The spino-bulbar-cerebellar pathway: organization and neurochemical properties of spinal cells that project to the lateral reticular nucleus in the rat

In addition to classical spinocerebellar pathways, the cerebellum receives information from the spinal cord indirectly via spino-bulbar-cerebellar systems. One of the structures in this pathway is the lateral reticular nucleus (LRt). We performed series of experiments to investigate the organization and neurotransmitter content of spinoreticular tract (SRT) neurons in the lumbar spinal cord that project to the LRt. Three rats received injections of the b subunit of Cholera toxin (CTb) or Fluorogold (FG) within the left and right LRt. The majority of SRT cells (56–61%) were found within the contralateral medial intermediate gray matter where small numbers (7–10%) of double-labeled cells were also present on both sides of the cord. Six rats received unilateral spinal injections of CTb to label spinal projections to the LRt. Injections of FG were made also into the anterior lobe of the cerebellum to label LRt pre-cerebellar neurons. Terminals were found mainly ipsilateral to spinal injection sites within the central and ventrolateral regions of the LRt. Immunocytochemical analysis of SRT terminals revealed that the majority (75%) were contained vesicular glutamate transporter 2 but a minority (20%) contained the vesicular GABA transporter. The inhibitory subpopulation was found to be GABAergic, glycinergic, or contained both transmitters. Inhibitory and excitatory terminals were present within overlapping regions of the nucleus. Most CTb terminals contacting LRt pre-cerebellar neurons were excitatory (80%) whereas a minority were inhibitory and most cells (88%) received contacts from both inhibitory and excitatory terminals. This study shows that SRT axons in the LRt have the capacity to exert direct excitatory and inhibitory actions on LRt pre-cerebellar neurons. Thus spinal cord input has the capacity to facilitate or depress the activity of individual LRt cells which in turn adjust activity in the cerebellum to produce coordinated motor behaviors

Distribution of glycinergic neurons in the brain of glycine transporter-2 Tg(glyt2:gfp) transgenic adult zebrafish:Relation with brain-spinal descending systems

We used a Tg(glyt2:gfp) transgenic zebrafish expressing the green fluorescent protein (GFP) under control of the glycine transporter 2 (GLYT2) regulatory sequences to study for the first time the glycinergic neurons in the brain of an adult teleost. We also performed in situ hybridization using a GLYT2 probe and glycine immunohistochemistry. This study was combined with biocytin tract tracing from the spinal cord to reveal descending glycinergic pathways. A few groups of GFP-positive/GLYT2 negative cells were observed in the midbrain and forebrain, including numerous pinealocytes. Conversely, a small nucleus of the midbrain tegmentum, was GLYT2 positive but GFP negative. Most of the GFP-positive and GLYT2-positive neurons were observed in the rhombencephalon and spinal cord, and a proportion of these cells showed double GLYT2/GFP labeling. In the hindbrain, GFP/GLYT2-positive populations were observed in the medial octavolateral nucleus, the secondary, magnocellular and descending octaval nuclei, the viscerosensory lobes and reticular populations distributed from trigeminal to vagal levels. No glycinergic cells were observed in the cerebellum. Tract tracing revealed three conspicuous pairs of GFP/GLYT2-positive reticular neurons projecting to the spinal cord. In the spinal cord, GFP/GLYT2-positive cells were observed in the dorsal and ventral horns. GFP-positive fibers were observed from the olfactory bulbs to the spinal cord, although its density varied among regions. The Mauthner neurons received very rich GFP-positive innervation, mainly around the axon cap. Comparison of the zebrafish glycinergic system with those of other adult vertebrates reveals shared patterns but also divergent traits in the evolution of this system. J. Comp. Neurol., 2012. © 2012 Wiley Periodicals, Inc

Medial Superior Olivary Neurons Receive Surprisingly Few Excitatory and Inhibitory Inputs with Balanced Strength and Short-Term Dynamics

Neurons in the medial superior olive (MSO) process microsecond interaural time differences, the major cue for localizing low-frequency sounds, by comparing the relative arrival time of binaural, glutamatergic excitatory inputs. This coincidence detection mechanism is additionally shaped by highly specialized glycinergic inhibition. Traditionally, it is assumed that the binaural inputs are conveyed by many independent fibers, but such an anatomical arrangement may decrease temporal precision. Short-term depression on the other hand might enhance temporal fidelity during ongoing activity. For the first time we show that binaural coincidence detection in MSO neurons may require surprisingly few but strong inputs, challenging long-held assumptions about mammalian coincidence detection. This study exclusively uses adult gerbils for in vitro electrophysiology, single-cell electroporation and immunohistochemistry to characterize the size and short-term plasticity of inputs to the MSO. We find that the excitatory and inhibitory inputs to the MSO are well balanced both in strength and short-term dynamics, redefining this fastest of all mammalian coincidence detector circuits

