The Vestibulospinal Nucleus Is a Locus of Balance Development.

Mature vertebrates maintain posture using vestibulospinal neurons that transform sensed instability into reflexive commands to spinal motor circuits. Postural stability improves across development. However, due to the complexity of terrestrial locomotion, vestibulospinal contributions to postural refinement in early life remain unexplored. Here we leveraged the relative simplicity of underwater locomotion to quantify the postural consequences of losing vestibulospinal neurons during development in larval zebrafish of undifferentiated sex. By comparing posture at two timepoints, we discovered that later lesions of vestibulospinal neurons led to greater instability. Analysis of thousands of individual swim bouts revealed that lesions disrupted movement timing and corrective reflexes without impacting swim kinematics, and that this effect was particularly strong in older larvae. Using a generative model of swimming, we showed how these disruptions could account for the increased postural variability at both timepoints. Finally, late lesions disrupted the fin/trunk coordination observed in older larvae, linking vestibulospinal neurons to postural control schemes used to navigate in depth. Since later lesions were considerably more disruptive to postural stability, we conclude that vestibulospinal contributions to balance increase as larvae mature. Vestibulospinal neurons are highly conserved across vertebrates; we therefore propose that they are a substrate for developmental improvements to postural control.

Neural populations within macaque early vestibular pathways are adapted to encode natural self-motion.

How the activities of large neural populations are integrated in the brain to ensure accurate perception and behavior remains a central problem in systems neuroscience. Here, we investigated population coding of naturalistic self-motion by neurons within early vestibular pathways in rhesus macaques (Macacca mulatta). While vestibular neurons displayed similar dynamic tuning to self-motion, inspection of their spike trains revealed significant heterogeneity. Further analysis revealed that, during natural but not artificial stimulation, heterogeneity resulted primarily from variability across neurons as opposed to trial-to-trial variability. Interestingly, vestibular neurons displayed different correlation structures during naturalistic and artificial self-motion. Specifically, while correlations due to the stimulus (i.e., signal correlations) did not differ, correlations between the trial-to-trial variabilities of neural responses (i.e., noise correlations) were instead significantly positive during naturalistic but not artificial stimulation. Using computational modeling, we show that positive noise correlations during naturalistic stimulation benefits information transmission by heterogeneous vestibular neural populations. Taken together, our results provide evidence that neurons within early vestibular pathways are adapted to the statistics of natural self-motion stimuli at the population level. We suggest that similar adaptations will be found in other systems and species.

Vestibular CCK signaling drives motion sickness-like behavior in mice.

Travel can induce motion sickness (MS) in susceptible individuals. MS is an evolutionary conserved mechanism caused by mismatches between motion-related sensory information and past visual and motion memory, triggering a malaise accompanied by hypolocomotion, hypothermia, hypophagia, and nausea. Vestibular nuclei (VN) are critical for the processing of movement input from the inner ear. Motion-induced activation of VN neurons recapitulates MS-related signs. However, the genetic identity of VN neurons mediating MS-related autonomic and aversive responses remains unknown. Here, we identify a central role of cholecystokinin (CCK)-expressing VN neurons in motion-induced malaise. Moreover, we show that CCK VN inputs onto the parabrachial nucleus activate Calca-expressing neurons and are sufficient to establish avoidance to novel food, which is prevented by CCK-A receptor antagonism. These observations provide greater insight into the neurobiological regulation of MS by identifying the neural substrates of MS and providing potential targets for treatment.

Reversible positioning head tilt observed in 14 cats with hypokalaemic myopathy.

