Evidence for Two Subpopulations of Cerebrospinal Fluid-Contacting Neurons with Opposite GABAergic Signaling in Adult Mouse Spinal Cord.

Spinal cerebrospinal fluid-contacting neurons (CSF-cNs) form an evolutionary conserved bipolar cell population localized around the central canal of all vertebrates. CSF-cNs were shown to express molecular markers of neuronal immaturity into adulthood; however, the impact of their incomplete maturation on the chloride (Cl-) homeostasis as well as GABAergic signaling remains unknown. Using adult mice from both sexes, in situ hybridization revealed that a proportion of spinal CSF-cNs (18.3%) express the Na+-K+-Cl- cotransporter 1 (NKCC1) allowing intracellular Cl- accumulation. However, we did not find expression of the K+-Cl- cotransporter 2 (KCC2) responsible for Cl- efflux in any CSF-cNs. The lack of KCC2 expression results in low Cl- extrusion capacity in CSF-cNs under high Cl- load in whole-cell patch clamp. Using cell-attached patch clamp allowing recordings with intact intracellular Cl- concentration, we found that the activation of ionotropic GABAA receptors (GABAA-Rs) induced both depolarizing and hyperpolarizing responses in CSF-cNs. Moreover, depolarizing GABA responses can drive action potentials as well as intracellular calcium elevations by activating voltage-gated calcium channels. Blocking NKCC1 with bumetanide inhibited the GABA-induced calcium transients in CSF-cNs. Finally, we show that metabotropic GABAB receptors have no hyperpolarizing action on spinal CSF-cNs as their activation with baclofen did not mediate outward K+ currents, presumably due to the lack of expression of G-protein-coupled inwardly rectifying potassium (GIRK) channels. Together, these findings outline subpopulations of spinal CSF-cNs expressing inhibitory or excitatory GABAA-R signaling. Excitatory GABA may promote the maturation and integration of young CSF-cNs into the existing spinal circuit.

Evidence for PKD2L1-positive neurons distant from the central canal in the ventromedial spinal cord and medulla of the adult mouse.

Neurons in contact with the cerebrospinal fluid (CSF) are found around the medullo-spinal central canal (CC) in adult mice. These neurons (CSF-cNs), located within or below the ependymal cell layer, known as the stem cell niche, present a characteristic morphology with a dendrite projecting to the CC and ending with a protrusion. They are GABAergic, present an intermediate neuronal maturity and selectively express PKD2L1, a member of the transient receptor potential channel superfamily with sensory properties. Using immunohistological and electrophysiological recording techniques in mice, we characterize the properties of a new population of PKD2L1 positive cells that is distant from the CC in a zone enriched with astrocytes and ependymal fibers of the ventro-medial spinal cord and medulla. They appear around embryonic day 16 and their number increases up to early postnatal days. With development and the reorganization of the CC region, they progressively become more distant from the CC, suggesting some migratory capabilities. These neurons share functional and phenotypical properties with CSF-cNs but appear subdivided in two groups. One group, present along the midline, has a bipolar morphology and extends a long dendrite along ependymal fibers and towards the CC. The second group, localized in more ventro-lateral regions, has a multipolar morphology and no apparent projection to the CC. Altogether, we describe a novel population of PKD2L1+ neurons distant from the CC but with properties similar to CSF-cNs that might serve to sense modification in the composition of either CSF or interstitial liquid, a function that will need to be confirmed.

GABAB receptors modulate Ca2+ but not G protein-gated inwardly rectifying K+ channels in cerebrospinal-fluid contacting neurones of mouse brainstem.

