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Patients with pathogenic variants in the TANGO2 gene suffer from severe and recurrent rhabdomyolysis (RM) episodes precipitated by fasting. Since starvation promotes autophagy induction, we wondered whether TANGO2-related muscle symptoms result from autophagy insufficiency to meet cellular demands in stress conditions. Autophagy functioning was analyzed in vitro, in primary skeletal muscle cells from TANGO2 patients in basal and fasting conditions. In addition, wce developed a tango2 morphant zebrafish model to assess the effect of tango2 knockdown (KD) on locomotor function and autophagy efficiency in vivo. We report that TANGO2 mutations are associated with decreased LC3-II levels upon starvation in primary muscle cells, but not in fibroblasts. In zebrafish larvae, tango2 knockdown induces locomotor defects characterized by reduced evoked movements which are exacerbated by exposure to atorvastatin, a compound known to cause RM. Importantly, RM features of tango2 KD are also associated with autophagy defects in zebrafish. Calpeptin treatment, a known activator of autophagy, is sufficient to rescue the locomotor function and improves autophagy in zebrafish. LC3-II levels of primary muscle cells of TANGO2 patients are also ameliorated by calpeptin treatment. Overall, we demonstrate that TANGO2 plays an important role in autophagy, and that autophagy efficiency is critical to prevent RM, thus giving rise to new therapeutic perspectives in the prevention of these life-threatening episodes in the context of TANGO2 pathology.

Epilepsy represents one of the most common neurological disorders, affecting an estimated 50 million people worldwide. Recent advances in genetic research have uncovered a large spectrum of genes implicated in various forms of epilepsy, highlighting the heterogeneous nature of this disorder. Appropriate animal models are essential for investigating the pathological mechanisms triggered by genetic mutations implicated in epilepsy and for developing specialized, targeted therapies. In recent years, zebrafish has emerged as a valuable vertebrate organism for modeling epilepsies, with the use of both genetic manipulation and exposure to known epileptogenic drugs, such as pentylenetetrazole (PTZ), to identify novel anti-epileptic therapeutics. Deleterious mutations in the mTOR regulator DEPDC5 have been associated with various forms of focal epilepsies and knock-down of the zebrafish orthologue causes hyperactivity associated with spontaneous seizure-like episodes, as well as enhanced electrographic activity and characteristic turn wheel swimming. Here, we described the method involved in generating the DEPDC5 loss-of-function model and illustrate the protocol for assessing motor activity at 28 and 48 h post fertilization (hpf), as well as a method for recording field activity in the zebrafish optic tectum. An illustration of the effect of the epileptogenic drug PTZ on neuronal activity over time is also provided.

Motor neuron diseases are untreatable with common pharmacological approaches. Spinal muscular atrophy (SMA) is caused by SMN1 gene mutations leading to lowered SMN expression. Symptoms are alleviated in infants with a higher copy number of the SMN2 gene, which, however, displays a splicing defect resulting in low SMN levels. Amyotrophic lateral sclerosis (ALS) is caused by a number of mutations, with C9orf72 repeat expansions the most common genetic cause and SOD1 gain-of-function mutations the first genetic cause identified for this disease. Genetic therapies based on oligonucleotides that enhance SMN2 splicing and SMN production or lower SOD1 expression have shown promise in initial clinical trials for individuals with SMA and ALS harboring SOD1 mutations, respectively. Gene addition/silencing approaches using adeno-associated viruses (AAVs) are also currently under clinical investigation in trials for SMA and ALS. Here we provide a brief overview of these efforts and their advantages and challenges. We also review genome editing approaches aimed at correcting the disease-causing mutations or modulating the expression of genetic modifiers, e.g., by repairing SOD1 mutations or the SMN2 splicing defect or deleting C9orf72 expanded repeats. These studies have shown promising results to approach therapeutic trials that should significantly lower the progression of these deadly disorders.

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Several genetic causes have been recently described for neurological diseases, increasing our knowledge of the common pathological mechanisms involved in these disorders. Mutation analysis has shown common causative factors for two major neurodegenerative disorders, ALS and FTD. Shared pathological and genetic markers as well as common neurological signs between these diseases have given rise to the notion of an ALS/FTD spectrum. This overlap among genetic factors causing ALS/FTD and the coincidence of mutated alleles (including causative, risk and modifier variants) have given rise to the notion of an oligogenic model of disease. In this review we summarize major advances in the elucidation of novel genetic factors in these diseases which have led to a better understanding of the common pathogenic factors leading to neurodegeneration.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressing, fatal disorder with no effective treatment. We used simple genetic models of ALS to screen phenotypically for potential therapeutic compounds. We screened libraries of compounds in C. elegans, validated hits in zebrafish, and tested the most potent molecule in mice and in a small clinical trial. We identified a class of neuroleptics that restored motility in C. elegans and in zebrafish, and the most potent was pimozide, which blocked T-type Ca2+ channels in these simple models and stabilized neuromuscular transmission in zebrafish and enhanced it in mice. Finally, a short randomized controlled trial of sporadic ALS subjects demonstrated stabilization of motility and evidence of target engagement at the neuromuscular junction. Simple genetic models are, thus, useful in identifying promising compounds for the treatment of ALS, such as neuroleptics, which may stabilize neuromuscular transmission and prolong survival in this disease.