CASE SERIES SUMMARY: Positioning head tilt (PHT) is a dynamic neurological sign in which the head tilts to the opposite side to which it is moving. This sign is triggered in response to head movement and is thought to be due to the lack of inhibition of vestibular nuclei by the cerebellar nodulus and uvula (NU). The occurrence of PHT in animals has been suggested to be an indicator of NU dysfunction. Here, we describe the acute onset of PHT in 14 cats. All the cats were diagnosed with hypokalaemic myopathy caused by a range of pathologies. The PHT resolved along with other signs related to myopathy, such as cervical flexion and generalised weakness, after electrolyte correction in all cats. RELEVANCE AND NOVEL INFORMATION: Hypokalaemic myopathy was the likely cause of PHT in the present feline cases.

Neuronal birthdate reveals topography in a vestibular brainstem circuit for gaze stabilization.

Across the nervous system, neurons with similar attributes are topographically organized. This topography reflects developmental pressures. Oddly, vestibular (balance) nuclei are thought to be disorganized. By measuring activity in birthdated neurons, we revealed a functional map within the central vestibular projection nucleus that stabilizes gaze in the larval zebrafish. We first discovered that both somatic position and stimulus selectivity follow projection neuron birthdate. Next, with electron microscopy and loss-of-function assays, we found that patterns of peripheral innervation to projection neurons were similarly organized by birthdate. Finally, birthdate revealed spatial patterns of axonal arborization and synapse formation to projection neuron outputs. Collectively, we find that development reveals previously hidden organization to the input, processing, and output layers of a highly conserved vertebrate sensorimotor circuit. The spatial and temporal attributes we uncover constrain the developmental mechanisms that may specify the fate, function, and organization of vestibulo-ocular reflex neurons. More broadly, our data suggest that, like invertebrates, temporal mechanisms may assemble vertebrate sensorimotor architecture.

Vestibular modulation of the tail of the rat striatum.

Fragmented and piecemeal evidence from animal and human studies suggests that vestibular information is transmitted to the striatum, a part of the basal ganglia that degenerates in Parkinson's Disease. Nonetheless, surprisingly little is known about the precise effects of activation of the vestibular system on the striatum. Electrophysiological studies have yielded inconsistent results, with many studies reporting only sparse responses to vestibular stimulation in the dorsomedial striatum. In this study, we sought to elucidate the effects of electrical stimulation of the peripheral vestibular system on electrophysiological responses in the tail of the rat striatum, a newly discovered region for sensory input. Rats were anaesthetised with urethane and a bipolar stimulating electrode was placed in the round window in order to activate the peripheral vestibular system. A recording electrode was positioned in the tail of the striatum. Local field potentials (LFPs) were recorded ipsilaterally and contralaterally to the stimulation using a range of current parameters. In order to confirm that the vestibular system was activated, video-oculography was used to monitor vestibular nystagmus. At current amplitudes that evoked vestibular nystagmus, clear triphasic LFPs were evoked in the bilateral tail of the striatum, with the first phase of the waveform exhibiting latencies of less than 22 ms. The LFP amplitude increased with increasing current amplitude (P ≤ 0.0001). In order to exclude the possibility that the LFPs were evoked by the activation of the auditory system, the cochlea was surgically lesioned in some animals. In these animals the LFPs persisted despite the cochlear lesions, which were verified histologically. Overall, the results obtained suggest that there are vestibular projections to the tail of the striatum, which could possibly arise from projections via the vestibular nucleus or cerebellum and the parafasicular nucleus of the thalamus.

The effects of vibration on the neuronal activity of lateral vestibular nuclei in unilaterally labyrinthectomized rats.

Unilateral labyrinthectomy causes distinct oculomotor and postural disorder syndromes that gradually deteriorate. Simultaneously, compensatory mechanisms for the suppression of pathological disorders were activated. The current study aimed to investigate the characteristics of impulse activity in the ipsilateral and contralateral neurons of the lateral vestibular nucleus of unilaterally labyrinthectomized rats during various periods of vibration exposure. A program analysis of the background impulse activity of the neurons in the right- and left-lateral vestibular nuclei of rats under normal condition and after right-sided labyrinthectomy was performed. The animals were subjected to different periods of vibration exposure 2 days after surgery (5-, 10-, and 15-day periods). A comparison of the characteristics of the background impulse activity of neurons in both nuclei of intact rats revealed an initial asymmetry in the values of the mean impulse frequency and coefficient of variation of interimpulse intervals. After 5 days of vibration exposure, the values of the mean impulse frequency of neurons in both Deiters' nuclei were almost equal in labyrinthectomized rats. The mean impulse frequency of neurons on the uninjured side was higher than that on the injured side on the days following vibration exposure. The characteristics and functional significance of the findings are discussed.