KEY POINTS: Medullo-spinal CSF contacting neurones (CSF-cNs) located around the central canal are conserved in all vertebrates and suggested to be a novel sensory system intrinsic to the CNS. CSF-cNs receive GABAergic inhibitory synaptic inputs involving ionotropic GABAA receptors, but the contribution of metabotropic GABAB receptors (GABAB -Rs) has not yet been studied. Here, we indicate that CSF-cNs express functional GABAB -Rs that inhibit postsynaptic calcium channels but fail to activate inhibitory potassium channel of the Kir3-type. We further show that GABAB -Rs localise presynaptically on GABAergic and glutamatergic synaptic inputs contacting CSF-cNs, where they inhibit the release of GABA and glutamate. Our data are the first to address the function of GABAB -Rs in CSF-cNs and show that on the presynaptic side they exert a classical synaptic modulation whereas at the postsynaptic level they have an atypical action by modulating calcium signalling without inducing potassium-dependent inhibition. ABSTRACT: Medullo-spinal neurones that contact the cerebrospinal fluid (CSF-cNs) are a population of evolutionary conserved cells located around the central canal. CSF-cN activity has been shown to be regulated by inhibitory synaptic inputs involving ionotropic GABAA receptors, but the contribution of the G-protein coupled GABAB receptors has not yet been studied. Here, we used a combination of immunofluorescence, electrophysiology and calcium imaging to investigate the expression and function of GABAB -Rs in CSF-cNs of the mouse brainstem. We found that CSF-cNs express GABAB -Rs, but their selective activation failed to induce G protein-coupled inwardly rectifying potassium (GIRK) currents. Instead, CSF-cNs express primarily N-type voltage-gated calcium (CaV 2.2) channels, and GABAB -Rs recruit Gßγ subunits to inhibit CaV channel activity induced by membrane voltage steps or under physiological conditions by action potentials. Moreover, using electrical stimulation, we indicate that GABAergic inhibitory (IPSCs) and excitatory glutamatergic (EPSCs) synaptic currents can be evoked in CSF-cNs showing that mammalian CSF-cNs are also under excitatory control by glutamatergic synaptic inputs. We further demonstrate that baclofen reversibly reduced the amplitudes of both IPSCs and EPSCs evoked in CSF-cNs through a presynaptic mechanism of regulation. In summary, these results are the first to demonstrate the existence of functional postsynaptic GABAB -Rs in medullar CSF-cNs, as well as presynaptic GABAB auto- and heteroreceptors regulating the release of GABA and glutamate. Remarkably, postsynaptic GABAB -Rs associate with CaV but not GIRK channels, indicating that GABAB -Rs function as a calcium signalling modulator without GIRK-dependent inhibition in CSF-cNs.

The Role of MicroRNA in the Modulation of the Melanocortinergic System.

The central control of energy balance involves a highly regulated neuronal network within the hypothalamus and the dorsal vagal complex. In these structures, pro-opiomelanocortin (POMC) neurons are known to reduce meal size and to increase energy expenditure. In addition, leptin, a peripheral signal that relays information regarding body fat content, modulates the activity of melanocortin pathway neurons including POMC-, Agouti-related peptide (AgRP)/Neuropeptide Y (NPY)-, melanocortin receptors (MC3R and MC4R)-expressing neurons. MicroRNAs (miRNAs) are short non-coding RNAs of 22-26 nucleotides that post-transcriptionally interfere with target gene expression by binding to their mRNAs. Evidence has demonstrated that miRNAs play important roles in the central regulation of energy balance. In this context, different studies identified miRNAs including miR-200 family, miR-103, or miR-488 that could target the genes of melanocortin pathway. More precisely, these different miRNAs can modulate energy homeostasis by affecting leptin transduction pathway in the POMC, or AgRP/NPY neurons. This article reviews the role of identified miRNAs in the modulation of melanocortin pathway in the context of energy homeostasis.

Postnatal maturation of mouse medullo-spinal cerebrospinal fluid-contacting neurons.

