Jean-Luc Dupont

Mammalian Scribble Forms a Tight Complex with the βPIX Exchange Factor

Drosophila Scribble is implicated in the development of normal synapse structure and epithelial tissues, but it remains unclear how it plays a role and which process it controls. The mammalian homolog of Scribble, hScrib, has a primary structure and subcellular localization similar to that of its fly homolog, but its function remains unknown. Here we have used tandem mass spectrometry to identify major components of the hScrib network. We show that it includes betaPIX (also called Cool-1), a guanine nucleotide exchange factor (GEF), and its partner GIT1 (also called p95-APP1), a GTPase activating protein (GAP). betaPIX directly binds to the hScrib PDZ domains, and the hScrib/betaPIX complex is efficiently recovered in epithelial and neuronal cells and tissues. In cerebellar granule cell cultures, hScrib and betaPIX are both partially localized at neuronal presynaptic compartments. Furthermore, we show that hScrib is required to anchor betaPIX at the cell cortex and that dominant-negative betaPIX or hScrib proteins can each inhibit Ca2+-dependent exocytosis in neuroendocrine PC12 cells, demonstrating a functional relationship between these proteins. These data reveal the existence of a tight hScrib/betaPIX interaction and suggest that this complex potentially plays a role in neuronal transmission.

A role for phospholipase D1 in neurotransmitter release

Phosphatidic acid produced by phospholipase D (PLD) as a result of signaling activity is thought to play a role in membrane vesicle trafficking, either as an intracellular messenger or as a cone-shaped lipid that promotes membrane fusion. We recently described that, in neuroendocrine cells, plasma membrane-associated PLD1 operates at a stage of Ca2+-dependent exocytosis subsequent to cytoskeletal-mediated recruitment of secretory granules to exocytotic sites. We show here that PLD1 also plays a crucial role in neurotransmitter release. Using purified rat brain synaptosomes subjected to hypotonic lysis and centrifugation, we found that PLD1 is associated with the particulate fraction containing the plasma membrane. Immunostaining of rat cerebellar granule cells confirmed localization of PLD1 at the neuronal plasma membrane in zones specialized for neurotransmitter release (axonal neurites, varicosities, and growth cone-like structures). To determine the potential involvement of PLD1 in neurotransmitter release, we microinjected catalytically inactive PLD1(K898R) into Aplysia neurons and analyzed its effects on evoked acetylcholine (ACh) release. PLD1(K898R) produced a fast and potent dose-dependent inhibition of ACh release. By analyzing paired-pulse facilitation and postsynaptic responses evoked by high-frequency stimulations, we found that the exocytotic inhibition caused by PLD1(K898R) was not the result of an alteration in stimulus-secretion coupling or in vesicular trafficking. Analysis of the fluctuations in amplitude of the postsynaptic responses revealed that the PLD1(K898R) blocked ACh release by reducing the number of active presynaptic-releasing sites. Our results provide evidence that PLD1 plays a major role in neurotransmission, most likely by controlling the fusogenic status of presynaptic release sites.

Spontaneous and GABA-evoked chloride channels on pituitary intermediate lobe cells and their internal Ca requirements.

On porcine intermediate lobe (IL) endocrine cells, spontaneously opening chloride channels have been studied and compared to GABA-A activated chloride channels. Elementary currents were recorded mainly from outside-out patches excised from IL cells maintained in culture for 1–4 weeks. Spontaneous inward currents were observed in Cs-loaded cells after replacing Na in the extracellular medium by the impermeant ion choline. This activity, at an internal calcium concentration of 10−8 M corresponded to a channel for chloride ions with a main conductance level of 26 pS, and substates around 11 pS. The sequence of permeabilities to halides was I>Br>Cl. These conductance characteristics were common to the GABA-operated channels which also showed a main conductance substate of 23–31 pS. The open time of the 26 pS level mostly encountered in spontaneous activity, was distributed along two modes: one, the most frequent, around 1 ms, and the other around 4 ms. This latter mode was the predominant one observed during GABA and isoguvacine applications but in addition a bursting activity of 19 ms duration was also seen. Specific GABA-A receptor antagonists (bicuculline and SR 42641, 1 μM) blocked activity evoked by GABA (1–10 μM), but did not affect spontaneous events. These spontaneous Cl events were only observed in a restricted range of internal Ca concentrations, i.e. between 1 nM and 0.1 μM, and were practically abolished at Cai 1 μM. The GABA-induced activity of Cl channels was also Ca-sensitive, being reduced when Cai reached 1 μM.