Tilt in Place Microscopy: a Simple, Low-Cost Solution to Image Neural Responses to Body Rotations.

Animals use information about gravity and other destabilizing forces to balance and navigate through their environment. Measuring how brains respond to these forces requires considerable technical knowledge and/or financial resources. We present a simple alternative-Tilt In Place Microscopy (TIPM), a low-cost and noninvasive way to measure neural activity following rapid changes in body orientation. Here, we used TIPM to study vestibulospinal neurons in larval zebrafish during and immediately after roll tilts. Vestibulospinal neurons responded with reliable increases in activity that varied as a function of ipsilateral tilt amplitude. TIPM differentiated tonic (i.e., sustained tilt) from phasic responses, revealing coarse topography of stimulus sensitivity in the lateral vestibular nucleus. Neuronal variability across repeated sessions was minor relative to trial-to-trial variability, allowing us to use TIPM for longitudinal studies of the same neurons across two developmental time points. There, we observed global increases in response strength and systematic changes in the neural representation of stimulus direction. Our data extend classical characterization of the body tilt representation by vestibulospinal neurons and establish the utility of TIPM to study the neural basis of balance, especially in developing animals.SIGNIFICANCE STATEMENT Vestibular sensation influences everything from navigation to interoception. Here, we detail a straightforward, validated, and nearly universal approach to image how the nervous system senses and responds to body tilts. We use our new method to replicate and expand on past findings of tilt sensing by a conserved population of spinal-projecting vestibular neurons. The simplicity and broad compatibility of our approach will democratize the study of the response of the brain to destabilization, particularly across development.

Properties of Deiters' neurons and inhibitory synaptic transmission in the mouse lateral vestibular nucleus.

Deiters' neurons, located exclusively in the lateral vestibular nucleus (LVN), are involved in vestibulospinal reflexes, innervate extensor motoneurons that drive antigravity muscles, and receive inhibitory inputs from the cerebellum. We investigated intrinsic membrane properties, short-term plasticity, and inhibitory synaptic inputs of mouse Deiters' and non-Deiters' neurons within the LVN. Deiters' neurons are distinguished from non-Deiters' neurons by their very low input resistance (105.8 vs. 521.8 MΩ, respectively), long axons that project as far as the ipsilateral lumbar spinal cord, and expression of the cytostructural protein nonphosphorylated neurofilament protein (NPNFP). Whole cell patch-clamp recordings in brain stem slices show that most Deiters' and non-Deiters' neurons were tonically active (>92%). Short-term plasticity was studied by examining discharge rate modulation following release from hyperpolarization [postinhibitory rebound firing (PRF)] and depolarization [firing rate adaptation (FRA)]. PRF and FRA gain were similar in Deiters' and non-Deiters' neurons (PRF 24.9 vs. 20.2 Hz and FRA gain 231.5 vs. 287.8 spikes/s/nA, respectively). Inhibitory synaptic input to both populations showed that GABAergic rather than glycinergic inhibition dominated. However, GABAA miniature inhibitory postsynaptic current (mIPSC) frequency was much higher in Deiters' neurons compared with non-Deiters' neurons (∼15.9 vs. 1.4 Hz, respectively). Our data suggest that Deiters' neurons can be reliably identified by their intrinsic membrane and synaptic properties. They are tonically active and glutamatergic, have low sensitivity or "gain," exhibit little adaptation, and receive strong GABAergic input. Deiters' neurons also have minimal short-term plasticity, and together these features suggest they are well suited to a role in encoding tonic signals for the vestibulospinal reflex.NEW & NOTEWORTHY Deiters' neurons within the lateral vestibular nucleus project the length of the spinal cord and activate antigravity extensor muscles. Deiters' neurons were characterized anatomically and physiologically in mice. Deiters' neurons are tonically active, have homogeneous intrinsic membrane properties, including low input resistance, and receive significant GABAAergic synaptic inputs. Deiters' neurons show little modulation in response to current injection. These features are consistent with Deiters' neurons responding to perturbations to maintain posture and balance.