The central canal along the spinal cord (SC.) and medulla is characterized by the presence of a specific population of neurons that contacts the cerebrospinal fluid (CSF). These medullo-spinal CSF-contacting neurons (CSF-cNs) are identified by the selective expression of the polycystin kidney disease 2-like 1 ionic channel (PKD2L1 or polycystin-L). In adult, they have been shown to express doublecortin (DCX) and Nkx6.1, two markers of juvenile neurons along with the neuron-specific nuclear protein (NeuN) typically expressed in mature neurons. They were therefore suggested to remain in a rather incomplete maturation state. The aim of this study was to assess whether such juvenile state is stable in postnatal animals or whether CSF-cNs may reach maturity at older stages than neurons in the parenchyma. We show, in the cervical SC. and the brainstem that, in relation to age, CSF-cN density declines and that their cell bodies become more distant from the cc, except in its ventral part. Moreover, in adults (from 1month) by comparison with neonatal mice, we show that CSF-cNs have evolved to a more mature state, as indicated by the increase in the percentage of cells positive for NeuN and of its level of expression. In parallel, CSF-cNs exhibit, in adult, lower DCX immunoreactivity and do not express PSA-NCAM and TUC4, two neurogenic markers. Nevertheless, CSF-cNs still share in adult characteristics of juvenile neurons such as the presence of phospho-CREB and DCX while NeuN expression remained low. This phenotype persists in 12-month-old animals. Thus, despite a pursuit of neuronal maturation during the postnatal period, CSF-cNs retain a durable low differentiated state.

An Emerging Role of micro-RNA in the Effect of the Endocrine Disruptors.

Endocrine-disrupting chemicals (EDCs) are diverse natural and synthetic chemicals that may alter various mechanisms of the endocrine system and produce adverse developmental, reproductive, metabolic, and neurological effects in both humans and wildlife. Research on EDCs has revealed that they use a variety of both nuclear receptor-mediated and non-receptor-mediated mechanisms to modulate different components of the endocrine system. The molecular mechanisms underlying the effects of EDCs are still under investigation. Interestingly, some of the effects of EDCs have been observed to pass on to subsequent unexposed generations, which can be explained by the gametic transmission of deregulated epigenetic marks. Epigenetics is the study of heritable changes in gene expression that occur without a change in the DNA sequence. Epigenetic mechanisms, including histone modifications, DNA methylation, and specific micro-RNAs (miRNAs) expression, have been proposed to mediate transgenerational transmission and can be triggered by environmental factors. MiRNAs are short non-coding RNA molecules that post-transcriptionally repress the expression of genes by binding to 3'-untranslated regions of the target mRNAs. Given that there is mounting evidence that miRNAs are regulated by hormones, then clearly it is important to investigate the potential for environmental EDCs to deregulate miRNA expression and action.

A single polycystic kidney disease 2-like 1 channel opening acts as a spike generator in cerebrospinal fluid-contacting neurons of adult mouse brainstem.

Cerebrospinal fluid contacting neurons (CSF-cNs) are found around the central canal of all vertebrates. They present a typical morphology, with a single dendrite that projects into the cavity and ends in the CSF with a protuberance. These anatomical features have led to the suggestion that CSF-cNs might have sensory functions, either by sensing CSF movement or composition, but the physiological mechanisms for any such role are unknown. This hypothesis was recently supported by the demonstration that in several vertebrate species medullo-spinal CSF-cNs selectively express Polycystic Kidney Disease 2-Like 1 proteins (PKD2L1). PKD2L1 are members of the 'transient receptor potential (TRP)' superfamily, form non-selective cationic channels of high conductance, are regulated by various stimuli including protons and are therefore suggested to act as sensory receptors. Using patch-clamp whole-cell recordings of CSF-cNs in brainstem slices obtained from wild type and mutant PKD2L1 mice, we demonstrate that spontaneously active unitary currents in CSF-cNs are due to PKD2L1 channels that are capable, with a single opening, of triggering action potentials. Thus PKD2L1 might contribute to the setting of CSF-cN spiking activity. We also reveal that CSF-cNs have the capacity of discriminating between alkalinization and acidification following activation of specific conductances (PKD2L1 vs. ASIC) generating specific responses. Altogether, this study reinforces the idea that CSF-cNs represent sensory neurons intrinsic to the central nervous system and suggests a role for PKD2L1 channels as spike generators.