IL1-receptor accessory protein-like 1 (IL1RAPL1), a protein involved in cognitive functions, regulates N-type Ca2+-channel and neurite elongation

Null mutations in the IL1-receptor accessory protein-like 1 gene (IL1RAPL1) are responsible for an inherited X-linked form of cognitive impairment. IL1RAPL1 protein physically interacts with neuronal calcium sensor-1 (NCS-1), but the functional impact of the IL1RAPL1/NCS-1 interaction remains unknown. Here, we demonstrate that stable expression of IL1RAPL1 in PC12 cells induces a specific silencing of N-type voltage-gated calcium channels (N-VGCC) activity that explains a secretion deficit observed in these IL1RAPL1 cells. Importantly, this modulation of VGCC activity is mediated by NCS-1. Indeed, a specific loss-of-function of N-VGCC was observed in PC12 cells overexpressing NCS-1, and a total recovery of N-VGCC activity was obtained by a down-regulation of NCS-1 in IL1RAPL1 cells. The functional relevance of the interaction between IL1RAPL1 and NCS-1 was also suggested by the reduction of neurite elongation observed in nerve growth factor (NGF)-treated IL1RAPL1 cells, a phenotype rescued by NCS-1 inactivation. Because both proteins are highly expressed in neurons, these results suggest that IL1RAPL1-related mental retardation could result from a disruption of N-VGCC and/or NCS-1-dependent synaptic and neuronal activities.

Clusters of cerebellar Purkinje cells control their afferent climbing fiber discharge

Significance The inferior olive, one of the major source of inputs to the cerebellum, sends climbing fibers to Purkinje cells, the key processing units of cerebellar-dependent motor control. Using an optogenetic strategy, we demonstrate that Purkinje cells disinhibit their climbing-fiber afferents via a poly-synaptic circuit. These findings identify a functional closed-loop organization in the olivo-cerebellar circuits that is potentially important for cerebellar motor learning. Climbing fibers, the projections from the inferior olive to the cerebellar cortex, carry sensorimotor error and clock signals that trigger motor learning by controlling cerebellar Purkinje cell synaptic plasticity and discharge. Purkinje cells target the deep cerebellar nuclei, which are the output of the cerebellum and include an inhibitory GABAergic projection to the inferior olive. This pathway identifies a potential closed loop in the olivo-cortico-nuclear network. Therefore, sets of Purkinje cells may phasically control their own climbing fiber afferents. Here, using in vitro and in vivo recordings, we describe a genetically modified mouse model that allows the specific optogenetic control of Purkinje cell discharge. Tetrode recordings in the cerebellar nuclei demonstrate that focal stimulations of Purkinje cells strongly inhibit spatially restricted sets of cerebellar nuclear neurons. Strikingly, such stimulations trigger delayed climbing-fiber input signals in the stimulated Purkinje cells. Therefore, our results demonstrate that Purkinje cells phasically control the discharge of their own olivary afferents and thus might participate in the regulation of cerebellar motor learning.

Polyethylenimine-Mediated DNA Transfection of Peripheral and Central Neurons in Primary Culture: Probing Ca2+Channel Structure and Function with Antisense Oligonucleotides

To study neuronal ion channel function with antisense oligonucleotides, a reliable method is needed which allows different neuronal cell types to be transfected without artifactual disruptive effects on their electrical properties. Here we report that use of the recently introduced transfecting agent, polyethylenimine, fulfills this requirement. Four days after transfection, in both central and peripheral neurons, an antisense designed to block the synthesis of the Ca2+ channel beta subunits induced a maximal decrease of the Ca2- current amplitude and modification of their kinetics and voltage-dependence. Controls with scrambled oligonucleotides, as well as Na+ current recordings of antisense transfected neurons, confirm both that the transfecting agent does not modify the electrophysiological properties of the neurons and that the effect of the antisense is sequence specific.

Effect of internal calcium concentration on calcium currents in rat sensory neurones

Using the patch-clamp technique in whole-cell configuration we have investigated the effect of increasing the internal calcium concentration (Cai) from below 10−8 M to 10−6 M on the three calcium currents: ICa, T (T for transient), ICa, S (S for sustained), ICa, N (N for neither), recently described in rat sensory neurones> Increasing Cai led to a dose-dependent reduction of the amplitude of ICa, S and, as Cai reached 5×10−7M ICa, S was nearly abolished. ICa, N is well evidenced from 5×10−10 M to 10−7 M where its is a large current. Preliminary observations indicate an increase of its inactivation rate following, as expected for a possible Cai dependent-inactivation, the increase of Cai from 5×10−10 M to 10−7 M. With Ca=5×10−7 M, all the cells displayed ICa, T and half of the cells in addition ICa, N, but it was of small amplitude. At Cai=10−6 M, most of the recorded cells only exhibited ICa, T.