The most common genetic cause for amyotrophic lateral sclerosis and frontotemporal dementia (ALS-FTD) is repeat expansion of a hexanucleotide sequence (GGGGCC) within the C9orf72 genomic sequence. To elucidate the functional role of C9orf72 in disease pathogenesis, we identified certain molecular interactors of this factor. We determined that C9orf72 exists in a complex with SMCR8 and WDR41 and that this complex acts as a GDP/GTP exchange factor for RAB8 and RAB39, 2 RAB GTPases involved in macroautophagy/autophagy. Consequently, C9orf72 depletion in neuronal cultures leads to accumulation of unresolved aggregates of SQSTM1/p62 and phosphorylated TARDBP/TDP-43. However, C9orf72 reduction does not lead to major neuronal toxicity, suggesting that a second stress may be required to induce neuronal cell death. An intermediate size of polyglutamine repeats within ATXN2 is an important genetic modifier of ALS-FTD. We found that coexpression of intermediate polyglutamine repeats (30Q) of ATXN2 combined with C9orf72 depletion increases the aggregation of ATXN2 and neuronal toxicity. These results were confirmed in zebrafish embryos where partial C9orf72 knockdown along with intermediate (but not normal) repeat expansions in ATXN2 causes locomotion deficits and abnormal axonal projections from spinal motor neurons. These results demonstrate that C9orf72 plays an important role in the autophagy pathway while genetically interacting with another major genetic risk factor, ATXN2, to contribute to ALS-FTD pathogenesis.

An intronic expansion of GGGGCC repeats within the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia (ALS-FTD). Ataxin-2 with intermediate length of polyglutamine expansions (Ataxin-2 Q30x) is a genetic modifier of the disease. Here, we found that C9ORF72 forms a complex with the WDR41 and SMCR8 proteins to act as a GDP/GTP exchange factor for RAB8a and RAB39b and to thereby control autophagic flux. Depletion of C9orf72 in neurons partly impairs autophagy and leads to accumulation of aggregates of TDP-43 and P62 proteins, which are histopathological hallmarks of ALS-FTD SMCR8 is phosphorylated by TBK1 and depletion of TBK1 can be rescued by phosphomimetic mutants of SMCR8 or by constitutively active RAB39b, suggesting that TBK1, SMCR8, C9ORF72, and RAB39b belong to a common pathway regulating autophagy. While depletion of C9ORF72 only has a partial deleterious effect on neuron survival, it synergizes with Ataxin-2 Q30x toxicity to induce motor neuron dysfunction and neuronal cell death. These results indicate that partial loss of function of C9ORF72 is not deleterious by itself but synergizes with Ataxin-2 toxicity, suggesting a double-hit pathological mechanism in ALS-FTD.

Summary: The maintenance of hydromineral homeostasis requires bidirectional detection of changes in extracellular fluid osmolality by primary osmosensory neurons (ONs) in the organum vasculosum laminae terminalis (OVLT). Hypertonicity excites ONs in part through the mechanical activation of a variant transient receptor potential vanilloid-1 channel (dn-Trpv1). However, the mechanism by which local hypotonicity inhibits ONs in the OVLT remains unknown. Here, we show that hypotonicity can reduce the basal activity of dn-Trpv1 channels and hyperpolarize acutely isolated ONs. Surprisingly, we found that mice lacking dn-Trpv1 maintain normal inhibitory responses to hypotonicity when tested in situ. In the intact setting, hypotonicity inhibits ONs through a non-cell-autonomous mechanism that involves glial release of the glycine receptor agonist taurine through hypotonicity activated anion channels (HAAC) that are activated subsequent to Ca2+ influx through Trpv4 channels. Our study clarifies how Trpv4 channels contribute to the inhibition of OVLT ONs during hypotonicity in situ. : Ciura et al. show that osmosensory neurons in organum vasculosum lamina terminalis are inhibited by hypotonicity. This effect is triggered by activation of Trpv4 channels and Ca2+ accumulation in astrocytes, causing these cells to release taurine through anion channels. Taurine inhibits firing by activating glycine receptors on the osmosensory neurons. Keywords: hyptonicity, taurine, TRPV, osmosensitive, gliotransmission, swelling

Contains fulltext : 167923.pdf (Publisher’s version ) (Open Access) Autosomal-recessive early-onset parkinsonism is clinically and genetically heterogeneous. The genetic causes of approximately 50% of autosomal-recessive early-onset forms of Parkinson disease (PD) remain to be elucidated. Homozygozity mapping and exome sequencing in 62 isolated individuals with early-onset parkinsonism and confirmed consanguinity followed by data mining in the exomes of 1,348 PD-affected individuals identified, in three isolated subjects, homozygous or compound heterozygous truncating mutations in vacuolar protein sorting 13C (VPS13C). VPS13C mutations are associated with a distinct form of early-onset parkinsonism characterized by rapid and severe disease progression and early cognitive decline; the pathological features were striking and reminiscent of diffuse Lewy body disease. In cell models, VPS13C partly localized to the outer membrane of mitochondria. Silencing of VPS13C was associated with lower mitochondrial membrane potential, mitochondrial fragmentation, increased respiration rates, exacerbated PINK1/Parkin-dependent mitophagy, and transcriptional upregulation of PARK2 in response to mitochondrial damage. This work suggests that loss of function of VPS13C is a cause of autosomal-recessive early-onset parkinsonism with a distinctive phenotype of rapid and severe progression.

