait jamais fait l'objet d’une caractérisation systématique en microscopie électronique. En raison de possibles différences entre le coeur et la coque du noyau accumbens, l'étude a donc porté sur les deux parties de ce noyau ainsi que le néostriatum et des souris jeunes (P15) et adultes (P70-90) de chaque lignée, préparées pour l'immunocytochimie de la TH, mais aussi pour le double marquage TH et VGLUT2, selon le protocole précédemment utilisé chez le rat. Les résultats ont surpris. Aux deux âges et quel que soit le génotype, les terminaisons axonales TH immunoréactives des trois régions sont apparues comparables quant à leur taille, leur contenu vésiculaire, le pourcentage contenant une mitochondrie et une très faible incidence synaptique (5% des varicosités, en moyenne). Ainsi, chez la souris, la régression du double phénotype pourrait être encore plus précoce que chez le rat, à moins que les deux protéines ne soient très tôt ségréguées dans des varicosités axonales distinctes des mêmes neurones DA. Ces données renforcent aussi l’hypothèse d’une transmission diffuse (volumique) et d’un niveau ambiant de DA comme élément déterminant du fonctionnement du système mésostriatal DA chez la souris comme chez le rat.

Increased 5-HT2C Receptor Binding in the Brain Stem And Cerebral Cortex During Liver Regeneration And Hepatic Neoplasia In Rats

In the present study, serotonin 2C (5-HT2c) receptor binding parameters in the brainstem and cerebral cortex were investigated during liver generation after partial hepatectomy (PH) and N-nitrosodiethylamine (NDEA) induced hepatic neoplasia in male Wistar rats. The serotonin content increased significantly (p<0.01) in the cerebral cortex after PH and in NDEA induced hepatic neoplasia. Brain stem serotonin content increased significantly (p<0.05) after PH and (p<0.001) in NDEA induced hepatic neoplasia. The number and affinity of the 5-HT2c receptors in the crude synaptic membrane preparations of the brain stem showed a significant (p<0.001) increase after PH and in NDEA induced hepatic neoplasia. The number and affinity of 5-HT2c receptors increased significantly (p<0.001) in NDEA induced hepatic neoplasia in the crude synaptic membrane preparations of the cerebral cortex. There was a significant (p<0.01) increase in plasma norepinephrine in PH and (p<0.001) in NDEA induced hepatic neoplasia, indicating sympathetic stimulation. Thus, our results suggest that during active hepatocyte proliferation 5-HT2c receptor in the brain stem and cerebral cortex are up-regulated which in turn induce hepatocyte proliferation mediated through sympathetic stimulation.

Infantile epileptic encephalopathy with hypsarrhythmia (infantile spasms/west syndrome) and immunity

West syndrome is a severe epilepsy, occurring in infancy, that comprises epileptic seizures known as spasms, in clusters, and a unique EEG pattern, hypsarrhythmia, with psychomotor regression. Maturation of the brain is a crucial component. The onset is within the first year of life, before 12 months of age. Patients are classified as cryptogenic (10 to 20%), when there are no known or diagnosed previous cerebral insults, and symptomatic (80 to 90%), when associated with pre-existing cerebral damages. The time interval from a brain insult to infantile spasms onset ranged from 6 weeks to 11 months. West syndrome has a time-limited natural evolutive course, usually disappearing by 3 or 4 years of age. In 62% of patients, there are transitions to another age-related epileptic encephalopathies, the Lennox-Gastaut Syndrome and severe epilepsy with multiple independent foci. Spontaneous remission and remission after viral infections may occur. Therapy with ACTH and corticosteroids are the most effective. Reports about intravenous immunoglobulins action deserve attention. There is also immune dysfunction, characterized mainly by anergy, impaired cell-mediated immunity, presence of immature thymocytes in peripheral blood, functional impairment of T lymphocytes induced by plasma inhibitory factors, and altered levels of immunoglobulins. Changes in B lymphocytes frequencies and increased levels of activated B cells have been reported. Sensitized lymphocytes to brain extract were also described. Infectious diseases are frequent and may, sometimes, cause fatal outcomes. Increase of pro-inflamatory cytokines in serum and cerebrospinal fluid of epileptic patients were reported. Association with specific HLA antigens was described by several authors (HLA-DR7, HLA-A7, HLA-DRw52, and HLA-DR5). Auto-antibodies to brain antigens, of several natures (N-methyl-d-aspartate glutamate receptor, gangliosides, brain tissue extract, synaptic membrane, and others), were described in epileptic patients and in epileptic syndromes. Experimental epilepsy studies with anti-brain antibodies demonstrated that epileptiform discharges can be obtained, producing hyperexcitability leading to epilepsy. We speculate that in genetically prone individuals, previous cerebral lesions may sensitize immune system and trigger an autoimmune disease. Antibody to brain antigens may be responsible for impairment of T cell function, due to plasma inhibitory effect and also cause epilepsy in immature brains. © 2008 Bentham Science Publishers Ltd.

