Pierre Giraud

Selective Blocking of Voltage-Gated K+ Channels Improves Experimental Autoimmune Encephalomyelitis and Inhibits T Cell Activation

Kaliotoxin (KTX), a blocker of voltage-gated potassium channels (Kv), is highly selective for Kv1.1 and Kv1.3. First, Kv1.3 is expressed by T lymphocytes. Blockers of Kv1.3 inhibit T lymphocyte activation. Second, Kv1.1 is found in paranodal regions of axons in the central nervous system. Kv blockers improve the impaired neuronal conduction of demyelinated axons in vitro and potentiate the synaptic transmission. Therefore, we investigated the therapeutic properties of KTX via its immunosuppressive and symptomatic neurological effects, using experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. The T line cells used to induce adoptive EAE were myelin basic protein (MBP)-specific, constitutively contained mRNA for Kv1.3. and expressed Kv1.3. These channels were shown to be blocked by KTX. Activation is a crucial step for MBP T cells to become encephalitogenic. The addition of KTX during Ag-T cell activation led to a great reduction in the MBP T cell proliferative response, in the production of IL-2 and TNF, and in Ca2+ influx. Furthermore, the addition of KTX during T cell activation in vitro led a decreased encephalitogenicity of MBP T cells. Moreover, KTX injected into Lewis rats impaired T cell function such as the delayed-type hypersensitivity. Lastly, the administration of this blocker of neuronal and lymphocyte channels to Lewis rats improved the symptoms of EAE. We conclude that KTX is a potent immunosuppressive agent with beneficial effects on the neurological symptoms of EAE.

Distribution of α-melanocyte-stimulating hormone in the rat brain: Evidence that α-MSH-containing cells in the arcuate region send projections to extrahypothalamic areas

Abstract The distribution and concentration of α-MSH in the rodent brain has been determined by radioimmunoassay. The limbic system contained substantial quantities of α-MSH. Forty per cent of the α-MSH present in the brain was localized in the hypothalamus, with the highest concentration of α-MSH in the arcuate nucleus. More than 40% of the extrahypothalamic α-MSH in the brain was found in the following areas: midbrain (16%), preoptic area (13%), septum (7%), and thalamus (7%). To determine the source of the hypothalamic and extrahypothalamic α-MSH, the anterior hypothalamic preoptic area of the brain was surgically separated from more caudal diencephalic structures, and the arcuate region of the hypothalamus was surgically isolated from the remainder of the brain. Following these deafferentations, no significant reduction in hypothalamic α-MSH levels was observed; however, a significant reduction in extrahypothalamic α-MSH levels was demonstrated. This dramatic decrease of α-MSH in extrahypothalamic areas of the rodent brain strongly suggests that the bulk of the extrahypothalamic α-MSH arises from neuronal perikarya in the arcuate region. These findings are consistent with the hypothesis that a population of neuronal cell bodies producing α-MSH originate in the arcuate region of the hypothalamus and that they send axonal projections to many areas of the limbic system and brain stem.

Identification of an axonal determinant in the C‐terminus of the sodium channel Nav1.2

To obtain a better understanding of how hippocampal neurons selectively target proteins to axons, we assessed whether any of the large cytoplasmic regions of neuronal sodium channel Nav1.2 contain sufficient information for axonal compartmentalization. We show that addition of the cytoplasmic C‐terminal region of Nav1.2 restricted the distribution of a dendritic–axonal reporter protein to axons. The analysis of mutants revealed that a critical segment of nine amino acids encompassing a di‐leucine‐based motif mediates axonal compartmentalization of chimera. In addition, the Nav1.2 C‐terminus is recognized by the clathrin endocytic pathway both in non‐neuronal cells and the somatodendritic domain of hippocampal neurons. The mutation of the di‐leucine motif located within the nine amino acid sequence to alanines resulted in the loss of chimera compartmentalization in axons and of internalization. These data suggest that selective elimination by endocytosis in dendrites may account for the compartmentalized distribution of some proteins in axons.