Systemic osmoregulation is an integrated physiological process through which water intake and excretion are continuously balanced against salt intake and excretion to maintain the osmolality of the extracellular fluid near an optimal 'set-point' value. The behaviors (that is, thirst and sodium appetite) and renal responses (diuresis and natriuresis) that are modulated to mediate osmoregulatory homeostasis are mainly controlled by the nervous system. Appropriate regulation of these parameters depends in large part on specialized osmosensitive neurons, termed osmoreceptors, which convert changes in plasma osmolality into electrical signals that ultimately modulate effector functions to achieve homeostasis. Previous work has shown that mechanosensitive cation channels expressed in osmoreceptor neurons play a key role in the process of osmosensory transduction. Although the molecular identity of these channels remains unknown, a growing body of evidence, reviewed here, indicates that members of the transient receptor potential vanilloid family of ion channels may contribute to osmosensory transduction and to homeostatic responses implicated in the control of water balance.

SummaryIncreases in core body temperature promote thermoregulatory cooling by stimulating sweat production and preemptive renal water reabsorption through the release of vasopressin (VP, antidiuretic hormone). The mechanism by which the hypothalamus orchestrates this anticipatory VP release during hyperthermia is unknown but has been linked to a central thermosensory mechanism. Here, we report that thermal stimuli spanning core body temperatures activate a calcium-permeable, ruthenium red- and SB366791-sensitive nonselective cation conductance in hypothalamic VP neurons. This response is associated with a depolarizing receptor potential and an increase in action potential firing rate, indicating that these neurons are intrinsically thermosensitive. The thermosensitivity of VP neurons isolated from trpv1 knockout (Trpv1−/−) mice was significantly lower than that of wild-type counterparts. Moreover, Trpv1−/− mice showed an impaired VP response to hyperthermia in vivo. Channels encoded by the trpv1 gene thus confer thermosensitivity in central VP neurons and contribute to the thermal control of VP release in vivo.

In mammals, the osmolality of the extracellular fluid is maintained near a predetermined set-point through a negative feedback regulation of thirst, diuresis, salt appetite and natriuresis. This homeostatic control is believed to be mediated by osmosensory neurones which synaptically regulate the electrical activity of command neurones that mediate each of these osmoregulatory effector responses. Our present understanding of the molecular, cellular and network basis that underlies the central control of osmoregulation is largely derived from studies on primary osmosensory neurones in the organum vasculosum lamina terminalis (OVLT) and effector neurones in the supraoptic nucleus (SON), which release hormones that regulate diuresis and natriuresis. Primary osmosensory neurones in the OVLT exhibit changes in action potential firing rate that vary in proportion with ECF osmolality. This effect results from the intrinsic depolarizing receptor potential which these cells generate via a molecular transduction complex that may comprise various members of the transient receptor potential vanilloid (TRPV) family of cation channel proteins, notably TRPV1 and TRPV4. Osmotically evoked changes in the firing rate of OVLT neurones then regulate the electrical activity of downstream neurones in the SON through graded changes in glutamate release.

Collagen VI (COLVI), a protein ubiquitously expressed in connective tissues, is crucial for structural integrity, cellular adhesion, migration and survival. Six different genes are recognized in mammalians, encoding six COLVI-chains that assemble as two 'short' (α1, α2) and one 'long' chain (theoretically any one of α3-6). In humans, defects in the most widely expressed heterotrimer (α123), due to mutations in the COL6A1-3 genes, cause a heterogeneous group of neuromuscular disorders, collectively termed COLVI-related muscle disorders. Little is known about the function(s) of the recently described α4-6 chains and no mutations have been detected yet. In this study, we characterized two novel COLVI long chains in zebrafish that are most homologous to the mammalian α4 chain; therefore, we named the corresponding genes col6a4a and col6a4b. These orthologues represent ancestors of the mammalian Col6a4-6 genes. By in situ hybridization and RT-qPCR, we unveiled a distinctive expression kinetics for col6a4b, compared with the other col6a genes. Using morpholino antisense oligonucleotides targeting col6a4a, col6a4b and col6a2, we modelled partial and complete COLVI deficiency, respectively. All morphant embryos presented altered muscle structure and impaired motility. While apoptosis was not drastically increased, autophagy induction was defective in all morphants. Furthermore, motoneuron axon growth was abnormal in these morphants. Importantly, some phenotypical differences emerged between col6a4a and col6a4b morphants, suggesting only partial functional redundancy. Overall, our results further confirm the importance of COLVI in zebrafish muscle development and may provide important clues for potential human phenotypes associated with deficiency of the recently described COLVI-chains.