Leptin is required for hypothalamic regulation of miRNAs targeting POMC 3'UTR.

The central nervous system (CNS) monitors modifications in metabolic parameters or hormone levels and elicits adaptive responses such as food intake regulation. Particularly, within the hypothalamus, leptin modulates the activity of pro-opiomelanocortin (POMC) neurons which are critical regulators of energy balance. Consistent with a pivotal role of the melanocortin system in the control of energy homeostasis, disruption of the POMC gene causes hyperphagia and obesity. MicroRNAs (miRNAs) are short noncoding RNA molecules that post-transcriptionally repress the expression of genes by binding to 3'-untranslated regions (3'UTR) of the target mRNAs. However, little is known regarding the role of miRNAs that target POMC 3'UTR in the central control energy homeostasis. Particularly, their interaction with the leptin signaling pathway remain unclear. First, we used common prediction programs to search for potential miRNAs target sites on 3'UTR of POMC mRNA. This screening identified a set of conserved miRNAs seed sequences for mir-383, mir-384-3p, and mir-488. We observed that mir-383, mir-384-3p, and mir-488 are up-regulated in the hypothalamus of leptin deficient ob/ob mice. In accordance with these observations, we also showed that mir-383, mir-384-3p, and mir-488 were increased in db/db mice that exhibit a non-functional leptin receptor. The intraperitoneal injection of leptin down-regulated the expression of these miRNAs of interest in the hypothalamus of ob/ob mice showing the involvement of leptin in the expression of mir-383, mir-384-3p, and mir-488. Finally, the evaluation of responsivity to intracerebroventricular administration of leptin exhibited that a chronic treatment with leptin decreased mir-488 expression in hypothalamus of C57BL/6 mice. In summary, these results suggest that leptin modulates the expression of miRNAs that target POMC mRNA in hypothalamus.

Morphology, distribution and phenotype of polycystin kidney disease 2-like 1-positive cerebrospinal fluid contacting neurons in the brainstem of adult mice.

The mammalian spinal cord and medulla oblongata harbor unique neurons that remain in contact with the cerebrospinal fluid (CSF-cNs). These neurons were shown recently to express a polycystin member of the TRP channels family (PKD2L1) that potentially acts as a chemo- or mechanoreceptor. Recent studies carried out in young rodents indicate that spinal CSF-cNs express immature neuronal markers that appear to persist even in adult cells. Nevertheless, little is known about the phenotype and morphological properties of medullar CSF-cNs. Using immunohistochemistry and confocal microscopy techniques on tissues obtained from three-month old PKD2L1:EGFP transgenic mice, we analyzed the morphology, distribution, localization and phenotype of PKD2L1(+) CSF-cNs around the brainstem and cervical spinal cord central canal. We show that PKD2L1(+) CSF-cNs are GABAergic neurons with a subependymal localization, projecting a dendrite towards the central canal and an axon-like process running through the parenchyma. These neurons display a primary cilium on the soma and the dendritic process appears to bear ciliary-like structures in contact with the CSF. PKD2L1(+) CSF-cNs present a conserved morphology along the length of the medullospinal central canal with a change in their density, localization and dendritic length according to the rostro-caudal axis. At adult stages, PKD2L1(+) medullar CSF-cNs appear to remain in an intermediate state of maturation since they still exhibit characteristics of neuronal immaturity (DCX positive, neurofilament 160 kDa negative) along with the expression of a marker representative of neuronal maturation (NeuN). In addition, PKD2L1(+) CSF-cNs express Nkx6.1, a homeodomain protein that enables the differentiation of ventral progenitors into somatic motoneurons and interneurons. The present study provides valuable information on the cellular properties of this peculiar neuronal population that will be crucial for understanding the physiological role of CSF-cNs in mammals and their link with the stem cells contained in the region surrounding the medullospinal central canal.

Gastric distension activates NUCB2/nesfatin-1-expressing neurons in the nucleus of the solitary tract.

