Catherine Tardivel

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

•  The brainstem is a major site of integration for autonomic information from neurones, blood and cerebrospinal fluid (CSF). Signals exchange from the CSF is limited by the ependymocytes forming the brain cavities. However neurones contacting the CSF (CSF‐cNs) are thought to integrate those signals. •  Using immunohistochemical and electrophysiological approaches, we characterise in the brainstem, subependymal CSF‐cNs projecting a process ending in the central canal with a protrusion and expressing a ‘transient receptor potential’ (TRP) channel subtype suggested to act as chemo‐ or mechanoreceptors: the polycystin kidney disease 2‐like 1 channels (PKD2L1). •  CSF‐cNs receive exclusively inhibitory synaptic inputs and express functional channels presenting all properties of PKD2L1: cationic non‐selective, large conductance and modulated by extracellular pH and osmolarity. •  Because medullar CSF‐cNs are strategically positioned between CSF and neuronal parenchyma, we hypothesise that they could play a role in the regulation of homeostasis by integrating CSF signals.

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.

BDNF-TrkB signaling interacts with the GABAergic system to inhibit rhythmic swallowing in the rat

Brain-derived neurotrophic factor (BDNF) acts as an anorexigenic factor in the dorsal vagal complex (DVC) of the adult rat brain stem. The DVC contains the premotoneurons controlling swallowing, a motor component of feeding behavior. Although rats with transected midbrain do not seek out food, they are able to swallow and to ingest food. Because BDNF and tropomyosin-related kinase B (TrkB) receptors are expressed in the DVC, this study hypothesized that BDNF could modify the activity of premotoneurons involved in swallowing. Repetitive electrical stimulation of the superior laryngeal nerve (SLN) induces rhythmic swallowing that can be recorded with electromyographic electrodes inserted in sublingual muscles. We show that a microinjection of BDNF in the swallowing network induced a rapid, transient, and dose-dependant inhibition of rhythmic swallowing. This BDNF effect appeared to be mediated via TrkB activation, since it no longer occurred when TrkB receptors were antagonized by K-252a. Interestingly, swallowing was inhibited when subthreshold doses of BDNF and GABA were coinjected, suggesting a synergistic interaction between these two signaling substances. Moreover, BDNF no longer had an inhibitory effect on swallowing when coinjected with bicuculline, a GABA(A) receptor antagonist. This blockade of BDNF inhibitory effect on swallowing was reversible, since it reappeared when BDNF was injected 15 min after bicuculline. Finally, we show that stimulation of SLN induced a decrease in BDNF protein within the DVC. Together, our results strongly suggest that BDNF inhibits swallowing via modulation of the GABAergic signaling within the central pattern generator of swallowing.

Dysregulation of energy balance by trichothecene mycotoxins: Mechanisms and prospects.

Trichothecenes are toxic metabolites produced by fungi that constitute a worldwide hazard for agricultural production and both animal and human health. More than 40 countries have introduced regulations or guidelines for food and feed contamination levels of the most prevalent trichothecene, deoxynivalenol (DON), on the basis of its ability to cause growth suppression. With the development of analytical tools, evaluation of food contamination and exposure revealed that a significant proportion of the human population is chronically exposed to DON doses exceeding the provisional maximum tolerable daily dose. Accordingly, a better understanding of trichothecene impact on health is needed. Upon exposure to low or moderate doses, DON and other trichothecenes induce anorexia, vomiting and reduced weight gain. Several recent studies have addressed the mechanisms by which trichothecenes induce these symptoms and revealed a multifaceted action targeting gut, liver and brain and causing dysregulation in neuroendocrine signaling, immune responses, growth hormone axis, and central neurocircuitries involved in energy homeostasis. Newly identified trichothecene toxicosis biomarkers are just beginning to be exploited and already open up new questions on the potential harmful effects of chronic exposure to DON at apparently asymptomatic very low levels. This review summarizes our current understanding of the effects of DON and other trichothecenes on food intake and weight growth.

Modification of energy balance induced by the food contaminant T-2 toxin: A multimodal gut-to-brain connection

T-2 toxin is one of the most toxic Fusarium-derived trichothecenes found on cereals and constitutes a widespread contaminant of agricultural commodities as well as commercial foods. Low doses toxicity is characterized by reduced weight gain. To date, the mechanisms by which this mycotoxin profoundly modifies feeding behavior remain poorly understood and more broadly the effects of T-2 toxin on the central nervous system (CNS) have received limited attention. Through an extensive characterization of sickness-like behavior induced by T-2 toxin, we showed that its per os (p.o.) administration affects not only feeding behavior but also energy expenditure, glycaemia, body temperature and locomotor activity. Using c-Fos expression mapping, we identified the neuronal structures activated in response to T-2 toxin and observed that the pattern of neuronal populations activated by this toxin resembled that induced by inflammatory signals. Interestingly, part of neuronal pathways activated by the toxin were NUCB-2/nesfatin-1 expressing neurons. Unexpectedly, while T-2 toxin induced a strong peripheral inflammation, the brain exhibited limited inflammatory response at a time point when anorexia was ongoing. Unilateral vagotomy partly reduced T-2 toxin-induced brainstem neuronal activation. On the other hand, intracerebroventricular (icv) T-2 toxin injection resulted in a rapid (<1h) reduction in food intake. Thus, we hypothesized that T-2 toxin could signal to the brain through neuronal and/or humoral pathways. The present work provides the first demonstration that T-2 toxin modifies feeding behavior by interfering with central neuronal networks devoted to central energy balance. Our results, with a particular attention to peripheral inflammation, strongly suggest that inflammatory mediators partake in the T-2 toxin-induced anorexia and other symptoms. In view of the broad human and breeding animal exposure to T-2 toxin, this new mechanism may lead to reconsider the impact of the consumption of this toxin on human health.