Mutations in SQSTM1, encoding for the protein SQSTM1/p62, have been recently reported in 1-3.5% of patients with amyotrophic lateral sclerosis and frontotemporal lobar degeneration (ALS/FTLD). Inclusions positive for SQSTM1/p62 have been detected in patients with neurodegenerative disorders, including ALS/FTLD. In order to investigate the pathogenic mechanisms induced by SQSTM1 mutations in ALS/FTLD, we developed a zebrafish model. Knock-down of the sqstm1 zebrafish ortholog, as well as impairment of its splicing, led to a specific phenotype, consisting of behavioral and axonal anomalies. Here, we report swimming deficits associated with shorter motor neuronal axons that could be rescued by the overexpression of wild-type human SQSTM1. Interestingly, no rescue of the loss-of-function phenotype was observed when overexpressing human SQSTM1 constructs carrying ALS/FTLD-related mutations. Consistent with its role in autophagy regulation, we found increased mTOR levels upon knock-down of sqstm1. Furthermore, treatment of zebrafish embryos with rapamycin, a known inhibitor of the mTOR pathway, yielded an amelioration of the locomotor phenotype in the sqstm1 knock-down model. Our results suggest that loss-of-function of SQSTM1 causes phenotypic features characterized by locomotor deficits and motor neuron axonal defects that are associated with a misregulation of autophagic processes.

The mechanisms regulating the generation of cell diversity in the mammalian cerebral cortex are beginning to be elucidated. In that regard, Hairy/Enhancer of split (Hes) 1 and 5 are basic helix-loop-helix (bHLH) factors that inhibit the differentiation of pluripotent cortical progenitors into neurons. In contrast, a related Hes family member termed Hes6 promotes neurogenesis. It is shown here that knockdown of endogenous Hes6 causes supernumerary cortical progenitors to differentiate into cells that exhibit an astrocytic morphology and express the astrocyte marker protein GFAP. Conversely, exogenous Hes6 expression in cortical progenitors inhibits astrocyte differentiation. The negative effect of Hes6 on astrocyte differentiation is independent of its ability to promote neuronal differentiation. We also show that neither its proneuronal nor its anti-gliogenic functions appear to depend on Hes6 ability to bind to DNA via the basic arm of its bHLH domain. Both of these activities require Hes6 to be localized to nuclei, but only its anti-gliogenic function depends on two short peptides, LNHLL and WRPW, that are conserved in all Hes6 proteins. These findings suggest that Hes6 is an important regulator of the neurogenic phase of cortical development by promoting the neuronal fate while suppressing astrocyte differentiation. They suggest further that separate molecular mechanisms underlie the proneuronal and anti-gliogenic activities of Hes6 in cortical progenitor cells.

Recent studies have indicated that members of the transient receptor potential vanilloid (TRPV) family of cation channels are required for the generation of normal osmoregulatory responses, yet the mechanism of osmosensory transduction in primary osmoreceptor neurons of the CNS remains to be defined. Indeed, despite ample evidence suggesting that the organum vasculosum lamina terminalis (OVLT) serves as the primary locus of the brain for the detection of osmotic stimuli, evidence that neurons in the OVLT are intrinsically osmosensitive has remained elusive. Here we show that murine OVLT neurons are intrinsically sensitive to increases in the osmolality of the extracellular fluid. Hypertonic conditions provoked increases in membrane cation conductance that resulted in the generation of an inward current, depolarizing osmoreceptor potentials, and enhanced action potential discharge. Moreover, we found that this osmosensory signal transduction cascade was absent in OVLT neurons from TRPV1 knock-out (TRPV1−/−) mice and that responses of wild type (WT) OVLT neurons could be blocked by ruthenium red, an inhibitor of TRPV channels. Finally,TRPV1−/−mice showed significantly attenuated water intake in response to systemic hypertonicity compared with WT controls. These findings indicate that OVLT neurons act as primary osmoreceptors and that a product of thetrpv1gene is required for osmosensory transduction.

SummaryThirst and antidiuretic hormone secretion occur during hyperthermia or hypertonicity to preserve body hydration. These vital responses are triggered when hypothalamic osmoregulatory neurons become depolarized by ion channels encoded by an unknown product of the transient receptor potential vanilloid-1 gene (Trpv1). Here, we show that rodent osmoregulatory neurons express a transcript of Trpv1 that mediates the selective translation of a TRPV1 variant that lacks a significant portion of the channel’s amino terminus (ΔN-TRPV1). The mRNA transcript encoding this variant (Trpv1dn) is widely expressed in the brains of osmoregulating vertebrates, including the human hypothalamus. Transfection of Trpv1dn into heterologous cells induced the expression of ion channels that could be activated by either hypertonicity or by heating in the physiological range. Moreover, expression of Trpv1dn rescued the osmosensory and thermosensory responses of single hypothalamic neurons obtained from Trpv1 knockout mice. ΔN-TRPV1 is therefore a co-detector of core body temperature and fluid tonicity.