Brainstem structures such as the nucleus of the solitary tract (NTS) and the dorsal motor nucleus of the vagus nerve (DMNX) are essential for the digestive function of the stomach. A large number of neurotransmitters including glutamate and gamma-aminobutyric acid (GABA) are involved in the central control of gastric functions. However, the neuropeptidergic systems implicated in this process remain undetermined. Nesfatin-1 was recently identified as a neuropeptide cleaved from the N-terminal part of NEFA/nucleobindin 2 precursor (NUCB2). Central administration of this neuropeptide inhibits food consumption and gastroduodenal motility in rodents. Interestingly, the NTS and the DMNX contain a dense population of NUCB2/nesfatin-1 cell bodies. These observations led us to investigate the possible involvement of NUCB2/nesfatin-1 neurons in the brainstem neuronal pathways that modulate gastric functions. We observed an activation of NTS NUCB2/nesfatinergic neurons after gastric distention in rats. In addition, we found that several NTS NUCB2/nesfatinergic neurons were GABAergic. Finally, when fluorogold was injected at the stomach level, many retrogradely labeled neurons were observed in the DMNX which were also positive for NUCB2/nesfatin-1. Taken together, these observations suggest for the first time that NUCB2/nesfatin-1 neurons of the NTS are sensitive to gastric distension and then may contribute to the satiety signal.

c-Fos immunoreactivity in the pig brain following deoxynivalenol intoxication: focus on NUCB2/nesfatin-1 expressing neurons.

Deoxynivalenol (DON), produced by the cereal-contaminating Fusarium fungi, is a major trichothecene responsible for mycotoxicoses in farm animals, including swine. The main effect of DON-intoxication is food intake reduction and the consequent body weight loss. The present study aimed to identify brain structures activated during DON intoxication in pigs. To this goal, we used c-Fos staining which constitutes a useful approach to identify activated neurons. We showed that per os administration of Fusarium graminearum extracts (containing the equivalent of 1mg DON per kg of body weight) induced an increase in c-Fos immunoreactivity in several central structures, including the ventrolateral medulla (VLM), dorsal vagal complex (DVC), paraventricular nucleus of the hypothalamus (PVN), arcuate nucleus (Arc), supraoptic nucleus (SON) and amygdala (CeA). Moreover, we coupled c-Fos staining with phenotypic markers detection in order to specify the neuronal populations activated during DON intoxication. This phenotypic characterization revealed the activation of catecholaminergic but not of serotoninergic neurons in response to the toxin. In this context, we also paid a particular attention to NUCB2/nesfatin-1 positive cells, since nesfatin-1 is known to exert a satiety effect. We report here, for the first time in the pig brain, the presence of NUCB2/nesfatin-1 neurons in the VLM, DVC, PVN, Arc and SON, and their activation during DON intoxication. Taken together, these data show that DON stimulates the main structures involved in food intake in pigs and suggest that catecholaminergic and NUCB2/nesfatin-1 neurons could contribute in the anorexigenic effects of the mycotoxin.

Properties of subependymal cerebrospinal fluid contacting neurones in the dorsal vagal complex of the mouse brainstem.

