The CSF-Contacting Nucleus Receives Anatomical Inputs From the Cerebral Cortex: A Combination of Retrograde Tracing and 3D Reconstruction Study in Rat.

OBJECTIVE: This study aimed to investigate the direct monosynaptic projections from cortical functional regions to the cerebrospinal fluid (CSF)-contacting nucleus for understanding the functions of the CSF-contacting nucleus. METHODS: The Sprague-Dawley rats received cholera toxin B subunit (CB) injections into the CSF-contacting nucleus. After 7-10 days of survival time, the rats were perfused, and the whole brain and spinal cord were sliced under a freezing microtome at 40 µm. All sections were treated with the CB immunofluorescence reaction. The retrogradely labeled neurons in different cortical areas were revealed under a confocal microscope. The distribution features were further illustrated under 3D reconstruction. RESULTS: The retrogradely labeled neurons were identified in the olfactory, orbital, cingulate, insula, retrosplenial, somatosensory, motor, visual, auditory, association, rhinal, and parietal cortical areas. A total of 12 functional areas and 34 functional subregions showed projections to the CSF-contacting nucleus in different cell intensities. CONCLUSION: According to the connectivity patterns, we conclude that the CSF-contacting nucleus participates in cognition, emotion, pain, visceral activity, etc. The present study firstly reveals the cerebral cortex→CSF-contacting nucleus connections, which implies the multiple functions of this special nucleus in neural and body fluid regulations.

Novel Projections to the Cerebrospinal Fluid-Contacting Nucleus From the Subcortex and Limbic System in Rat.

Objective: To identify the novel projections received by the cerebrospinal fluid (CSF)-contacting nucleus from the subcortex and limbic system to understand the biological functions of the nucleus. Methods: The cholera toxin subunit B (CB), a retrograde tracer, was injected into the CSF-contacting nucleus in Sprague-Dawley rats. After 7-10 days, the surviving rats were perfused, and the whole brain and spinal cord were sliced for CB immunofluorescence detection. The CB-positive neurons in the subcortex and limbic system were observed under a fluorescence microscope, followed by 3D reconstructed with the imaris software. Results: CB-positive neurons were found in the basal forebrain, septum, periventricular organs, preoptic area, and amygdaloid structures. Five functional areas including 46 sub-regions sent projections to the CSF-contacting nucleus. However, the projections had different densities, ranging from sparse to moderate, to dense. Conclusions: According to the projections from the subcortex and limbic system, we hypothesize that the CSF-contacting nucleus participates in emotion, cognition, homeostasis regulation, visceral activity, pain, and addiction. In this study, we illustrate the novel projections from the subcortex and limbic system to the CSF-contacting nucleus, which underlies the diverse and complicated circuits of the nucleus in body regulations.

A Special Cranial Nucleus (CSF-Contacting Nucleus) in Primates.

BACKGROUND: There is a unique nucleus (CSF-contacting nucleus) in the brain of rat. It has been demonstrated in our previous research. The extraordinary feature of this nucleus is that it is not connected to any parenchymal organ but to the CSF. In primates, however, the presence or absence of this nucleus has not been proven. Confirmation of the presence of this nucleus in primates will provide the structural basis for brain-CSF communication and help to understand the neurohumoral regulatory mechanisms in humans. METHODS: The tracer cholera toxin B subunit conjugated to horseradish peroxidase (CB-HRP) was injected into the CSF in the lateral ventricle (LV) of primate rhesus monkeys. After 48 h, the monkeys were perfused and the brain was dissected out, and sectioned for CB-HRP staining. The CB-HRP positive structures were observed under confocal and electron microscopy. The three-dimensional (3D) structure of the CB-HRP positive neurons cluster was reconstructed by computer software. RESULTS: (1) CB-HRP labeling is confined within the ventricle, but not leakage into the brain parenchyma. (2) From the midbrain inferior colliculus superior border plane ventral to the aqueduct to the upper part of the fourth ventricle (4V) floor, a large number of CB-HRP positive neurons are consistently located, form a cluster, and are symmetrically located on both sides of the midline. (3) 3D reconstruction shows that the CB-HRP positive neurons cluster in the monkey brain occupies certain space. The rostral part is large and caudal part is thin appearing a "rivet"-like shape. (4) Under electron microscopy, the CB-HRP positive neurons show different types of synaptic connections with the non-CSF-contacting structures in the brain. Some of the processes stretch directly into the ventricle cavity. CONCLUSION: Same as we did in rats, the CSF-contacting nucleus is also existed in the primate brain parenchyma. We also recommend listing it as the XIII pair of cranial nucleus, which is specialized in the communications between the brain and the CSF. It is significant to the completing of innervation in the organism.

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.

Monosynaptic Input Mapping of Diencephalic Projections to the Cerebrospinal Fluid-Contacting Nucleus in the Rat.

Objective: To investigate the projections the cerebrospinal fluid-contacting (CSF-contacting) nucleus receives from the diencephalon and to speculate on the functional significance of these connections. Methods: The retrograde tracer cholera toxin B subunit (CB) was injected into the CSF-contacting nucleus in SD rats according to the experimental formula of the stereotaxic coordinates. Animals were perfused 7-10 days after the injection, and the diencephalon was sliced at 40 µm with a freezing microtome. CB-immunofluorescence was performed on all diencephalic sections. The features of CB-positive neuron distribution in the diencephalon were observed with a fluorescence microscope. Results: The retrograde labeled CB-positive neurons were found in the epithalamus, subthalamus, and hypothalamus. Three functional diencephalic areas including 43 sub-regions revealed projections to the CSF-contacting nucleus. The CB-positive neurons were distributed in different density ranges: sparse, moderate, and dense. Conclusion: Based on the connectivity patterns of the CSF-contacting nucleus that receives anatomical inputs from the diencephalon, we preliminarily assume that the CSF-contacting nucleus participates in homeostasis regulation, visceral activity, stress, emotion, pain and addiction, and sleeping and arousal. The present study firstly illustrates the broad projections of the CSF-contacting nucleus from the diencephalon, which implies the complicated functions of the nucleus especially for the unique roles of coordination in neural and body fluids regulations.