L-Thyroxine Improves Vestibular Compensation in a Rat Model of Acute Peripheral Vestibulopathy: Cellular and Behavioral Aspects.

Unilateral vestibular lesions induce a vestibular syndrome, which recovers over time due to vestibular compensation. The therapeutic effect of L-Thyroxine (L-T4) on vestibular compensation was investigated by behavioral testing and immunohistochemical analysis in a rat model of unilateral vestibular neurectomy (UVN). We demonstrated that a short-term L-T4 treatment reduced the vestibular syndrome and significantly promoted vestibular compensation. Thyroid hormone receptors (TRα and TRß) and type II iodothyronine deiodinase (DIO2) were present in the vestibular nuclei (VN), supporting a local action of L-T4. We confirmed the T4-induced metabolic effects by demonstrating an increase in the number of cytochrome oxidase-labeled neurons in the VN three days after the lesion. L-T4 treatment modulated glial reaction by decreasing both microglia and oligodendrocytes in the deafferented VN three days after UVN and increased cell proliferation. Survival of newly generated cells in the deafferented vestibular nuclei was not affected, but microglial rather than neuronal differentiation was favored by L-T4 treatment.

Integration of vestibular and hindlimb inputs by vestibular nucleus neurons: multisensory influences on postural control.

We recently demonstrated in decerebrate and conscious cat preparations that hindlimb somatosensory inputs converge with vestibular afferent input onto neurons in multiple central nervous system (CNS) locations that participate in balance control. Although it is known that head position and limb state modulate postural reflexes, presumably through vestibulospinal and reticulospinal pathways, the combined influence of the two inputs on the activity of neurons in these brainstem regions is unknown. In the present study, we evaluated the responses of vestibular nucleus (VN) neurons to vestibular and hindlimb stimuli delivered separately and together in conscious cats. We hypothesized that VN neuronal firing during activation of vestibular and limb proprioceptive inputs would be well fit by an additive model. Extracellular single-unit recordings were obtained from VN neurons. Sinusoidal whole body rotation in the roll plane was used as the search stimulus. Units responding to the search stimulus were tested for their responses to 10° ramp-and-hold roll body rotation, 60° extension hindlimb movement, and both movements delivered simultaneously. Composite response histograms were fit by a model of low- and high-pass filtered limb and body position signals using least squares nonlinear regression. We found that VN neuronal activity during combined vestibular and hindlimb proprioceptive stimulation in the conscious cat is well fit by a simple additive model for signals with similar temporal dynamics. The mean R2 value for goodness of fit across all units was 0.74 ± 0.17. It is likely that VN neurons that exhibit these integrative properties participate in adjusting vestibulospinal outflow in response to limb state.NEW & NOTEWORTHY Vestibular nucleus neurons receive convergent information from hindlimb somatosensory inputs and vestibular inputs. In this study, extracellular single-unit recordings of vestibular nucleus neurons during conditions of passively applied limb movement, passive whole body rotations, and combined stimulation were well fit by an additive model. The integration of hindlimb somatosensory inputs with vestibular inputs at the first stage of vestibular processing suggests that vestibular nucleus neurons account for limb position in determining vestibulospinal responses to postural perturbations.

Inter-fastigial projections along the roof of the fourth ventricle.

