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.

Melanin-concentrating hormone (MCH) immunoreactivity in non-neuronal cells within the raphe nuclei and subventricular region of the brainstem of the cat.

Neurons that utilize melanin-concentrating hormone (MCH) as a neuromodulator are localized within the postero-lateral hypothalamus and zona incerta. These neurons project diffusely throughout the central nervous system and have been implicated in critical physiological processes such as energy homeostasis and sleep. In the present report, we examined the distribution of MCH immunoreactivity in the brainstem of the cat. In addition to MCH+ axons, we found MCH-immunoreactive cells that have not been previously described either in the midbrain raphe nuclei or in the periaqueductal and periventricular areas. These MCH+ cells constituted: 1. ependymal cells that lined the fourth ventricle and aqueduct, 2. ependymal cells with long basal processes that projected deeply into the subventricular (subaqueductal) parenchyma, and, 3. cells in subventricular regions and the midbrain raphe nuclei. The MCH+ cells in the midbrain raphe nuclei were closely related to neuronal processes of serotonergic neurons. Utilizing Neu-N and GFAP immunohistochemistry we determined that the preceding MCH+ cells were neither neurons nor astrocytes. However, we found that vimentin, an intermediate-filament protein that is used as a marker for tanycytes, was specifically co-localized with MCH in these cells. We conclude that MCH is present in tanycytes whose processes innervate the midbrain raphe nuclei and adjacent subependymal regions. Because tanycytes are specialized cells that transport substances from the cerebrospinal fluid (CSF) to neural parenchyma, we suggest that MCH is absorbed from the CSF by tanycytes and subsequently liberate to act upon neurons of brainstem nuclei.

Localization of myosin II regulatory light chain in the cerebral vasculature.

The cytoskeleton of cerebral microvascular endothelial cells is a critical determinant of blood-brain barrier (BBB) function. Barrier integrity appears to be particularly sensitive to the phosphorylation state of specific residues within myosin regulatory light chain (RLC), one of two accessory light chains of the myosin II motor complex. Phosphorylation of myosin RLC by myosin light chain kinase (MLCK) has been implicated in BBB dysfunction associated with alcohol abuse and hypoxia, whereas dephosphorylation may enhance BBB integrity following exposure to lipid-lowering statin drugs. Using immunohistochemistry we provide evidence of widespread myosin II RLC distribution throughout the cerebral vasculature of the mouse. Light microscopy revealed immunolocalization of myosin II RLC protein in the endothelium of brain capillaries, the endothelial cell layer of arterioles and in association with venules. Immunolabeling of myosin RLC in non-muscle endothelial cells could be distinguished from myosin RLC immunoreactivity associated with the smooth muscle layer of the tunica media in larger muscular arterioles. These findings support an emerging role for myosin II RLC as a component of the actomyosin cytoskeleton of cerebral endothelial cells with the potential to contribute to the selective vulnerability of the brain in vivo.

Subcommissural organ/Reissner's fiber complex: characterization of SCO-spondin, a glycoprotein with potent activity on neurite outgrowth.

In the developing vertebrate nervous system, several proteins of the thrombospondin superfamily act on axonal pathfinding. By successive screening of a SCO-cDNA library, we have characterized a new member of this superfamily, which we call SCO-spondin. This extracellular matrix glycoprotein of 4,560 amino acids is expressed and secreted early in development by the subcommissural organ (SCO), an ependymal differentiation located in the roof of the Sylvian aqueduct. Furthermore, SCO-spondin makes part of Reissner's fiber (RF), a thread-like structure present in the central canal of the spinal cord. This novel protein shows a unique arrangement of several conserved domains, including 26 thrombospondin type 1 repeats (TSR), nine low-density lipoprotein receptor (LDLr) type A domains, two epidermal growth factor (EGF)-like domains, and N- and C-terminal von Willebrand factor (vWF) cysteine-rich domains, all of which are potent sites of protein-protein interaction. Regarding the huge number of TSR, the putative function of SCO-spondin on axonal guidance is discussed in comparison with other developmental molecules of the CNS exhibiting TSR. To correlate SCO-spondin molecular feature and function, we tested the effect of oligopeptides, whose sequences include highly conserved amino acids of the consensus domains on a neuroblastoma cell line B 104. One of these peptides (WSGWSSCSRSCG) markedly increased neurite outgrowth of B 104 cells and this effect was dose dependent. Thus, SCO-spondin is a favorable substrate for neurite outgrowth and may participate in the posterior commissure formation and spinal cord differentiation during ontogenesis of the central nervous system.