Objective: To define the role that repeat expansions of a GGGGCC hexanucleotide sequence of the C9orf72 gene play in the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). A genetic model for ALS was developed to determine whether loss of function of the zebrafish orthologue of C9orf72 (zC9orf72) leads to abnormalities in neuronal development. Methods: C9orf72 mRNA levels were quantified in brain and lymphoblasts derived from FTLD and ALS=FTLD patients and in zebrafish. Knockdown of the zC9orf72 was performed using 2 specific antisense morpholino oligonucleotides to block transcription. Quantifications of spontaneous swimming and tactile escape response, as well as measurements of axonal projections from the spinal cord, were performed. Results: Significantly decreased expression of C9orf72 transcripts in brain and lymphoblasts was found in sporadic FTLD and ALS=FTLD patients with normal-size or expanded hexanucleotide repeats. The zC9orf72 is selectively expressed in the developing nervous system at developmental stages. Loss of function of the zC9orf72 transcripts causes both behavioral and cellular deficits related to locomotion without major morphological abnormalities. These deficits were rescued upon overexpression of human C9orf72 mRNA transcripts. Interpretation: Our results indicate C9orf72 haploinsufficiency could be a contributing factor in the spectrum of ALS=FTLD neurodegenerative disorders. Loss of function of the zebrafish orthologue of zC9orf72 expression in zebrafish is associated with axonal degeneration of motor neurons that can be rescued by expressing human C9orf72 mRNA, highlighting the specificity of the induced phenotype. These results reveal a pathogenic consequence of decreased C9orf72 levels, supporting a loss of function mechanism of disease. ANN NEUROL 2013;74:180–187

Homozygous mutations in TREM2 have been recently identified by exome sequencing in families presenting with frontotemporal dementia (FTD)–like phenotype. No study has evaluated the exact frequency of TREM2 mutations in cohorts of FTD patients so far. We sequenced TREM2 in 175 patients with pure FTD, mostly French, to test whether mutations could be implicated in the pathogenesis of the disease. No disease-causing mutation was identified in 175 individuals from the French cohort of FTD patients. We did not identify the polymorphism p.R47H (rs75932628), strongly associated with an increased risk of developing Alzheimer's disease. We conclude that TREM2 mutations are extremely rare in patients with pure FTD, although further investigation in larger populations is needed.

The DNA/RNA-binding proteins TDP-43 and FUS are found in protein aggregates in a growing number of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and related dementia, but little is known about the neurotoxic mechanisms. We have generated Caenorhabditis elegans and zebrafish animal models expressing mutant human TDP-43 (A315T or G348C) or FUS (S57Δ or R521H) that reflect certain aspects of ALS including motor neuron degeneration, axonal deficits, and progressive paralysis. To explore the potential of our humanized transgenic C. elegans and zebrafish in identifying chemical suppressors of mutant TDP-43 and FUS neuronal toxicity, we tested three compounds with potential neuroprotective properties: lithium chloride, methylene blue and riluzole. We identified methylene blue as a potent suppressor of TDP-43 and FUS toxicity in both our models. Our results indicate that methylene blue can rescue toxic phenotypes associated with mutant TDP-43 and FUS including neuronal dysfunction and oxidative stress.

Primary osmosensory neurons in the mouse organum vasculosum lamina terminalis (OVLT) transduce hypertonicity via the activation of nonselective cation channels that cause membrane depolarization and increased action potential discharge, and this effect is absent in mice lacking expression of the transient receptor potential vanilloid 1 (Trpv1) gene (Ciura and Bourque, 2006). However other experiments have indicated that channels encoded byTrpv4also contribute to central osmosensation in mice (Liedtke and Friedman, 2003; Mizuno et al., 2003). At present, the mechanism by which hypertonicity modulates cation channels in OVLT neurons is unknown, and it remains unclear whetherTrpv1andTrpv4both contribute to this process. Here, we show that physical shrinking is necessary and sufficient to mediate hypertonicity sensing in OVLT neurons isolated from adult mice. Steps coupling progressive decreases in cell volume to increased neuronal activity were quantitatively equivalent whether shrinking was evoked by osmotic pressure or mechanical aspiration. Finally, modulation of OVLT neurons by tonicity or mechanical stimulation was unaffected by deletion oftrpv4but was abolished in cells lackingTrpv1or wild-type neurons treated with the TRPV1 antagonist SB366791. Thus, hypertonicity sensing is a mechanical process requiringTrpv1, but notTrpv4.