The fastigial nucleus (FN) is a bilateral cerebellar integrative center for saccadic and vestibular control associated with non-motor functions such as feeding and cardiovascular regulation. In a previous study, we identified a tract of myelinated axons embedded in the subventricular zone (SVZ) that is located between the ependymal cells that form the dorsal wall of the ventricle and the glia limitans at the roof of the fourth ventricle González-González (Sci Rep 2017, 7:40768). Here, we show that this tract of axons, named subventricular axons or SVa, contains projection neurons that bilaterally interconnect both FNs. The approach consisted of the use of a battery of fluorescent neuronal tracers, transgenic mouse lines, and immunohistofluorescence. Our observations show that the SVa belong to a wide network of GABAergic projection neurons mainly located in the medial and caudal region of the FN. The SVa should be considered a part of a continuum of the cerebellar white matter that follows an alternative pathway through the SVZ, a region closely associated with the physiology of the fourth ventricle. This finding adds to our understanding of the complex organization of the FN; however, the function of the interconnection remains to be elucidated.

Adult and endemic neurogenesis in the vestibular nuclei after unilateral vestibular neurectomy.

We previously revealed adult reactive neurogenesis in deafferented vestibular nuclei following unilateral vestibular neurectomy (UVN) in the feline model. We recently replicated the same surgery in a rodent model and aimed to elucidate the origin and fate of newly generated cells following UVN. We used specific markers of cell proliferation, glial reaction, and cell differentiation in the medial vestibular nucleus (MVN) of adult rats. UVN induced an intense cell proliferation and glial reaction with an increase of GFAP-Immunoreactive (Ir), IBA1-Ir and Olig2-Ir cells 3 days after the lesion in the deafferented MVN. Most of the newly generated cells survived after UVN and differentiated into oligodendrocytes, astrocytes, microglial cells and GABAergic neurons. Interestingly, UVN induced a significant increase in a population of cells colocalizing SOX2 and GFAP 3 days after lesion in the deafferented MVN indicating the probable presence of multipotent cells in the vestibular nuclei. The concomitant increase in BrdU- and SOX2-Ir cells with the presence of SOX2 and GFAP colocalization 3 days after UVN in the deafferented MVN may support local mitotic activity of endemic quiescent neural stem cells in the parenchyma of vestibular nuclei.

Why the cerebellar shutdown/clampdown hypothesis of vestibular compensation is inconsistent with neurophysiological evidence.

BACKGROUND: Vestibular compensation is the process by which the central nervous system (CNS) attempts to adapt to the loss of vestibular sensory inputs. As such, the compensation process is critically involved in the vestibular rehabilitation programs that are implemented by physical therapists for patients with vestibular disorders. One hypothesis regarding vestibular compensation, which has persisted in some of the published vestibular compensation literature and particularly on some vestibular and physical therapy websites, is the 'cerebellar shutdown' or 'cerebellar clampdown' hypothesis proposed by McCabe and Ryu in 1969. This hypothesis proposes that the cerebellum inhibits neuronal activity in the bilateral vestibular nuclei (VN) following unilateral vestibular loss (UVL), causing the VN contralateral to the UVL to be electrically silent during the early phases of vestibular compensation. Despite a wealth of evidence against this idea, it has gained traction amongst some physical therapists and has implications for vestibular rehabilitation early in the compensation process. CONCLUSIONS: In this paper it is argued that the 'cerebellar shutdown' or 'clampdown' hypothesis is inconsistent with well accepted neurophysiological and imaging evidence and that it is also logically flawed.

Neural variability determines coding strategies for natural self-motion in macaque monkeys.