QSAR e dinâmica molecular no estudo de sistemas biomoleculares: predição da atividade biológica de antagonistas do receptor sigma-1 e simulações de bicamadas lipídicas

Diferentes abordagens teóricas têm sido utilizadas em estudos de sistemas biomoleculares com o objetivo de contribuir com o tratamento de diversas doenças. Para a dor neuropática, por exemplo, o estudo de compostos que interagem com o receptor sigma-1 (Sig-1R) pode elucidar os principais fatores associados à atividade biológica dos mesmos. Nesse propósito, estudos de Relações Quantitativas Estrutura-Atividade (QSAR) utilizando os métodos de regressão por Mínimos Quadrados Parciais (PLS) e Rede Neural Artificial (ANN) foram aplicados a 64 antagonistas do Sig-1R pertencentes à classe de 1-arilpirazóis. Modelos PLS e ANN foram utilizados com o objetivo de descrever comportamentos lineares e não lineares, respectivamente, entre um conjunto de descritores e a atividade biológica dos compostos selecionados. O modelo PLS foi obtido com 51 compostos no conjunto treinamento e 13 compostos no conjunto teste (r² = 0,768, q² = 0,684 e r²teste = 0,785). Testes de leave-N-out, randomização da atividade biológica e detecção de outliers confirmaram a robustez e estabilidade dos modelos e mostraram que os mesmos não foram obtidos por correlações ao acaso. Modelos também foram gerados a partir da Rede Neural Artificial Perceptron de Multicamadas (MLP-ANN), sendo que a arquitetura 6-12-1, treinada com as funções de transferência tansig-tansig, apresentou a melhor resposta para a predição da atividade biológica dos compostos (r²treinamento = 0,891, r²validação = 0,852 e r²teste = 0,793). Outra abordagem foi utilizada para simular o ambiente de membranas sinápticas utilizando bicamadas lipídicas compostas por POPC, DOPE, POPS e colesterol. Os estudos de dinâmica molecular desenvolvidos mostraram que altas concentrações de colesterol induzem redução da área por lipídeo e difusão lateral e aumento na espessura da membrana e nos valores de parâmetro de ordem causados pelo ordenamento das cadeias acil dos fosfolipídeos. As bicamadas lipídicas obtidas podem ser usadas para simular interações entre lipídeos e pequenas moléculas ou proteínas contribuindo para as pesquisas associadas a doenças como Alzheimer e Parkinson. As abordagens usadas nessa tese são essenciais para o desenvolvimento de novas pesquisas em Química Medicinal Computacional.