A scanning and transmission electron microscopic analysis of the cerebral aqueduct in the rabbit.

An examination of the surface of the cerebral aqueduct with the scanning electron microscope revealed that the walls of the cerebral aqueduct were so heavily ciliated that most of the ependymal surface was obscured, yet certain specialized supraependymal structures could be discerned lying on (or embedded within) this matt of cilia. These structures were determined by transmission electron microscopy and Golgi analysis to be either macrophages, supraependymal neurons, dendrites from medial periaqueductal gray neurons, or axons of unknown origin. Some axons, which were found to contain vesicles, appeared to make synaptic contacts with ependymal cells. Using the transmission electron microscope, the ependymal lining was found to consist of two different cell types: normal ependymal cells and tanycytes which have a long tapering basal process that was observed to contact blood vessels or, more rarely, seemed to terminate in relation to neuronal elements. While there have been previous reports on the structure of the third and lateral ventricles in other species, there are limited reports in the rabbit. The present report is not only the first description for the rabbit, but it is the first complete scanning and transmission electron microscopic analysis of the cerebral aqueduct in any species.

Extrastriate cortical neurons correlated with ocular convergence in the cat.

Responses of neurons in the lateral suprasylvian area to visual stimulation in association with ocular convergence were studied in eight alert cats trained to track a visual target moving in depth. Activities of 18 (3%) of 659 cells were related to ocular convergence. These 18 neurons were divided into two groups: activities of seven neurons (40%) correlated with peak velocities of convergence eye movement at both fast and slow target speeds (group I), while those of five neurons (30%) correlated with them only at faster target speed (group II). Activities of six other neurons correlated with peak velocity of ocular convergence at faster target speed but were not tested at slower speed. Activities of four group-I neurons (60%) did not or only weakly correlate with lens accommodation, while those of four group-II neurons (80%) correlated with peak velocity or amplitude of lens accommodation. It can therefore be concluded that the four group-I neurons are primarily related to ocular convergence. Other cells were either convergence-related, lens accommodation-related or both. It is suggested that these different types of neurons contribute in combination to optimal control of convergence eye movement.

Ocular convergence-related neuronal responses in the lateral suprasylvian area of alert cats.

Neuronal spike discharges were recorded from the lateral suprasylvian (LS) area while ocular convergence was elicited in five alert cats. Ocular convergence was elicited by presenting a visual target moving in depth. Cats were rewarded for convergence eye movement. In 9 out of 426 cells sampled in the caudal postero-medial LS area, the number of spikes was positively correlated with the peak eye velocities during ocular convergence. Significant correlation was found mostly within 400 ms preceding the moment at which the maximum velocity of ocular convergence was obtained. The result favors the hypothesis that the LS area plays an important role in the integrative control of ocular convergence.

Functional roles of the lateral suprasylvian cortex in ocular near response in the cat.

The lateral suprasylvian (LS) area, an extrastriate visual area in the cat, has been suggested to play an important role in processing motion in 3-dimensional visual space. In addition, the LS area is related to all three components of the ocular near response, i.e. lens accommodation, pupillary constriction, and ocular convergence: microstimulation in this area evoked these intra- and extraocular movements, and neuronal discharges associated with these movements were also found. Anatomical pathways, direct and indirect, from this area to premotor nuclei in the brainstem are known to exist. The present paper reviews studies useful for assessing the functional roles played by the LS area in triggering and modulating component movements in the ocular near response.

Regressive changes among corticocortical neurons projecting from the lateral suprasylvian cortex to area 18 of the kitten's visual cortex.