Spinal V2b neurons reveal a role for ipsilateral inhibition in speed control

The spinal cord contains a diverse array of interneurons that govern motor output. Traditionally, models of spinal circuits have emphasized the role of inhibition in enforcing reciprocal alternation between left and right sides or flexors and extensors. However, recent work has shown that inhibition also increases coincident with excitation during contraction. Here, using larval zebrafish, we investigate the V2b (Gata3+) class of neurons, which contribute to flexor-extensor alternation but are otherwise poorly understood. Using newly generated transgenic lines we define two stable subclasses with distinct neurotransmitter and morphological properties. These V2b subclasses synapse directly onto motor neurons with differential targeting to speed-specific circuits. In vivo, optogenetic manipulation of V2b activity modulates locomotor frequency: suppressing V2b neurons elicits faster locomotion, whereas activating V2b neurons slows locomotion. We conclude that V2b neurons serve as a brake on axial motor circuits. Together, these results indicate a role for ipsilateral inhibition in speed control

Anatomical and molecular properties of long descending propriospinal neurons in mice

Long descending propriospinal neurons (LDPNs) are interneurons that form direct connections between cervical and lumbar spinal circuits. LDPNs are involved in interlimb coordination and are important mediators of functional recovery after spinal cord injury (SCI). Much of what we know about LDPNs comes from a range of species, however, the increased use of transgenic mouse lines to better define neuronal populations calls for a more complete characterisation of LDPNs in mice. In this study, we examined the cell body location, inhibitory neurotransmitter phenotype, developmental provenance, morphology and synaptic inputs of mouse LDPNs throughout the cervical and upper thoracic spinal cord. LDPNs were retrogradely labelled from the lumbar spinal cord to map cell body locations throughout the cervical and upper thoracic segments. Ipsilateral LDPNs were distributed throughout the dorsal, intermediate and ventral grey matter as well as the lateral spinal nucleus and lateral cervical nucleus. In contrast, contralateral LDPNs were more densely concentrated in the ventromedial grey matter. Retrograde labelling in GlyT2GFP and GAD67GFP mice showed the majority of inhibitory LDPNs project either ipsilaterally or adjacent to the midline. Additionally, we used several transgenic mouse lines to define the developmental provenance of LDPNs and found that V2b positive neurons form a subset of ipsilaterally projecting LDPNs. Finally, a population of Neurobiotin (NB) labelled LDPNs were assessed in detail to examine morphology and plot the spatial distribution of contacts from a variety of neurochemically distinct axon terminals. These results provide important baseline data in mice for future work on their role in locomotion and recovery from SCI

Glycine transporters GlyT1 and GlyT2 are differentially modulated by glycogen synthase kinase 3β

© 2014 Elsevier Ltd. All rights reserved. Inhibitory glycinergic neurotransmission is terminated by the specific glycine transporters GlyT1 and GlyT2 which actively reuptake glycine from the synaptic cleft. GlyT1 is associated with both glycinergic and glutamatergic pathways, and is the main regulator of the glycine levels in the synapses. GlyT2 is the main supplier of glycine for vesicle refilling, a process that is vital to preserve the quantal glycine content in synaptic vesicles. Therefore, to control glycinergic neurotransmission efficiently, GlyT1 and GlyT2 activity must be regulated by diverse neuronal and glial signaling pathways. In this work, we have investigated the possible functional modulation of GlyT1 and GlyT2 by glycogen synthase kinase 3 (GSK3β). This kinase is involved in mood stabilization, neurodegeneration and plasticity at excitatory and inhibitory synapses. The co-expression of GSK3β with GlyT1 or GlyT2 in COS-7 cells and Xenopus laevis oocytes, leads to inhibition and stimulation of GlyT1 and GlyT2 activities, respectively, with a decrease of GlyT1, and an increase in GlyT2 levels at the plasma membrane. The specificity of these changes is supported by the antagonism exerted by a catalytically inactive form of the kinase and through inhibitors of GSK3β such as lithium chloride and TDZD-8. GSK3β also increases the incorporation of 32Pi into GlyT1 and decreases that of GlyT2. The pharmacological inhibition of the endogenous GSK3β in neuron cultures of brainstem and spinal cord leads to an opposite modulation of GlyT1 and GlyT2.Our results suggest that GSK3β is important for stabilizing and/or controlling the expression of functional GlyTs on the neural cell surface.Spanish Dirección General de Investigación Científica y Tecnica SAF2011-29961 and by an institutional grant from the ‘Fundación Ramón ArecesPeer Reviewe

Ventromedial medulla inhibitory neuron inactivation induces REM sleep without atonia and REM sleep behavior disorder

Despite decades of research, there is a persistent debate regarding the localization of GABA/glycine neurons responsible for hyperpolarizing somatic motoneurons during paradoxical (or REM) sleep (PS), resulting in the loss of muscle tone during this sleep state. Combining complementary neuroanatomical approaches in rats, we first show that these inhibitory neurons are localized within the ventromedial medulla (vmM) rather than within the spinal cord. We then demonstrate their functional role in PS expression through local injections of adeno-associated virus carrying specific short-hairpin RNA in order to chronically impair inhibitory neurotransmission from vmM. After such selective genetic inactivation, rats display PS without atonia associated with abnormal and violent motor activity, concomitant with a small reduction of daily PS quantity. These symptoms closely mimic human REM sleep behavior disorder (RBD), a prodromal parasomnia of synucleinopathies. Our findings demonstrate the crucial role of GABA/glycine inhibitory vmM neurons in muscle atonia during PS and highlight a candidate brain region that can be susceptible to α-synuclein-dependent degeneration in RBD patients