Neurobiological alterations in the human alcoholic brain: effect of genotype

Long-term alcohol abuse by human subjects leads to selective brain damage that is restricted in extent and variable in severity. Within the cerebral cortex, neuronal loss is most marked in the superior frontal cortex and relatively mild in motor cortex. Cirrhotic alcoholics and subjects with alcohol-related Wernicke-Korsakoff syndrome show more severe and more extensive damage than do uncomplicated cases. Accumulating evidence suggests that the likelihood of developing alcohol dependency is associated with one or more genetic markers. In previous work we showed that GABAA receptor functionality, and the subunit isoform expression that underlies this, differed in region- and disease-specific ways between alcoholics and controls. By contrast, glutamate receptor (NMDA, KA, AMPA) differences were muted or absent. Here we asked if genotype differentiated the form, pharmacology, or expression of glutamate and GABA receptors in pathologically vulnerable and spared cortical regions, with a view to determining whether such subject factors might influence the severity of alcohol-induced brain damage. Cerebrocortical tissue was obtained at autopsy under informed, written consent from uncomplicated and alcoholic-cirrhotic Caucasian (predominantly Anglo-Celtic) cases, together with matched controls and cases with cirrhosis of non-alcoholic origin. All subjects had pathological confirmation of liver and brain diagnosis; none had been polydrug abusers. Samples were processed for synaptic membrane receptor binding, mRNA analysis by quantitative RT-PCR, and protein analysis by Western blot. Genotyping was performed by PCR methods, in the main using published primers. Several genetic markers differentiated between our alcoholic and control subjects, including the GABAA receptor 2 subunit (GABB2) gene ( 2 (3) 10.329, P 0.01), the dopamine D2 receptor B1 (DRD2B) allele ( 2 (3) 10.109, P 0.01) and a subset of the alcohol dehydrogenase-3 (ADH3) alleles ( 2 (2) 4.730, P 0.05). Although neither the type-2 glutamate transporter (EAAT2) nor the serotonin transporter (5HTT) genes were significantly associated with alcoholism, only EAAT2 heterozygotes showed a significant association between ADH3 genotype and alcoholism ( 2 (3) 7.475, P 0.05). Other interactions between genotypes were also observed. DRD2A, DRD2B, GABB2, EAAT2 and 5HTT genotypes did not divide alcoholic cases and controls on NMDA receptor parameters, although in combined subjects there was a significant DRD2B X Area Interaction with glutamateNMDA receptor efficacy (F(1,57) 4.67; P 0.05), measured as the extent of glutamate-enhanced MK801 binding. In contrast, there was a significant Case-group X ADH3 X Area Interaction with glutamateNMDA receptor efficacy (F(3,57) 2.97; P 0.05). When GABAA receptor subunit isoform expression was examined, significant Case-group X Genotype X Area X Isoform interactions were found for EAAT2 with subunit mRNA (F(1,37) 4.22; P0.05), for GABB2 with isoform protein (F(1,37) 5.69; P 0.05), and for DRD2B with isoform protein (F(2,34)5.69; P0.05). The results suggest that subjects’ genetic makeup may modulate the effectiveness of amino acid-mediated transmission in different cortical regions, and thereby influence neuronal vulnerability to excitotoxicity.

Eps15 and Dap160 control synaptic vesicle membrane retrieval and synapse development

Epidermal growth factor receptor pathway substrate clone 15 (Eps15) is a protein implicated in endocytosis, endosomal protein sorting, and cytoskeletal organization. Its role is, however, still unclear, because of reasons including limitations of dominant-negative experiments and apparent redundancy with other endocytic proteins. We generated Drosophila eps15-null mutants and show that Eps15 is required for proper synaptic bouton development and normal levels of synaptic vesicle (SV) endocytosis. Consistent with a role in SV endocytosis, Eps15 moves from the center of synaptic boutons to the periphery in response to synaptic activity. The endocytic protein, Dap160/intersectin, is a major binding partner of Eps15, and eps15 mutants phenotypically resemble dap160 mutants. Analyses of eps15 dap160 double mutants suggest that Eps15 functions in concert with Dap160 during SV endocytosis. Based on these data, we hypothesize that Eps15 and Dap160 promote the efficiency of endocytosis from the plasma membrane by maintaining high concentrations of multiple endocytic proteins, including dynamin, at synapses.