Clostridium perfringens Epsilon Toxin Targets Granule Cells in the Mouse Cerebellum and Stimulates Glutamate Release

Epsilon toxin (ET) produced by C. perfringens types B and D is a highly potent pore-forming toxin. ET-intoxicated animals express severe neurological disorders that are thought to result from the formation of vasogenic brain edemas and indirect neuronal excitotoxicity. The cerebellum is a predilection site for ET damage. ET has been proposed to bind to glial cells such as astrocytes and oligodendrocytes. However, the possibility that ET binds and attacks the neurons remains an open question. Using specific anti-ET mouse polyclonal antibodies and mouse brain slices preincubated with ET, we found that several brain structures were labeled, the cerebellum being a prominent one. In cerebellar slices, we analyzed the co-staining of ET with specific cell markers, and found that ET binds to the cell body of granule cells, oligodendrocytes, but not astrocytes or nerve endings. Identification of granule cells as neuronal ET targets was confirmed by the observation that ET induced intracellular Ca2+ rises and glutamate release in primary cultures of granule cells. In cultured cerebellar slices, whole cell patch-clamp recordings of synaptic currents in Purkinje cells revealed that ET greatly stimulates both spontaneous excitatory and inhibitory activities. However, pharmacological dissection of these effects indicated that they were only a result of an increased granule cell firing activity and did not involve a direct action of the toxin on glutamatergic nerve terminals or inhibitory interneurons. Patch-clamp recordings of granule cell somata showed that ET causes a decrease in neuronal membrane resistance associated with pore-opening and depolarization of the neuronal membrane, which subsequently lead to the firing of the neuronal network and stimulation of glutamate release. This work demonstrates that a subset of neurons can be directly targeted by ET, suggesting that part of ET-induced neuronal damage observed in neuronal tissue is due to a direct effect of ET on neurons.

Glia-induced neuronal differentiation by transcriptional regulation

There is increasing evidence that different phases of brain development depend on neuron–glia interactions including postnatal key events like synaptogenesis. To address how glial cells influence synapse development, we analyzed whether and how glia‐derived factors affect gene expression in primary cultures of immunoisolated rat retinal ganglion cells (RGCs) by oligonucleotide microarrays. Our results show that the transcript pattern matched the developmental stage and characteristic properties of RGCs in vitro. Glia‐conditioned medium (GCM) and cholesterol up‐ and downregulated a limited number of genes that influence the development of dendrites and synapses and regulate cholesterol and fatty acid metabolism. The oligonucleotide microarrays detected the transcriptional regulation of neuronal cholesterol homeostasis in response to GCM and cholesterol treatment. Surprisingly, our study revealed neuronal expression and glial regulation of matrix gla protein (Mgp). Together, our results suggest that glial cells promote different aspects of neuronal differentiation by regulating transcription of distinct classes of genes.

Synaptic organization of the mouse cerebellar cortex in organotypic slice cultures.

The cellular and synaptic organization of new born mouse cerebellum maintained in organotypic slice cultures was investigated using immunohistochemical and patch-clamp recording approaches. The histological organization of the cultures shared many features with that observed in situ. Purkinje cells were generally arranged in a monolayer surrounded by a molecular-like neuropil made of Purkinje cell dendritic arborizations. Purkinje cell axons ran between clusters of small round cells identified as granule cells by Kv3.1b potassium channel immunolabelling. The terminal varicosities of the Purkinje cells axons enwrapped presumptive neurons of the cerebellar nuclei whereas their recurrent collaterals were in contact with Purkinje cells and other neurons. Granule cell axons established contacts with Purkinje cell somata and dendrites. Parvalbumin and glutamine acid decarboxylase (GAD) immunohistochemistry revealed the presence of presumptive interneurons throughout the culture. The endings of granule cell axons were observed to be in contact with these interneurons. Similarly, interneurons endings were seen close to Purkinje cells and granule cells. Whole cell recordings from Purkinje cell somata showed AMPA receptor-mediated spontaneous excitatory post-synaptic currents (sEPSCs) and GABAA receptor-mediated spontaneous inhibitory post-synaptic currents (sIPSCs). Similar events were recorded from granule cell somata except that in this neuronal type EPSPs have both a NMDA component and an AMPA component. In addition, pharmacological experiments demonstrated a GABAergic control of granule cell activity and a glutamatergic control of GABAergic neurons by granule cells. This study shows that a functional neuronal network is established in such organotypic cultures even in the absence of the two normal excitatory afferents, the mossy fibers and the climbing fibers.