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.

The role of intraspinal sensory neurons in the control of quadrupedal locomotion.

From swimming to walking and flying, animals have evolved specific locomotor strategies to thrive in different habitats. All types of locomotion depend on the integration of motor commands and sensory information to generate precisely coordinated movements. Cerebrospinal-fluid-contacting neurons (CSF-cN) constitute a vertebrate sensory system that monitors CSF composition and flow. In fish, CSF-cN modulate swimming activity in response to changes in pH and bending of the spinal cord; however, their role in mammals remains unknown. We used mouse genetics to study their function in quadrupedal locomotion. We found that CSF-cN are directly integrated into spinal motor circuits. The perturbation of CSF-cN function does not affect general motor activity nor the generation of locomotor rhythm and pattern but results in specific defects in skilled movements. These results identify a role for mouse CSF-cN in adaptive motor control and indicate that this sensory system evolved a novel function to accommodate the biomechanical requirements of limb-based locomotion.

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.

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.

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.

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.

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.

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.

Contrasting the functional properties of GABAergic axon terminals with single and multiple synapses in the thalamus.

Diverse sources of GABAergic inhibition are a major feature of cortical networks, but distinct inhibitory input systems have not been systematically characterized in the thalamus. Here, we contrasted the properties of two independent GABAergic pathways in the posterior thalamic nucleus of rat, one input from the reticular thalamic nucleus (nRT), and one "extrareticular" input from the anterior pretectal nucleus (APT). The vast majority of nRT-thalamic terminals formed single synapses per postsynaptic target and innervated thin distal dendrites of relay cells. In contrast, single APT-thalamic terminals formed synaptic contacts exclusively via multiple, closely spaced synapses on thick relay cell dendrites. Quantal analysis demonstrated that the two inputs displayed comparable quantal amplitudes, release probabilities, and multiple release sites. The morphological and physiological data together indicated multiple, single-site contacts for nRT and multisite contacts for APT axons. The contrasting synaptic arrangements of the two pathways were paralleled by different short-term plasticities. The multisite APT-thalamic pathway showed larger charge transfer during 50-100 Hz stimulation compared with the nRT pathway and a greater persistent inhibition accruing during stimulation trains. Our results demonstrate that the two inhibitory systems are morpho-functionally distinct and suggest and that multisite GABAergic terminals are tailored for maintained synaptic inhibition even at high presynaptic firing rates. These data explain the efficacy of extrareticular inhibition in timing relay cell activity in sensory and motor thalamic nuclei. Finally, based on the classic nomenclature and the difference between reticular and extrareticular terminals, we define a novel, multisite GABAergic terminal type (F3) in the thalamus.

Cholinergic axons modulate GABAergic signaling among hippocampal interneurons via postsynaptic alpha 7 nicotinic receptors.

Homopentameric alpha7 nicotinic receptors have a high affinity for acetylcholine (ACh), are permeable to Ca2+ ions, and are abundant in hippocampal interneurons. Although nicotinic agonists evoke inward currents and Ca2+ transients in stratum radiatum interneurons, the role of endogenous ACh in modulating synaptic integration by interneurons is incompletely understood. Many cholinergic axonal varicosities do not have postsynaptic specializations, but alpha7 receptors frequently occur close to synaptic GABA(A) receptors. These observations raise the possibility that alpha7 nicotinic receptors activated by ACh released from cholinergic axons modulate GABAergic transmission in interneurons. We show that agonists of alpha7 receptors profoundly depress GABAergic IPSCs recorded in stratum radiatum interneurons in the CA1 region of the hippocampus. This depression is accompanied by a small increase in GABA release. Alpha7 nicotinic receptor agonists also depress GABA- or muscimol-evoked currents in interneurons, indicating that the major effect is a postsynaptic modulation of GABA(A) receptors. The depression of GABA-evoked currents is abolished by chelating Ca2+ in the recorded interneuron and attenuated by inhibitors of PKC. We also show that stimuli designed to release endogenous ACh from cholinergic axons evoke an alpha7 receptor-dependent heterosynaptic depression of GABAergic IPSCs in interneurons. This heterosynaptic modulation is amplified by blocking cholinesterases. These results reveal a novel mechanism by which cholinergic neurons modulate information processing in the hippocampus.