Fast subplasma membrane Ca2+ transients control exo-endocytosis of synaptic-like microvesicles in astrocytes

Astrocytes are the most abundant glial cell type in the brain. Although not apposite for long-range rapid electrical communication, astrocytes share with neurons the capacity of chemical signaling via Ca(2+)-dependent transmitter exocytosis. Despite this recent finding, little is known about the specific properties of regulated secretion and vesicle recycling in astrocytes. Important differences may exist with the neuronal exocytosis, starting from the fact that stimulus-secretion coupling in astrocytes is voltage independent, mediated by G-protein-coupled receptors and the release of Ca(2+) from internal stores. Elucidating the spatiotemporal properties of astrocytic exo-endocytosis is, therefore, of primary importance for understanding the mode of communication of these cells and their role in brain signaling. We here take advantage of fluorescent tools recently developed for studying recycling of glutamatergic vesicles at synapses (Voglmaier et al., 2006; Balaji and Ryan, 2007); we combine epifluorescence and total internal reflection fluorescence imaging to investigate with unprecedented temporal and spatial resolution, the stimulus-secretion coupling underlying exo-endocytosis of glutamatergic synaptic-like microvesicles (SLMVs) in astrocytes. Our main findings indicate that (1) exo-endocytosis in astrocytes proceeds with a time course on the millisecond time scale (tau(exocytosis) = 0.24 +/- 0.017 s; tau(endocytosis) = 0.26 +/- 0.03 s) and (2) exocytosis is controlled by local Ca(2+) microdomains. We identified submicrometer cytosolic compartments delimited by endoplasmic reticulum tubuli reaching beneath the plasma membrane and containing SLMVs at which fast (time-to-peak, approximately 50 ms) Ca(2+) events occurred in precise spatial-temporal correlation with exocytic fusion events. Overall, the above characteristics of transmitter exocytosis from astrocytes support a role of this process in fast synaptic modulation.

Membrane properties and synaptic integration of identified neuronal populations in the developing rodent neocortex

In my PhD work I concentrated on three elementary questions that are essential to understand the interactions between the different neuronal cell populations in the developing neocortex. The questions regarded the identity of Cajal-Retzius (CR) cells, the ubiquitous expression of glycine receptors in all major cell populations of the immature neocortex, and the role of taurine in the modulation of immature neocortical network activity.rnTo unravel whether CR cells of different ontogenetic origin have divergent functions I investigated the electrophysiological properties of YFP+ (derived from the septum and borders of the pallium) and YFP− CR cells (derived from other neocortical origins). This study demonstrated that the passive and active electrophysiological properties as well as features of GABAergic PSCs and glutamatergic currents are similar between both CR cell populations. These findings suggest that CR cells of different origins most probably support similar functions within the neuronal networks of the early postnatal cerebral cortex.rnTo elucidate whether glycine receptors are expressed in all major cell populations of the developing neocortex I analyzed the functional expression of glycine receptors on subplate (SP) cells. Activation of glycine receptors by glycine, -alanine and taurine elicited membrane responses that could be blocked by the selective glycinergic antagonist strychnine. Pharmacological experiments suggest that SP cells express functional heteromeric glycine receptors that do not contain 1 subunits. The activation of glycine receptors by glycine and taurine induced a membrane depolarization, which mediated excitatory effects. Considering the key role of SP cells in immature cortical networks and the development of thalamocortical connections, this glycinergic excitation may influence the properties of early cortical networks and the formation of cortical circuits.rnIn the third part of my project I demonstrated that tonic taurine application induced a massive increase in the frequency of PSCs. Based on their reversal potential and their pharmacological properties these taurine-induced PSCs are exclusively transmitted via GABAA receptors to the pyramidal neurons, while both GABAA and glycine receptors were implicated in the generation of the presynaptic activity. Accordingly, whole-cell and cell-attached recordings from genetically labeled interneurons revealed the expression of glycine and GABAA receptors, which mediated an excitatory action on these cells. These findings suggest that low taurine concentrations can tonically activate exclusively GABAergic networks. The activity level maintained by this GABAergic activity in the immature nervous system may contribute to network properties and can facilitate the activity dependent formation of adequate synaptic projections.rnIn summary, the results of my studies complemented the knowledge about neuronal interactions in the immature neocortex and improve our understanding of cellular processes that guide neuronal development and thus shape the brain.rn

Secretagogue-triggered Transfer of Membrane Proteins from Neuroendocrine Secretory Granules to Synaptic-like Microvesicles