The release of vasopressin (antidiuretic hormone) plays a key role in the osmoregulatory response of mammals to changes in salt or water intake and in the rate of water loss through evaporation during thermoregulatory cooling. Previous work has shown that the hypothalamus encloses the sensory elements that modulate vasopressin release during systemic changes in fluid osmolality or body temperature. These responses depend in part on a synaptic regulation of vasopressin neurons by afferent inputs arising from osmosensory and thermosensory neurons in the preoptic area. However, recent studies in rats and mice have shown that vasopressin neurons in the supraoptic nucleus also display intrinsic osmosensory and thermosensory properties. Isolated vasopressin neurons exposed to increases in perfusate temperature or osmolality generate increases in non-selective cation channel activity that cause membrane depolarization and increase neuronal excitability. These channels are calcium-permeable and can be blocked by ruthenium red. Moreover, intrinsic responses to osmotic and thermal stimuli are absent in magnocellular neurosecretory cells isolated from mice lacking the transient receptor potential vanilloid-1 (trpv1) gene, which encodes the capsaicin receptor. Immunostaining of vasopressin-releasing neurons with anti-TRPV1 antibodies reveals the presence of amino acids present in the carboxy terminus of the protein, but not those lying in the amino terminal domain. Thus, magnocellular neurosecretory neurons appear to express an N-terminal variant of trpv1 which lacks sensitivity to capsaicin, but which enables osmosensing and thermosensing.

The neuropeptides oxytocin (OXT) and arginine vasopressin (AVP) contribute to the regulation of diverse cognitive and physiological functions including nociception. Indeed, OXT has been reported to be analgesic when administered directly into the brain, the spinal cord, or systemically. Here, we characterized the phenotype of oxytocin receptor (OTR) and vasopressin-1A receptor (V1AR) null mutant mice in a battery of pain assays. Surprisingly, OTR knock-out mice displayed a pain phenotype identical to their wild-type littermates. Moreover, systemic administration of OXT dose-dependently produced analgesia in both wild-type and OTR knock-out mice in three different assays, the radiant-heat paw withdrawal test, the von Frey test of mechanical sensitivity, and the formalin test of inflammatory nociception. In contrast, OXT-induced analgesia was completely absent in V1AR knock-out mice. In wild-type mice, OXT-induced analgesia could be fully prevented by pretreatment with a V1AR but not an OTR antagonist. Receptor binding studies demonstrated that the distribution of OXT and AVP binding sites in mouse lumbar spinal cord resembles the pattern observed in rat. AVP binding sites diffusely label the lumbar spinal cord, whereas OXT binding sites cluster in the substantia gelatinosa of the dorsal horn. In contrast, quantitative real-time reverse transcription (RT)-PCR revealed that V1AR but not OTR mRNA is abundantly expressed in mouse dorsal root ganglia, where it localizes to small- and medium-diameter cells as shown by single-cell RT-PCR. Hence, V1ARs expressed in dorsal root ganglia might represent a previously unrecognized target for the analgesic action of OXT and AVP.

Osmotic control of arginine vasopressin (AVP) and oxytocin (OXT) release from magnocellular neurosecretory cells (MNCs) of the supraoptic (SON) and paraventricular (PVN) nuclei is essential for body fluid homeostasis. The electrical activity of MNCs, which is regulated by intrinsic and extrinsic osmosensitive factors, is a primary determinant of blood AVP and OXT levels. Although we now understand many of the cellular mechanisms that mediate the osmotic control of electrical activity and secretion from MNCs, further insight is likely to emerge from a molecular analysis of these mechanisms. An important step towards this goal could be made through the use of mouse genetic models. However, the electrophysiological properties of MNCs in mice have not been characterized, making direct comparisons with the rat model somewhat difficult. In this study, we examined the electrical properties of MNCs from the mouse SON. Extracellular recordings from neurons in superfused explants revealed modes of basal and osmotically modulated firing very similar to those observed previously in rats. Recordings in hypothalamic slices confirmed that SON neurons receive kynurenic-acid-sensitive excitatory synaptic inputs from the organum vasculosum laminae terminalis (OVLT). Current-clamp recordings from acutely dissociated SON neurons showed proportional changes in membrane cation conductance during changes in fluid osmolality. We conclude, therefore, that MNCs in the mouse SON display intrinsic osmosensitive properties and firing patterns that are very similar to those reported in the rat. Mouse MNCs therefore represent a useful model for the study of molecular factors contributing to the osmotic control of AVP and OXT release.

Osmotic control of arginine vasopressin (AVP) and oxytocin (OXT) release from magnocellular neurosecretory cells (MNCs) of the supraoptic (SON) and paraventricular (PVN) nuclei is essential for body fluid homeostasis. The electrical activity of MNCs, which is regulated by intrinsic and extrinsic osmosensitive factors, is a primary determinant of blood AVP and OXT levels. Although we now understand many of the cellular mechanisms that mediate the osmotic control of electrical activity and secretion from MNCs, further insight is likely to emerge from a molecular analysis of these mechanisms. An important step towards this goal could be made through the use of mouse genetic models. However, the electrophysiological properties of MNCs in mice have not been characterized, making direct comparisons with the rat model somewhat difficult. In this study, we examined the electrical properties of MNCs from the mouse SON. Extracellular recordings from neurons in superfused explants revealed modes of basal and osmotically modulated firing very similar to those observed previously in rats. Recordings in hypothalamic slices confirmed that SON neurons receive kynurenic-acid-sensitive excitatory synaptic inputs from the organum vasculosum laminae terminalis (OVLT). Current-clamp recordings from acutely dissociated SON neurons showed proportional changes in membrane cation conductance during changes in fluid osmolality. We conclude, therefore, that MNCs in the mouse SON display intrinsic osmosensitive properties and firing patterns that are very similar to those reported in the rat. Mouse MNCs therefore represent a useful model for the study of molecular factors contributing to the osmotic control of AVP and OXT release.