We have previously reported that central neurons mediating vestibulo-spinal reflexes and self-motion perception optimally encode natural self-motion (Mitchell et al., 2018). Importantly however, the vestibular nuclei also comprise other neuronal classes that mediate essential functions such as the vestibulo-ocular reflex (VOR) and its adaptation. Here we show that heterogeneities in resting discharge variability mediate a trade-off between faithful encoding and optimal coding via temporal whitening. Specifically, neurons displaying lower variability did not whiten naturalistic self-motion but instead faithfully represented the stimulus' detailed time course, while neurons displaying higher variability displayed temporal whitening. Using a well-established model of VOR pathways, we demonstrate that faithful stimulus encoding is necessary to generate the compensatory eye movements found experimentally during naturalistic self-motion. Our findings suggest a novel functional role for variability toward establishing different coding strategies: (1) faithful stimulus encoding for generating the VOR; (2) optimized coding via temporal whitening for other vestibular functions.

Central Vestibular Tuning Arises from Patterned Convergence of Otolith Afferents.

As sensory information moves through the brain, higher-order areas exhibit more complex tuning than lower areas. Though models predict that complexity arises via convergent inputs from neurons with diverse response properties, in most vertebrate systems, convergence has only been inferred rather than tested directly. Here, we measure sensory computations in zebrafish vestibular neurons across multiple axes in vivo. We establish that whole-cell physiological recordings reveal tuning of individual vestibular afferent inputs and their postsynaptic targets. Strong, sparse synaptic inputs can be distinguished by their amplitudes, permitting analysis of afferent convergence in vivo. An independent approach, serial-section electron microscopy, supports the inferred connectivity. We find that afferents with similar or differing preferred directions converge on central vestibular neurons, conferring more simple or complex tuning, respectively. Together, these results provide a direct, quantifiable demonstration of feedforward input convergence in vivo.

[The adult brain produces new neurons to restore balance after vestibular loss]. / Le cerveau adulte produit de nouveaux neurones pour restaurer l'équilibre après une perte vestibulaire.

Following partial or total loss of peripheral vestibular inputs, a phenomenon called central vestibular compensation takes place in the hours and days following the injury. This neuroplasticity process involves a mosaic of profound rearrangements within the brain stem vestibular nuclei. Among them, the setting of a new neuronal network is maybe the most original and unexpected, as it involves an adult reactive neurogenesis in a brain area not reported as neurogenic so far. Both the survival and functionality of this newly generated neuronal network will depend on its integration to pre-existing networks in the deafferented structure. Far from being aberrant, this new structural organization allows the use of inputs from other sensory modalities (vision and proprioception) to promote the restoration of the posture and equilibrium. We choose here to detail this model, which does not belong to the traditional niches of adult neurogenesis, but it is the best example so far of the reparative role of the adult neurogenesis. Not only it represents an original neuroplasticity mechanism, interesting for basic neuroscience, but it also opens new medical perspectives for the development of therapeutic approaches to alleviate vestibular disorders.

TITLE: Le cerveau adulte produit de nouveaux neurones pour restaurer l'équilibre après une perte vestibulaire. ABSTRACT: Un phénomène appelé « compensation vestibulaire ¼ se produit après une atteinte vestibulaire périphérique. Ce processus, qui permet un retour progressif de l'équilibre, se produit principalement au sein des noyaux vestibulaires du tronc cérébral, et met en jeu une mosaïque de réarrangements structurels. Parmi ceux-ci, la neurogenèse vestibulaire réactionnelle (NGVR) adulte est peut-être la plus inattendue, car elle se produit dans une région du cerveau qui n'a jamais été signalée auparavant comme neurogène. La survie et la fonctionnalité de ce réseau neuronal nouvellement généré dépendent de son intégration dans les réseaux préexistants des noyaux désafférentés. Cette organisation permet au cerveau d'utiliser les apports d'autres modalités sensorielles pour faciliter le rétablissement de la posture et de l'équilibre. C'est à ce jour le meilleur exemple du rôle réparateur de la neurogenèse adulte. Ces observations soulèvent de nombreuses questions sur la pertinence physiologique de la NGVR.

Repeated Galvanic Vestibular Stimulation Modified the Neuronal Potential in the Vestibular Nucleus.