Cerebrospinal fluid (CSF) contacting neurones have been observed in various brain regions such as the hypothalamus, the dorsal nucleus of the raphe and around the central canal (cc) of the spinal cord but their functional role remains unclear. At the level of the spinal cord, subependymal cerebrospinal fluid contacting neurones (S-CSF-cNs) present a peculiar morphology with a soma close to the ependymal layer, a process projecting towards the cc and ending with a bud and a cilium. These neurones were recently shown to express polycystin kidney disease 2-like 1 (PKD2L1 or TRPP3) channels that are members of the polycystin subtype of the transient receptor potential (TRP) channel superfamily and that have been proposed as either chemo- or mechanoreceptors in several tissues. Using immunohistological techniques and whole-cell electrophysiological recordings in brain slices obtained from PKD2L1:EGFP transgenic adult mice, we looked for and determined the functional properties of S-CSF-cNs in the dorsal vagal complex (DVC), a hindbrain structure controlling autonomic functions such as blood pressure, energy balance and food intake. Here, we demonstrate that S-CSF-cNs received GABAergic and/or glycinergic synaptic entries and were also characterised by the presence of non-selective cationic channels of large conductance that could be detected even under whole-cell configuration. The channel activity was not affected by Psalmopoeus cambridgei toxin 1, a blocker of acid sensing ion channels (ASICs), but was blocked by amiloride and by a strong extracellular acidification. In contrast, extracellular alkalinisation and hypo-osmotic shocks increased channel activity. Based on these properties, we suggest that the single-channel activity recorded in medullar S-CSF-cNs is carried by PKD2L1 channels. Our study therefore reinforces the idea that PKD2L1 is a marker of S-CSF-cNs and points toward a role for S-CSF-cNs in the detection of circulating signals and of modifications in the extracellular environment.

The food-contaminant deoxynivalenol modifies eating by targeting anorexigenic neurocircuitry.

Physiological regulations of energy balance and body weight imply highly adaptive mechanisms which match caloric intake to caloric expenditure. In the central nervous system, the regulation of appetite relies on complex neurocircuitry which disturbance may alter energy balance and result in anorexia or obesity. Deoxynivalenol (DON), a trichothecene, is one of the most abundant mycotoxins found on contaminated cereals and its stability during processing and cooking explains its widespread presence in human food. DON has been implicated in acute and chronic illnesses in both humans and farm animals including weight loss. Here, we provide the first demonstration that DON reduced feeding behavior and modified satiation and satiety by interfering with central neuronal networks dedicated to food intake regulation. Moreover, our results strongly suggest that during intoxication, DON reaches the brain where it modifies anorexigenic balance. In view of the widespread human exposure to DON, the present results may lead to reconsider the potential consequences of chronic DON consumption on human eating disorders.

Central inflammation and sickness-like behavior induced by the food contaminant deoxynivalenol: a PGE2-independent mechanism.

Deoxynivalenol (DON), one of the most abundant trichothecenes found on cereals, has been implicated in mycotoxicoses in both humans and farm animals. Low-dose toxicity is characterized by reduced weight gain, diminished nutritional efficiency, and immunologic effects. The levels and patterns of human food commodity contamination justify that DON consumption constitutes a public health issue. DON stability during processing and cooking explains its large presence in human food. We characterized here DON intoxication by showing that the toxin concomitantly affects feeding behavior, body temperature, and locomotor activity after both per os and central administration. Using c-Fos expression mapping, we identified the neuronal structures activated in response to DON and observed that the pattern of neuronal populations activated by the toxin resembled those induced by inflammatory signals. By real-time PCR, we report the first evidences for a DON-induced central inflammation, attested by the strong upregulation of interleukin-1ß, interleukin-6, tumor necrosis factor-α, cyclooxygenase-2, and microsomal prostaglandin synthase-1 (mPGES-1) messenger RNA. However, silencing prostaglandins E2 signaling pathways using mPGES-1 knockout mice, which are resistant to cytokine-induced sickness behavior, did not modify the responses to the toxin. These results reveal that, despite strong similarities, behavioral changes observed after DON intoxication differ from classical sickness behavior evoked by inflammatory cytokines.

Glial cells of the nucleus tractus solitarius as partners of the dorsal hindbrain regulation of energy balance: a proposal for a working hypothesis.

While the evidences emphasizing the role of astroglial cells in numerous aspects of information processing within the brain merges, the literature dealing with the involvement of this cell population in the signalization involved in feeding behavior and energetic homeostasis remains scarce. Nevertheless, some clues are now available indicating that glia could play a dynamic role in the regulation of energy balance, and that strengthening research effort in this field may further our understanding of the mechanisms controlling feeding behaviour. In the present review, we have summarized recent data indicating that the multifaceted glial compartment of the brainstem should be considered in future research aimed at identifying feeding-related processes operating at this level.