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.

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.

Brain‐derived neurotrophic factor/TrkB signaling regulates daily astroglial plasticity in the suprachiasmatic nucleus: Electron‐microscopic evidence in mouse

Synchronization of circadian rhythms to the 24‐h light/dark (L/D) cycle is associated with daily rearrangements of the neuronal‐glial network of the suprachiasmatic nucleus of the hypothalamus (SCN), the central master clock orchestrating biological functions in mammals. These anatomical plastic events involve neurons synthesizing vasoactive intestinal peptide (VIP), known as major integrators of photic signals in the retinorecipient region of the SCN. Using an analog‐sensitive kinase allele murine model (TrkBF616A), we presently show that the pharmacological blockade of the tropomyosin‐related kinase receptor type B (TrkB), the high‐affinity receptor of brain‐derived neurotrophic factor (BDNF), abolished day/night changes in the dendrite enwrapping of VIP neurons by astrocytic processes (glial coverage), used as an index of SCN plasticity on electron‐microscopic sections. Therefore, the BDNF/TrkB signaling pathway exerts a permissive role on the ultrastructural rearrangements that occur in SCN under L/D alternance, an action that could be a critical determinant of the well‐established role played by BDNF in the photic regulation of the SCN. In contrast, the extent of glial coverage of non‐VIP neighboring dendrites was not different at daytime and nighttime in TrkBF616A mice submitted to TrkB inactivation or not receiving any pharmacological treatment. These data not only show that BDNF regulates SCN structural plasticity across the 24‐h cycle but also reinforce the view that the daily changes in SCN architecture subserve the light synchronization process.

Modulation of orexigenic and anorexigenic peptides gene expression in the rat DVC and hypothalamus by acute immobilization stress

We studied the long term effects of a single exposure to immobilization stress (IS) (1 h) on the expression of anorexigenic (Pro-opiomelanocortin: POMC and cocaine amphetamine related transcript: CART) and orexigenic (neuropeptide Y:NPY, Agouti related peptide: AgRP) factors in hypothalamus and dorso vagal complex (DVC). We showed, by using RT-PCR that in the hypothalamus, that the mRNAs of POMC and CART were up-regulated at the end of IS and up to 24 h. This up regulation persists until 48–72 h after IS for CART only. In the DVC, their expressions peak significantly at 24 h post stress and decline afterwards; CART mRNA is down regulated after 48 h post stress. NPY and AgRP mRNAs show a gradual increase just after the end of IS. The up regulation is significant only at 24 h after stress for AgRP but remains significantly higher for NPY compared to controls. In DVC, the mRNAs of the two factors show generally a similar post stress pattern. A significant increase jut after the end of IS of rats which persists up to 24 h after is firstly noticed. The levels tend then to reach the basal levels although, they were slightly but significantly higher up to 72 h after stress for mRNA NPY. The comparison between the expression profiles of anorexigenic and the two orexigenic peptides investigated shows the presence of a parallelism between that of POMC and AgRP and that of CART and NPY when each brain region (hypothalamus and DVC) is considered separately. It seems that any surge in the expression of each anorexigenic factor stimulates the expression of those of corresponding and appropriated orexigenic one. These last reactions from orexigenic peptides tend to attenuate the anorexigenic effects of CART and POMC and by consequent to abolish the anorexia state generated by stress.

The Food Contaminant Mycotoxin Deoxynivalenol Inhibits the Swallowing Reflex in Anaesthetized Rats

Deoxynivalenol (DON), one of the most abundant mycotoxins found on cereals, is known to be implicated in acute and chronic illnesses in both humans and animals. Among the symptoms, anorexia, reduction of weight gain and decreased nutrition efficiency were described, but the mechanisms underlying these effects on feeding behavior are not yet totally understood. Swallowing is a major motor component of ingestive behavior which allows the propulsion of the alimentary bolus from the mouth to the esophagus. To better understand DON effects on ingestive behaviour, we have studied its effects on rhythmic swallowing in the rat, after intravenous and central administration. Repetitive electrical stimulation of the superior laryngeal nerve or of the tractus solitarius, induces rhythmic swallowing that can be recorded using electromyographic electrodes inserted in sublingual muscles. Here we provide the first demonstration that, after intravenous and central administration, DON strongly inhibits the swallowing reflex with a short latency and in a dose dependent manner. Moreover, using c-Fos staining, a strong neuronal activation was observed in the solitary tract nucleus which contains the central pattern generator of swallowing and in the area postrema after DON intravenous injection. Our data show that DON modifies swallowing and interferes with central neuronal networks dedicated to food intake regulation.