Vestibular nucleus (VN) and cerebellar flocculus are known as the core candidates for the neuroplasticity of vestibular system. However, it has been still elusive how to induce the artificial neuroplasticity, especially caused by an electrical stimulation, and assess the neuronal information related with the plasticity. To understand the electrically induced neuroplasticity, the neuronal potentials in VN responding to the repeated electrical stimuli were examined. Galvanic vestibular stimulation (GVS) was applied to excite the neurons in VN, and their activities were measured by an extracellular neural recording technique. Thirty-eight neuronal responses (17 for the regular and 21 for irregular neurons) were recorded and examined the potentials before and after stimulation. Two-third of the population (63.2%, 24/38) modified the potentials under the GVS repetition before stimulation (p = 0.037), and more than half of the population (21/38, 55.3%) changed the potentials after stimulation (p = 0.209). On the other hand, the plasticity-related neuronal modulation was hardly observed in the temporal responses of the neurons. The modification of the active glutamate receptors was also investigated to see if the repeated stimulation changed the number of both types of glutamate receptors, and the results showed that AMPA and NMDA receptors decreased after the repeated stimuli by 28.32 and 16.09%, respectively, implying the modification in the neuronal amplitudes.

VGLUT2-expressing neurons in the vestibular nuclear complex mediate gravitational stress-induced hypothermia in mice.

The vestibular system, which is essential for maintaining balance, contributes to the sympathetic response. Although this response is involved in hypergravity load-induced hypothermia in mice, the underlying mechanism remains unknown. This study showed that hypergravity (2g) decreased plasma catecholamines, which resulted in hypoactivity of the interscapular brown adipose tissue (iBAT). Hypothermia induced by 2g load was significantly suppressed by administration of beta-adrenergic receptor agonists, suggesting the involvement of decrease in iBAT activity through sympathoinhibition. Bilateral chemogenetic activation of vesicular glutamate transporter 2 (VGLUT2)-expressing neurons in the vestibular nuclear complex (VNC) induced hypothermia. The VGLUT2-expressing neurons contributed to 2g load-induced hypothermia, since their deletion suppressed hypothermia. Although activation of vesicular gamma-aminobutyric acid transporter-expressing neurons in the VNC induced slight hypothermia instead of hyperthermia, their deletion did not affect 2g load-induced hypothermia. Thus, we concluded that 2g load-induced hypothermia resulted from sympathoinhibition via the activation of VGLUT2-expressing neurons in the VNC.

Connection Input Mapping and 3D Reconstruction of the Brainstem and Spinal Cord Projections to the CSF-Contacting Nucleus.

Objective: To investigate whether the CSF-contacting nucleus receives brainstem and spinal cord projections and to understand the functional significance of these connections. Methods: The retrograde tracer cholera toxin B subunit (CB) was injected into the CSF-contacting nucleus in Sprague-Dawley rats according the previously reported stereotaxic coordinates. After 7-10 days, these rats were perfused and their brainstem and spinal cord were sliced (thickness, 40 µm) using a freezing microtome. All the sections were subjected to CB immunofluorescence staining. The distribution of CB-positive neuron in different brainstem and spinal cord areas was observed under fluorescence microscope. Results: The retrograde labeled CB-positive neurons were found in the midbrain, pons, medulla oblongata, and spinal cord. Four functional areas including one hundred and twelve sub-regions have projections to the CSF-contacting nucleus. However, the density of CB-positive neuron distribution ranged from sparse to dense. Conclusion: Based on the connectivity patterns of the CSF-contacting nucleus receives anatomical inputs from the brainstem and spinal cord, we preliminarily conclude and summarize that the CSF-contacting nucleus participates in pain, visceral activity, sleep and arousal, emotion, and drug addiction. The present study firstly illustrates the broad projections of the CSF-contacting nucleus from the brainstem and spinal cord, which implies the complicated functions of the nucleus especially for the unique roles of coordination in neural and body fluids regulation.