Glycinergic input to the mouse basal forebrain cholinergic neurons

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Neurones glycinergiques et transmission inhibitrice dans les noyaux cérébelleux

The cerebellum is composed of a three-layered cortex and of nuclei and is responsible for the learned fine control of posture and movements. I combined a genetic approach (based on the use of transgenic mouse lines) with anatomical tracings, immunohistochemical stainings, electrophysiological recordings and optogenetic stimulations to establish the distinctive characteristics of the inhibitory neurons of the cerebellar nuclei and to detail their connectivity and their role in the cerebellar circuitry.We showed that the glycinergic inhibitory neurons of the cerebellar nuclei constitute a distinct neuronal population and are characterized by their mixed inhibitory GABAergic/glycinergic phenotype. Those inhibitory neurons are also distinguished by their axonal plexus which includes a local arborization with the cerebellar nuclei where they contact principal output neurons and a projection to the granular layer of the cerebellar cortex where they end onto Golgi cells dendrites. Finally, the inhibitory neurons of the cerebellar nuclei receive inhibitory afferents from Purkinje cells and may be contacted by mossy fibers or climbing fibers.We provided the first evidence of functional mixed transmission in the cerebellar nuclei and the first demonstration of a mixed inhibitory nucleo-cortical projection. Overall, our data establish the inhibitory neurons as the third cellular component of the cerebellar nuclei. Their importance in the modular organization of the cerebellum and their impact on sensory-motor integration need to be confirmed by optogenetic experiments in vivo.Le cervelet, composé d'un cortex et de noyaux, est responsable du contrôle moteur fin des mouvements et de la posture. En combinant une approche génétique (basée sur l'utilisation de lignées de souris transgéniques) avec des traçages anatomiques, des marquages immunohistochimiques et des expériences d'électrophysiologie et d'optogénétique, nous établissons les caractères distinctifs des neurones inhibiteurs des noyaux cérébelleux et en détaillons la connectivité ainsi que les fonctions dans le circuit cérébelleux. Les neurones inhibiteurs glycinergiques des noyaux profonds constituent une population de neurones distincts des autres types cellulaires identifiables par leur phénotype inhibiteur mixte GABAergique/glycinergique. Ces neurones se distinguent également par leur plexus axonal qui comporte une arborisation locale dans les noyaux cérébelleux où ils contactent les neurones principaux et une projection vers le cortex cérébelleux où ils contactent les cellules de Golgi. Ces neurones inhibiteurs reçoivent également des afférences inhibitrices des cellules de Purkinje et pourraient être contactés par les fibres moussues ou les fibres grimpantes.Nous apportons ainsi la première étude d'une transmission mixte fonctionnelle par les neurones inhibiteurs des noyaux cérébelleux, projetant à la fois dans les noyaux et le cortex cérébelleux. L'ensemble de nos données établissent les neurones inhibiteurs mixtes des noyaux cérébelleux comme la troisième composante cellulaire des noyaux profonds. Leur importance dans l'organisation modulaire du cervelet, ainsi que leur impact sur l'intégration sensori-motrice, devront être confirmés par des études optogénétiques in vivo

The Mammalian Interaural Time Difference Detection Circuit Is Differentially Controlled by GABAB Receptors during Development

Throughout development GABAB receptors (GABABRs) are widely expressed in the mammalian brain. In mature auditory brainstem neurons, GABABRs are involved in the short-term regulation of the strength and dynamics of excitatory and inhibitory inputs, thus modulating sound analysis. During development, GABABRs also contribute to long-term changes in input strength. Using a combination of whole-cell patch-clamp recordings in acute brain slices and immunostainings in gerbils, we characterized developmental changes in GABABR-mediated regulation of synaptic inputs to neurons in the medial superior olive (MSO), an auditory brainstem nucleus that analyzes interaural time differences (ITDs). Here, we show that, before hearing onset, GABABR-mediated depression of transmitter release is much stronger for excitation than inhibition, whereas in mature animals GABABRs mainly control the inhibition. During the same developmental period, GABABR immunoreactivity shifts from the dendritic to the somatic region of the MSO. Furthermore, only before hearing onset (postnatal day 12), stimulation of the fibers originating in the medial and the lateral nucleus of the trapezoid body (MNTB and LNTB) activates GABABRs on both the inhibitory and the excitatory inputs. After hearing onset, GAD65-positive endings devoid of glycine transporter reactivity suggest GABA release from sources other than the MNTB and LNTB. At this age, pharmacological increase of spontaneous synaptic release activates GABABRs only on the inhibitory inputs. This indicates not only a profound inhibitory effect of GABABRs on the major inputs to MSO neurons in neonatal animals but also a direct modulatory role of GABABRs for ITD analysis in the MSO of adult animals