The membrane proteins of all regulated secretory organelles (RSOs) recycle after exocytosis. However, the recycling of those membrane proteins that are targeted to both dense core granules (DCGs) and synaptic-like microvesicles (SLMVs) has not been addressed. Since neuroendocrine cells contain both RSOs, and the recycling routes that lead to either organelle overlap, transfer between the two pools of membrane proteins could occur during recycling. We have previously demonstrated that a chimeric protein containing the cytosolic and transmembrane domains of P-selectin coupled to horseradish peroxidase is targeted to both the DCG and the SLMV in PC12 cells. Using this chimera, we have characterized secretagogue-induced traffic in PC12 cells. After stimulation, this chimeric protein traffics from DCGs to the cell surface, internalizes into transferrin receptor (TFnR)-positive endosomes and thence to a population of secretagogue-responsive SLMVs. We therefore find a secretagogue-dependent rise in levels of HRP within SLMVs. In addition, the levels within SLMVs of the endogenous membrane protein, synaptotagmin, as well as a green fluorescent protein-tagged version of vesicle-associated membrane protein (VAMP)/synaptobrevin, also show a secretagogue-dependent increase.

Simultaneous estimation of global background synaptic inhibition and excitation from membrane potential fluctuations in layer III neurons of the rat entorhinal cortex in vitro

It is becoming clear that the detection and integration of synaptic input and its conversion into an output signal in cortical neurons are strongly influenced by background synaptic activity or "noise." The majority of this noise results from the spontaneous release of synaptic transmitters, interacting with ligand-gated ion channels in the postsynaptic neuron [Berretta N, Jones RSG (1996); A comparison of spontaneous synaptic EPSCs in layer V and layer II neurones in the rat entorhinal cortex in vitro. J Neurophysiol 76:1089-1110; Jones RSG, Woodhall GL (2005) Background synaptic activity in rat entorhinal cortical neurons: differential control of transmitter release by presynaptic receptors. J Physiol 562:107-120; LoTurco JJ, Mody I, Kriegstein AR (1990) Differential activation of glutamate receptors by spontaneously released transmitter in slices of neocortex. Neurosci Lett 114:265-271; Otis TS, Staley KJ, Mody I (1991) Perpetual inhibitory activity in mammalian brain slices generated by spontaneous GABA release. Brain Res 545:142-150; Ropert N, Miles R, Korn H (1990) Characteristics of miniature inhibitory postsynaptic currents in CA1 pyramidal neurones of rat hippocampus. J Physiol 428:707-722; Salin PA, Prince DA (1996) Spontaneous GABAA receptor-mediated inhibitory currents in adult rat somatosensory cortex. J Neurophysiol 75:1573-1588; Staley KJ (1999) Quantal GABA release: noise or not? Nat Neurosci 2:494-495; Woodhall GL, Bailey SJ, Thompson SE, Evans DIP, Stacey AE, Jones RSG (2005) Fundamental differences in spontaneous synaptic inhibition between deep and superficial layers of the rat entorhinal cortex. Hippocampus 15:232-245]. The function of synaptic noise has been the subject of debate for some years, but there is increasing evidence that it modifies or controls neuronal excitability and, thus, the integrative properties of cortical neurons. In the present study we have investigated a novel approach [Rudolph M, Piwkowska Z, Badoual M, Bal T, Destexhe A (2004) A method to estimate synaptic conductances from membrane potential fluctuations. J Neurophysiol 91:2884-2896] to simultaneously quantify synaptic inhibitory and excitatory synaptic noise, together with postsynaptic excitability, in rat entorhinal cortical neurons in vitro. The results suggest that this is a viable and useful approach to the study of the function of synaptic noise in cortical networks. © 2007 IBRO.