Central nesfatin-1-expressing neurons are sensitive to peripheral inflammatory stimulus.

Recently, a novel factor with anorexigenic properties was identified and called nesfatin-1. This protein (82 aac) is not only expressed in peripheral organs but it is also found in neurons located in specific structures including the hypothalamus and the brainstem, two sites strongly involved in food intake regulation. Here, we studied whether some of the neurons that become activated following an injection of an anorectic dose of lipopolysaccharides (LPS) exhibit a nesfatin-1 phenotype. To this end, we used double immunohistochemistry to target the expression of the immediate-early gene c-fos and of nesfatin-1 on coronal frozen sections of the rat brain. The number of c-Fos+/nesfatin-1+ neurons was evaluated in the immunosensitive structures reported to contain nesfatin-1 neurons; i.e. paraventricular hypothalamic nucleus (PVN), supraoptic nucleus (SON), arcuate nucleus (ARC) and nucleus of the solitary tract (NTS). LPS strongly increased the number of c-Fos+/nesfatin-1+ neurons in the PVN, SON and NTS, and to a lesser extent in the ARC. Triple labeling showed that a portion of the nesfatin-1 neurons activated in response to LPS within the NTS are catecholaminergic since they co-express tyrosine hydroxylase (TH). Our data therefore indicate that a portion of nesfatin-1 neurons of both the hypothalamus and brainstem are sensitive to peripheral inflammatory signals, and provide the first clues suggesting that centrally released nesfatin-1 may contribute to the neural mechanisms leading to endotoxaemic anorexia.

Depolarization-induced release of endocannabinoids by murine dorsal motor nucleus of the vagus nerve neurons differentially regulates inhibitory and excitatory neurotransmission.

Numerous studies, focused on the hypothalamus, have recently implicated endocannabinoids (EC) as orexigenic factors in the central control of food intake. However, the EC system is also highly expressed in the hindbrain autonomic integrator of food intake regulation, i.e. the dorsal vagal complex (DVC). Previous studies have shown that exogenous cannabinoids, by acting on cannabinoid 1 receptor (CB1R), suppress GABAergic and glutamatergic neuronal transmission in adult rat dorsal motor nucleus of the vagus nerve (DMNV), the principal efferent compartment of the DVC. However, no endogenous release of EC has been demonstrated in DVC to date. Using patch-clamp techniques on mouse coronal brainstem slices, we confirmed that both inhibitory and excitatory neurotransmission were depressed by WIN 55,212-2, a CB1R agonist. We demonstrated that DMNV neurons exhibited a rapid and reversible depolarization-induced suppression of electrically evoked GABAergic IPSCs (eIPSCs), classically known as DSI (depolarization-induced suppression of inhibition), while spontaneous or miniature IPSCs activity remained unaltered. Further, no depolarization-induced suppression of glutamatergic eEPSCs (DSE) occurred. Our results indicate that DSI was blocked by SR141716A (Rimonabant), a selective CB1R antagonist, and was dependent on calcium elevation in DMNV neurons, suggesting a release of EC in the DVC. Moreover, the analysis of the paired-pulse ratio, of the coefficient of variation and of the failure rate of eIPSCs support the fact that EC-mediated suppression of GABAergic inhibition takes place at the presynaptic level. These results show for the first time that DMNV neurons release EC in an activity-dependent manner, which in turn differentially regulates their inhibitory and excitatory synaptic inputs.

Brain-derived neurotrophic factor/tropomyosin-related kinase receptor type B signaling is a downstream effector of the brainstem melanocortin system in food intake control.