The postnatal development of corticocortical neurons projecting from the medial bank of the lateral suprasylvian cortex to area 18 of the kitten's visual cortex was examined using retrograde fluorescent tracers. Area 18 was injected in young kittens aged nine days or less and in older kittens aged 30 days or more. Many of the injected kittens were perfused with fixative four to five days later, but some of the youngest were killed after longer survival periods of 35-50 days (long-survival animals). Labelled neurons in the medial bank of the lateral suprasylvian cortex were densely distributed in both superficial layers (II and III) and deep layers (V and VI) in the kittens injected less than nine days postnatal, irrespective of whether survival was short or long, but they were found almost exclusively in layers V and VI in the old, short-survival animals. Only in the group of old kittens did we find a clear topographical arrangement of projections in the rostrocaudal direction and a correlation between the rostrocaudal lengths of the injection sites and labelled areas. In the other two groups, for a similarly sized injection site, the labelled areas were much longer rostrocaudally than in the old, short-survival kittens, and occupied roughly the posterior two-thirds of the medial bank of the lateral suprasylvian cortex, irrespective of the positions of the injections. In the frontal plane, topography was unclear in all groups. These findings demonstrate that there is considerable postnatal refinement of the projection from the medial bank of the lateral suprasylvian cortex to area 18. This involves a loss of connections originating from superficial layers and a decrease of convergence with the appearance of topography. Our results from long-survival kittens suggest that most of the early exuberant population of corticocortical neurons projecting from the medial bank of the lateral suprasylvian cortex to area 18 survive beyond the first postnatal month but undergo axonal elimination during this period.

Dopaminergic cells align along radial glia in the developing mesencephalon of the rat.

Studies were performed to examine the relation of dopaminergic cells and radial glia in the developing mesencephalon of the rat at ages E12-E20. Dopaminergic cells were immunolabelled with an antiserum which recognizes tyrosine hydroxylase, and radial glia were immunolabelled with a monoclonal antibody which recognizes vimentin. The vimentin-immunoreactive fibres of radial glia were noted at E12. At E12, and more clearly at later time points, the radial glia extended from the aqueduct to the pial surface, and this pattern persisted throughout the prenatal period. Tyrosine hydroxylase-immunoreactive cells were located along the ventral surface of the mesencephalon at age E13. At age E15, E16, and E18 the tyrosine hydroxylase-immunoreactive cells were present from the aqueduct to the ventral pial surface of the mesencephalon and were aligned along radial glia. Our study suggests that radial glia provide paths for migration of dopaminergic cells in the mantle layer from E15 to E18 of the developing mesencephalon. It also suggests that some dopaminergic cells between E15 and E18 may express tyrosine hydroxylase during their migration through the mantle layer and prior to reaching the location they occupy in the adult brain.

Immunocytochemical localization of serotonin in the rat periaqueductal gray: a quantitative light and electron microscopic study.

The distribution of serotonin-like immunoreactivity in five regions of the rodent midbrain periaqueductal gray (PAG) was studied by using light and electron microscopic immunohistochemistry in combination with quantitative analysis. Light microscopic analysis revealed the presence of serotonin-like immunoreactive cell bodies located in the ventrolateral and ventromedial regions of the caudal PAG and serotonin-like immunoreactive processes throughout the PAG. Ultrastructural analysis showed dendritic profiles that stained positively for serotonin primarily in ventral regions, although an occasional profile was seen dorsally. Numerous synaptic contacts between unstained axon terminals and ventral dendritic profiles were seen. Axonal profiles that contained reaction product were identified throughout the PAG, but were rarely observed to make any type of specialized contact. Ultrastructural quantification of serotonin-like immunoreactive processes indicated that the highest volume fraction of serotonin immunoreactivity occurred caudoventrally where stained processes constituted 2.6% of the neuropil volume. Rostroventrally stained processes constituted only 0.14% of the neuropil volume at the level of the posterior commissure. By contrast the amount of serotonin-like immunoreactivity found dorsally remained relatively constant at all rostrocaudal levels. Analysis of serotonin staining among PAG regions demonstrated the lowest overall volume fraction in the dorsal region and the highest overall volume fraction in the ventromedial region. No significant differences were observed between medial and lateral regions. A comparison of the results of light microscopic quantitative analysis of serotoninergic processes with electron microscopic quantitative analysis indicated that both techniques produce comparable results.