Endocytotic elimination and domain-selective tethering constitute a potential mechanism of protein segregation at the axonal initial segment.

The axonal initial segment is a unique subdomain of the neuron that maintains cellular polarization and contributes to electrogenesis. To obtain new insights into the mechanisms that determine protein segregation in this subdomain, we analyzed the trafficking of a reporter protein containing the cytoplasmic II–III linker sequence involved in sodium channel targeting and clustering (Garrido, J.J., P. Giraud, E. Carlier, F. Fernandes, A. Moussif, M.P. Fache, D. Debanne, and B. Dargent. 2003. Science. 300:2091–2094). Here, we show that this reporter protein is preferentially inserted in the somatodendritic domain and is trapped at the axonal initial segment by tethering to the cytoskeleton, before its insertion in the axonal tips. The nontethered population in dendrites, soma, and the distal part of axons is subsequently eliminated by endocytosis. We provide evidence for the involvement of two independent determinants in the II–III linker of sodium channels. These findings indicate that endocytotic elimination and domain-selective tethering constitute a potential mechanism of protein segregation at the axonal initial segment of hippocampal neurons.

Effect of nicotine on in vivo secretion of melanocorticotropic hormones in the rat

Abstract Corticosterone, ACTH, β-endorphin and α-MSH were measured in rat plasma by radioimmunoassay before and 2,5,15,30 minutes after an intraperitoneal injection of nicotine (500 μg/Kg b.w.). Nicotine induced an increase of plasma corticosterone (p

Influence of haloperidol on ACTH and β-endorphin secretion in the rat☆

The injection of haloperidol, a dopamine receptor blocker, was followed by a large increase of plasma ACTH and beta-endorphin-like immunoreactivity (beta-EI) in the rat. This effect was prevented when the rats were previously treated with corticosteroids. These results suggest that catecholamines inhibit ACTH and beta-endorphin secretion in the rat.

Spike-Time Precision and Network Synchrony Are Controlled by the Homeostatic Regulation of the D-Type Potassium Current

Homeostatic plasticity of neuronal intrinsic excitability (HPIE) operates to maintain networks within physiological bounds in response to chronic changes in activity. Classically, this form of plasticity adjusts the output firing level of the neuron through the regulation of voltage-gated ion channels. Ion channels also determine spike timing in individual neurons by shaping subthreshold synaptic and intrinsic potentials. Thus, an intriguing hypothesis is that HPIE can also regulate network synchronization. We show here that the dendrotoxin-sensitive D-type K+ current (I D) disrupts the precision of AP generation in CA3 pyramidal neurons and may, in turn, limit network synchronization. The reduced precision is mediated by the sequence of outward I D followed by inward Na+ current. The homeostatic downregulation of I D increases both spike-time precision and the propensity for synchronization in iteratively constructed networks in vitro. Thus, network synchronization is adjusted in area CA3 through activity-dependent remodeling of I D.

Dynamic compartmentalization of the voltage-gated sodium channels in axons.