In mammals, the osmolality of the extracellular fluid is maintained near a predetermined set-point through a negative feedback regulation of thirst, diuresis, salt appetite and natriuresis. This homeostatic control is believed to be mediated by osmosensory neurones which synaptically regulate the electrical activity of command neurones that mediate each of these osmoregulatory effector responses. Our present understanding of the molecular, cellular and network basis that underlies the central control of osmoregulation is largely derived from studies on primary osmosensory neurones in the organum vasculosum lamina terminalis (OVLT) and effector neurones in the supraoptic nucleus (SON), which release hormones that regulate diuresis and natriuresis. Primary osmosensory neurones in the OVLT exhibit changes in action potential firing rate that vary in proportion with ECF osmolality. This effect results from the intrinsic depolarizing receptor potential which these cells generate via a molecular transduction complex that may comprise various members of the transient receptor potential vanilloid (TRPV) family of cation channel proteins, notably TRPV1 and TRPV4. Osmotically evoked changes in the firing rate of OVLT neurones then regulate the electrical activity of downstream neurones in the SON through graded changes in glutamate release.

Research in Epilepsy relies strongly on animal models, either for describing genetic conditions involved in the disease or for testing potential drugs that might alleviate the symptoms [1]. In this study, we looked at the differential effect of Pentylenetetrazole (PTZ), a well-known epileptogenic drug [2], on 5-6 dpf zebrafish larvae of two different genetic conditions: a mutant line, where a gene known to be involved in focal epilepsy was specifically knocked down, as well as control mismatch oligonucleotide-injected zebrafish. The zebrafish larvae were scored for phenotypic features, including hyperactivity and aberrant locomotion. These phenotypic features were present in all mutant fish recorded, but were absent in the mismatch control. We acquired electrophysiological field recordings from the optic tectum before and after PTZ application and selected a low frequency band of the signal (0.05 - 0.5 Hz). Troughs, representing epileptic events, were extracted and a wide range of analyses were applied: total event count, event time histogram, event duration distributions, inter-event-interval (IEI) distribution, classical and scaled autocorrelations on the field and event signals [3]. Traditional analysis provided a quantitative evaluation of PTZ-induced epileptic events: an increase in number of epileptic events was observed in mutant fish with respect to control, and the event time histogram showed a more abrupt increase at 32-37 min. after PTZ application, whereas in the control case, the increase was constant throughout the response period. Compared to controls, in mutant fish, PTZ application led to a larger increase in the number of short (0.5-1.5 s) events with small IEI (< 10s). Correlation analysis revealed qualitative information about the effect of PTZ on field recordings: The scaled autocorrelation (scale segment of 1s) on the field signal exhibited oscillatory components around 0.4 Hz in all conditions, but mutant fish exhibited frequency variability with time after PTZ application and variability across animals, leading to a low average oscillation power. In controls the frequency was robustly locked at 0.4 Hz. Autocorrelation histograms computed on the extracted events evidenced a wide refractory period (~ 4 s, see Figure ​Figure1A),1A), followed by baseline in mutant fish recordings, whereas control animals exhibited a narrow refractory period (~ 2 s, see Figure ​Figure1B),1B), followed by a secondary peak and a slow modulation (~ 35 s). Scaled autocorrelation histograms (scale segment of 1000s) showed constant correlation decrease with increased lag in the control fish, but not in mutant fish, where correlation for larger lags fluctuated around zero (see Figure ​Figure1C1C and ​and1D1D). Figure 1 Correlation histograms on events from zebrafish field recordings: Top: Autocorrelation of mutant (A) versus control (B) animals; Bottom: Scaled autocorrelation with scale segment of 1000s of mutant (C) versus control (D) animals. The characteristics revealed by correlation analysis suggest potentially different mechanisms underlying PTZ-induced epileptic events. We therefore propose that, in addition to the traditional statistics on epileptic events, looking at the temporal characteristics and correlation structure of field recordings may lead to a better classification and understanding of the mechanisms underlying epileptogenesis in various models of epilepsy.