It has been shown that the neurotropin brain-derived neurotrophic factor (BDNF) and its high-affinity receptor, tropomyosin-related kinase receptor type B (TrkB), contribute to the central control of food intake. BDNF has previously been implicated as a probable downstream effector of melanocortinergic signaling within the ventromedial hypothalamus, and we have shown its implication as an anorexigenic factor within the brainstem autonomic integrator of food intake control, namely the dorsal vagal complex (DVC). In the brainstem, the melanocortinergic signaling pathway is known to integrate phasic responses to satiety signals, such as cholecystokinin. In this study, we explored the interactions between melanocortin and BDNF/TrkB signaling within the DVC. First, we tested the effect of a local pharmacological activation or inhibition of melanocortin receptors type 3/4 (MC3/4R) on BDNF protein content in the DVC of adult rats. We showed that fourth intracerebroventricular delivery of MC3/4R agonist and antagonist increased and decreased the BDNF protein content within the DVC, respectively. Second, we showed that the orexigenic effect of a selective MC4R antagonist delivered fourth-icv can be blocked by a coadministration of BDNF. We also tested the causal role of BDNF/TrkB signaling in the anorexigenic effect of melanocortinergic signaling by using a recently developed analog-sensitive kinase allele murine model (TrkB(F616A) mice) and showed that the pharmacological blockade of TrkB abolished the anorexigenic effect of a selective MC4R agonist and of cholecystokinin. Our results provide strong evidence for a role of BDNF as a downstream effector of melanocortinergic signaling pathway within the DVC.

Corelease of GABA/glycine in lamina-X of the spinal cord of neonatal rats.

Spinal-cord slices from neonatal rats were used to record lamina-X neurons using the patch-clamp technique under whole cell recording configuration. Lamina-X surrounds the central canal of the spinal cord and contains sympathetic preganglionic neurons of the central autonomic nucleus. Miniature inhibitory postsynaptic currents were recorded in the presence of tetrodotoxin and kynurenic acid to block action potential-dependent transmitter release and glutamatergic transmissions, respectively. We recorded mixed gamma-amino-n-butyric acid/glycine miniature synaptic currents suggesting that gamma-amino-n-butyric acid and glycine can be coreleased from the same single synaptic vesicles, and that this corelease can be detected by the postsynaptic cell. In addition, acetylcholine can induce the release of gamma-amino-n-butyric acid/glycine by acting presynaptically at nicotinic receptors located on the gamma-amino-n-butyric acid ergic/glycinergic terminals.

Modulation of GABAergic synaptic transmission by terminal nicotinic acetylcholine receptors in the central autonomic nucleus of the neonatal rat spinal cord.

Using patch clamp recordings from an in vitro spinal cord slice preparation of neonatal rats (9-15days old), we characterized the GABAergic synaptic transmission in sympathetic preganglionic neurones (SPN) of the central autonomic nucleus (CA) of lamina X. Local applications of isoguvacine (100microM), a selective agonist at GABA(A) receptors, induced in all cells tested a chloride current which was abolished by bicuculline, a competitive antagonist at GABA(A) receptors. In addition, 25% of the recorded cells displayed spontaneous tetrodotoxin-insensitive and bicuculline-sensitive chloride miniature inhibitory postsynaptic currents (mIPSCs). Acetylcholine (100microM) increased the frequency of GABAergic mIPSCs without affecting their amplitudes or their kinetic properties indicating a presynaptic site of action. The presynaptic effect of ACh was restricted to GABAergic neurones synapsing onto sympathetic preganglionic neurones. The facilitatory effect of ACh was abolished in the absence of external calcium or in the presence of 100microM cadmium added to the bath solution. Choline 10mM, an agonist at alpha7 nicotinic acetylcholine receptors (nAChRs) or muscarine (10microM), a muscarinic receptor agonist, did not reproduce the presynaptic effect of ACh. The presynaptic effect of ACh was blocked by 1microM of dihydro-beta-erythroidine (DHbetaE), an antagonist of non-alpha7 nAChRs but was insensitive to alpha7 nAChRs antagonists (strychnine, alpha-bungarotoxin and methyllycaconitine) or to the muscarinic receptor antagonist atropine (10microM). It was concluded that SPNs of the central autonomic nucleus displayed a functional GABAergic transmission which is facilitated by terminal non alpha7 nAChRs.