Functional stabilization of weakened thalamic pacemaker channel regulation in rat absence epilepsy.

Aberrant function of pacemaker currents (Ih), carried by hyperpolarization-activated cation non-selective (HCN) channels, affects neuronal excitability and accompanies epilepsy, but its distinct roles in epileptogenesis and chronic epilepsy are unclear. We probed Ih function and subunit composition during both pre- and chronically epileptic stages in thalamocortical (TC) neurones of the Genetic Absence Epilepsy Rat from Strasbourg (GAERS). Voltage gating of Ih was unaltered in mature somatosensory TC cells, both in vivo and in vitro. However, the enhancement of Ih by phasic, near-physiological, cAMP pulses was diminished by approximately 40% and the half-maximal cAMP concentration increased by approximately 5-fold. This decreased responsiveness of Ih to its major cellular modulator preceded epilepsy onset in GAERS, persisted throughout the chronic state, and was accompanied by an enhanced expression of the cAMP-insensitive HCN1 channel mRNA (> 50%), without changes in the mRNA levels of HCN2 and HCN4. To assess for alterations in TC cell excitability, we monitored the slow up-regulation of Ih that is induced by Ca2+-triggered cAMP synthesis and important for terminating in vitro synchronized oscillations. Remarkably, repetitive rebound Ca2+ spikes evoked normal slow Ih up-regulation in mature GAERS neurones; that sufficed to attenuate spontaneous rhythmic burst discharges. These adaptive mechanisms occurred upstream of cAMP turnover and involved enhanced intracellular Ca2+ accumulation upon repetitive low-threshold Ca2+ discharges. Therefore, HCN channels appear to play a dual role in epilepsy. Weakened cAMP binding to HCN channels precedes, and likely promotes, epileptogenesis in GAERS, whereas compensatory mechanisms stabilizing Ih function contribute to the termination of spike-and-wave discharges in chronic epilepsy.

AKAP150 signaling complex promotes suppression of the M-current by muscarinic agonists.

M-type (KCNQ2/3) potassium channels are suppressed by activation of G(q/11)-coupled receptors, thereby increasing neuronal excitability. We show here that rat KCNQ2 can bind directly to the multivalent A-kinase-anchoring protein AKAP150. Peptides that block AKAP150 binding to the KCNQ2 channel complex antagonize the muscarinic inhibition of the currents. A mutant form of AKAP150, AKAP(DeltaA), which is unable to bind protein kinase C (PKC), also attenuates the agonist-induced current suppression. Analysis of recombinant KCNQ2 channels suggests that targeting of PKC through association with AKAP150 is important for the inhibition. Phosphorylation of KCNQ2 channels was increased by muscarinic stimulation; this was prevented either by coexpression with AKAP(DeltaA) or pretreatment with PKC inhibitors that compete with diacylglycerol. These inhibitors also reduced muscarinic inhibition of M-current. Our data indicate that AKAP150-bound PKC participates in receptor-induced inhibition of the M-current.

Signaling microdomains define the specificity of receptor-mediated InsP(3) pathways in neurons.

M(1) muscarinic (M(1)AChRs) and B(2) bradykinin (B(2)Rs) receptors are two PLCbeta-coupled receptors that mobilize Ca(2+) in nonexcitable cells. In many neurons, however, B(2)Rs but not M(1)AChRs mobilize intracellular Ca(2+). We have studied the membrane organization and dynamics underlying this coupling specificity by using Trp channels as biosensors for real-time detection of PLCbeta products. We found that, in sympathetic neurons, although both receptors rapidly produced DAG and InsP(3) as messengers, only InsP(3) formed by B(2)Rs has the ability to activate IP(3)Rs. This exclusive coupling results from spatially restricted complexes linking B(2)Rs to IP(3)Rs, a missing partnership for M(1)AChRs. These complexes allow fast and localized rises of InsP(3), necessary to activate the low-affinity neuronal IP(3)R. Thus, these signaling microdomains are of critical importance for the induction of selective responses, discriminating proinflammatory information associated with B(2)Rs from cholinergic neurotransmission.