Mammalian osmosensory neurons exposed to hypertonicity display a shrinking-induced increase in non-selective cation conductance. That causes membrane depolarization and excitation triggering homeostatic responses such as thirst and antidiuretic hormone release. The molecular identity of the osmoreceptor channel is not known, but appears to involve a capsaicin-insensitive N-terminal variant of the transient receptor potential vanilloid type 1 (TRPV1) channel (Ciura and Bourque, 2006; Sharif-Naeini et al., 2006). Here, we report that heterologous expression of TRPV1 variant Δ1-283, which lacks exons 1-5, confers osmosensory characteristics similar to those found in native osmosensory neurons. Whole cell voltage clamp recordings were used to examine the effects of hyperosmolality (+mannitol) on human embryonic kidney (HEK293) cells expressing Δ1-283 TRPV1, GFP or wild type TRPV1. Dynamic imaging confirmed that all HEK cells exposed to hypertonicity underwent visible shrinking. However in contrast to GFP (n = 10) and TRPV1 (n = 32) transfected cells, which were unresponsive, ∼41% (84/206) of cells transfected with Δ1-283 TRPV1 showed a progressive and sustained increase in non-selective cation current and membrane conductance when exposed to a hyperosmotic stimuli lasting 2-5 minutes. The effects of osmolality were dose-dependent (+5 to +80 mosmol/kg), mimicked by suction induced cell shrinking (n=6), and were abolished by Ruthenium Red. Moreover, addition of capsaicin caused robust responses in cells expressing TRPV1, but had no effect on those expressing Δ1-283 TRPV1. Imaging experiments on cells loaded with Fura-2 revealed that ∼21% of the cells transfected with Δ1-283 TRPV1 display reversible increases in intracellular [Ca2+], whereas ∼17% shown an increase without reversion when exposed to a +40 mosmol/kg hyperosmotic stimulus. These results suggest Δ1-283 TRPV1 channels could mediate mechanically-induced increases in cation current associated with hypertonicity-induced shrinking in osmosensory neurons.

To investigate the pathogenic mechanisms induced by SQSTM1 mutations in ALS, we developed a zebrafish model of sqstm1 haploinsufficiency. We observed that loss of function of sqstm1 leads to a specific motor phenotype. To elucidate the common cellular mechanisms underlying motor neuron degeneration in ALS, we analyzedc9orf72 and sqstm1 epistatic interactions inzebrafish. C9orf72 and sqstm1 partial inhibitions have an additive effect and C9ORF72 rescues the phenotype induced by sqstm1 knockdown. Thus, both proteins belong to the same pathway and c9orf72 acts downstream of sqstm1. Also, we observed that depletion of these genes in mouse motor neurons primary cultures leads to the early death of motor neurons associated with autophagy impairment. To develop a vertebrate model that recapitulates the different mechanisms associated withthe C9ORF72 HRE pathogenicity in ALS, we combined the partial inhibition of c9orf72 with the expression of the DPRs in zebrafish. This induces a robust motor phenotype characterized by locomotor defects and paralysis. Focusing on GP repeats, we observed that the loss of function of c9orf72 is essential to inhibitpoly(GP) clearance.This is associated with SQSTM1/p62 accumulation, severe motor neurons abnormalities and loss. These phenotypes are rescued by the inhibition of caspase 9, a regulator of apoptosis. Also, rapamycinis able to improve the clearance of poly(GP) and p62, with restored swim and motor neurons features, thus confirming the role of C9ORF72 in autophagy.These results show that DPR toxicity is related to lowered expression of C9ORF72, suggesting that both gain and loss of function synergize in the C9ORF72 HRE pathogenicity., Pour analyser les mécanismes pathogéniques de la mutation de SQSTM1 dans la SLA, nous avons développé un modèle d’haploinsuffisance de sqstm1 chez le poisson zèbre. La perte de fonction de ce gène induit un phénotype moteur caractéristique de la SLA. Pour élucider les mécanismes entraînant la dégénérescence des motoneurones dans cette maladie, nous avons étudié les interactions épistatiques de c9orf72 et de sqstm1 chez le poisson zèbre et nous avons observé que les deux protéines appartiennent à une voie cellulaire commune, avec c9orf72 agissant en aval de sqstm1. Dans des cultures primaires de motoneurones de souris, nous avons démontré que la perte de ces gènes entraîne la mort des motoneurones associée à des défauts de l’autophagie. Afin de développer un modèle qui récapitulerait les différents mécanismes de la mutation de C9ORF72 dans la SLA, nous avons combiné l’inhibition partielle de ce gène avec l’expression des DPRs chez le poisson zèbre. Cela induit un phénotype moteur caractérisé par des défauts locomoteurs et une paralysie. En particulier, nous avons observé que les répétitions de GP s’accumulent lorsque c9orf72 est inhibé. Ceci est associé à une accumulation de SQSTM1/p62 ainsi qu’à la perte des motoneurones. Ces phénotypes sont limités lors de l’inhibition de la caspase 9, un régulateur essentiel de l’apoptose. De plus, en activant l’autophagie avec la rapamycine, nous avons amélioré la clairance des GP et de p62, restaurant ainsi les paramètres moteurs. Ces résultats montrent que la toxicité des DPRs est relative au niveau de de l’expression de C9ORF72, suggérant que le gain et la perte de fonction synergisent dans la pathogénicité de C9ORF72.