Quantitative interactions between the A-type K+ current and inositol trisphosphate receptors regulate intraneuronal Ca2+ waves and synaptic plasticity

Key points center dot Active calcium signal propagation occurs when an initial calcium trigger elicits calcium release through endoplasmic reticulum (ER) receptors. A high concentration of the calcium trigger in thin-calibre dendrites would suppress release of calcium through hippocampal inositol trisphosphate receptors (InsP3Rs). center dot Could the high-density expression of A-type K+ channels in thin-calibre dendrites be a mechanism for inhibiting this suppression, thereby restoring the utility of the ER as a substrate for active calcium propagation? center dot Quantitative analyses involving experimentally constrained models reveal a bell-shaped dependence of calcium released through InsP3Rs on the A-type K+ channel density, during the propagation of a calcium wave. center dot A-type K+ channels regulated the relative contribution of ER calcium to the induction of synaptic plasticity in the presence of model metabotropic glutamate receptors. center dot These results identify a novel form of interaction between active dendrites and the ER membrane and suggest that A-type K+ channels are ideally placed for inhibiting the suppression of InsP3Rs in thin-calibre dendrites. Abstract The A-type potassium current has been implicated in the regulation of several physiological processes. Here, we explore a role for the A-type potassium current in regulating the release of calcium through inositol trisphosphate receptors (InsP3R) that reside on the endoplasmic reticulum (ER) of hippocampal pyramidal neurons. To do this, we constructed morphologically realistic, conductance-based models equipped with kinetic schemes that govern several calcium signalling modules and pathways, and constrained the distributions and properties of constitutive components by experimental measurements from these neurons. Employing these models, we establish a bell-shaped dependence of calcium release through InsP3Rs on the density ofA-type potassium channels, during the propagation of an intraneuronal calcium wave initiated through established protocols. Exploring the sensitivities of calcium wave initiation and propagation to several underlying parameters, we found that ER calcium release critically depends on dendritic diameter and that wave initiation occurred at branch points as a consequence of a high surface area to volume ratio of oblique dendrites. Furthermore, analogous to the role ofA-type potassium channels in regulating spike latency, we found that an increase in the density ofA-type potassium channels led to increases in the latency and the temporal spread of a propagating calcium wave. Next, we incorporated kinetic models for the metabotropic glutamate receptor (mGluR) signalling components and a calcium-controlled plasticity rule into our model and demonstrate thatthe presence of mGluRs induced a leftward shift in a BienenstockCooperMunro-like synaptic plasticity profile. Finally, we show that the A-type potassium current could regulate the relative contribution of ER calcium to synaptic plasticity induced either through 900 pulses of various stimulus frequencies or through theta burst stimulation. Our results establish a novel form of interaction between active dendrites and the ER membrane, uncovering a powerful mechanism that could regulate biophysical/biochemical signal integration and steer the spatiotemporal spread of signalling microdomains through changes in dendritic excitability.

Botulinum neurotoxin type-A enters a non-recycling pool of synaptic vesicles

Neuronal communication relies on synaptic vesicles undergoing regulated exocytosis and recycling for multiple rounds of fusion. Whether all synaptic vesicles have identical protein content has been challenged, suggesting that their recycling ability may differ greatly. Botulinum neurotoxin type-A (BoNT/A) is a highly potent neurotoxin that is internalized in synaptic vesicles at motor nerve terminals and induces flaccid paralysis. Recently, BoNT/A was also shown to undergo retrograde transport, suggesting it might enter a specific pool of synaptic vesicles with a retrograde trafficking fate. Using high-resolution microscopy techniques including electron microscopy and single molecule imaging, we found that the BoNT/A binding domain is internalized within a subset of vesicles that only partially co-localize with cholera toxin B-subunit and have markedly reduced VAMP2 immunoreactivity. Synaptic vesicles loaded with pHrodo-BoNT/A-Hc exhibited a significantly reduced ability to fuse with the plasma membrane in mouse hippocampal nerve terminals when compared with pHrodo-dextran-containing synaptic vesicles and pHrodo-labeled anti-GFP nanobodies bound to VAMP2-pHluorin or vGlut-pHluorin. Similar results were also obtained at the amphibian neuromuscular junction. These results reveal that BoNT/A is internalized in a subpopulation of synaptic vesicles that are not destined to recycle, highlighting the existence of significant molecular and functional heterogeneity between synaptic vesicles.