One of the major physiological roles of the neuronal voltage‐gated sodium channel is to generate action potentials at the axon hillock/initial segment and to ensure propagation along myelinated or unmyelinated fibers to nerve terminal. These processes require a precise distribution of sodium channels accumulated at high density in discrete subdomains of the nerve membrane. In neurons, information relevant to ion channel trafficking and compartmentalization into sub‐domains of the plasma membrane is far from being elucidated. Besides, whereas information on dendritic targeting is beginning to emerge, less is known about the mechanisms leading to the polarized distribution of proteins in axon. To obtain a better understanding of how neurons selectively target sodium channels to discrete subdomains of the nerve, we addressed the question as to whether any of the large intracellular regions of Nav1.2 contain axonal sorting and/or clustering signals. We first obtained evidence showing that addition of the cytoplasmic carboxy‐terminal region of Nav1.2 restricted the distribution of a dendritic‐axonal reporter protein to axons of hippocampal neurons. The analysis of mutants revealed that a di‐leucine‐based motif mediates chimera compartmentalization in axons and its elimination in soma and dendrites by endocytosis. The analysis of the others generated chimeras showed that the determinant conferring sodium channel clustering at the axonal initial segment is contained within the cytoplasmic loop connecting domains II‐III of Nav1.2. Expression of a soluble Nav1.2 II‐III linker protein led to the disorganization of endogenous sodium channels. The motif was sufficient to redirect a somatodendritic potassium channel to the axonal initial segment, a process involving association with ankyrin G. Thus, it is conceivable that concerted action of the two determinants is required for sodium channel compartmentalization in axons.

Molecular determinants of emerging excitability in rat embryonic motoneurons

Molecular determinants of excitability were studied in pure cultures of rat embryonic motoneurons. Using RT‐PCR, we have shown here that the spike‐generating Na+ current is supported by Nav1.2 and/or Nav1.3 α‐subunits. Nav1.1 and Nav1.6 transcripts were also identified. We have demonstrated that alternatively spliced isoforms of Nav1.1 and Nav1.6, resulting in truncated proteins, were predominant during the first week in culture. However, Nav1.6 protein could be detected after 12 days in vitro. The Navβ2.1 transcript was not detected, whereas the Nav β1.1 transcript was present. Even in the absence of Navβ2.1, α‐subunits were correctly inserted into the initial segment. RT‐PCR (at semi‐quantitative and single‐cell levels) and immunocytochemistry showed that transient K+ currents result from the expression of Kv4.2 and Kv4.3 subunits. This is the first identification of subunits responsible for a transient K+ current in spinal motoneurons. The blockage of Kv4.2/Kv4.3 using a specific toxin modified the shape of the action potential demonstrating the involvement of these conductance channels in regulating spike repolarization and the discharge frequency. Among the other Kv α‐subunits (Kv1.3, 1.4, 1.6, 2.1, 3.1 and 3.3), we showed that the Kv1.6 subunit was partly responsible for the sustained K+ current. In conclusion, this study has established the first correlation between the molecular nature of voltage‐dependent Na+ and K+ channels expressed in embryonic rat motoneurons in culture and their electrophysiological characteristics in the period when excitability appears.

Interaction of the Nav1.2a subunit of the voltage-dependent sodium channel with nodal ankyrinG. In vitro mapping of the interacting domains and association in synaptosomes.

Voltage-dependant sodium channels at the axon initial segment and nodes of Ranvier colocalize with the nodal isoforms of ankyrinG (AnkG node). Using fusion proteins derived from the intracellular regions of the Nav1.2a subunit and the Ank repeat domain of AnkG node, we mapped a major interaction site in the intracellular loop separating α subunit domains I-II. This 57-amino acid region binds the Ank repeat region with a K D value of 69 nm. We identified another site in intracellular loop III-IV, and we mapped both Nav1.2a binding sites on the ankyrin repeat domain to the region encompassing repeats 12–22. The ankyrin repeat domain did not bind the β1 and β2 subunit cytoplasmic regions. We showed that in cultured embryonic motoneurons, expression of the β2 subunit is not necessary for the colocalization of AnkG node with functional sodium channels at the axon initial segment. Antibodies directed against the β1subunit intracellular region, α subunit loop III-IV, and AnkG node could not co-immunoprecipitate AnkGnode and sodium channels from Triton X-100 solubilisates of rat brain synaptosomes. Co-immunoprecipitation of sodium channel α subunit and of the 270- and 480-kDa AnkG node isoforms was obtained when solubilization conditions that maximize membrane protein extraction were used. However, we could not find conditions that allowed for co-immunoprecipitation of ankyrin with the sodium channel β1 subunit.