Binocular properties of lateral suprasylvian cortex are not affected by neonatal corpus callosum section.

The lateral suprasylvian cortex (LS) was physiologically examined in cats with neonatal section of the posterior corpus callosum (CC) at 2 or 3 weeks after birth. All receptive field properties of the LS cells, including ocular dominance, were found to be normal, despite a reduction in binocular activation in area 17 in the 2 week CC cats. Therefore, binocular activation of LS cells is not dependent on callosal input at any age, nor is it dependent on normal levels of binocular activation of striate cortex.

Aqueductal tanycytes in the rabbit brain: A Golgi study.

Utilizing Golgi-Cox impregnation, tanycytes were found in the ependyma of the cerebral aqueduct of the neonatal and adult rabbit. These tanycyte somas showed a variety of shapes, apical projections into the aqueduct, and basal processes (shafts) projecting into the mesencephalon, particularly into the periaqueductal gray (PAG). The shafts showed a variety of branching patterns, and sometimes abutted or terminated on capillaries or on specific neuronal elements. Other shafts coiled within the PAG or terminated within the neuropil of the mesencephalon. It is possible that these tanycytes provide a route for transport of cerebrospinal-fluid-borne substances from the aqueduct to the neuronal regions and vasculature of the mesencephalon. The presence of these tanycytes with complex branching patterns in proximity to neural and vascular structures suggests a permanent, active role for these specialized ependymal cells.

Properties of visually sensitive neurons in lateral suprasylvian area of the cat.

Functional properties of neurons in lateral suprasylvian area were investigated by single unit recordings in unanesthetized cats with the brain stem pretrigeminal transection. Majority of cells in the lateral suprasylvian area responded vigorously to moving visual stimuli. Many of them were responsive only to the movement of black objects, without any reaction to the light stimuli, 78 percent of the observed neurons revealed direction selective properties, the remainder being direction nonselective. Seventy eight percent of neurons revealed well defined responses to stationary flashes of light and 26 percent showed multimodal type of responses to moving and stationary visual stimuli. The vertical organization of neurons in the lateral suprasylvian area was investigated. The present results indicate that a regular vertical organization of neurons exists in the lateral suprasylvian cortex of the cat.

The raphe nuclei of the cat brain stem: a topographical atlas of their efferent projections as revealed by autoradiography.

Stereotaxic injections of [14C]leucine were made in nulei raphe centralis superior, raphe dorsalis, raphe magnus and raphe pontis of the cat. The organization of the regional connections was outlined in a stereotaxic atlas using the autoradiographic tracing method: the majority of the ascending pathways from the rostral raphe nuclei are directed mainly through a ventrolateral bundle via the ventral tegmental area of Tsai, with some lateral extensions to the substantia nigra, and then through the fields of Forel and the zona incerta. More rostrally the fibers are joined to the medial forebrain bundle through the hypothalamic region up to the preoptic area or the diagonal band of Broca. Multiple divisions leave this tract towards the epithalamic or the intralaminar thalamic nuclei, the stria terminalis, the septum, the capsula interna and the ansa lenticularis. The bulk of the rostral projections terminates in the frontal lobe, while some labeling is scarcely distributed throughout the rest of the neocortex. The projections of nucleus (n.) raphe centralis superior are specifically associated with the n. interpeduncularis, the mammillary bodies and the hippocampal formation while the n. raphe dorsalis innervates selectively the lateral geniculate bodies, striatus, piriform lobes, olfactory bulb and amygdala. The rest of the ascending fibers form the centrolateral or the dorsal ascending tracts radiating either in the reticular mesencephalic formation or in the periventricular gray matter. On the contrary there are heavy descending projections from n. raphe centralis superior which distribute to the main nuclei of the brain stem, the central gray matter and the cerebellum. The ascending projections form the caudal raphe nuclei are much less dense. They disseminate mainly in the colliculus superior, the pretectum, the nucleus of the posterior commissure, the preoculomotor complex and the intralaminar nuclei of the thalamus. From n. raphe pontis, a dense labeling is selectively localized at the n. paraventricularis hypothalami with some rostral extensions to limbic areas. Diffuse caudal and rostral projections from both nuclei are observed in the mesencephalic, pontobulbar reticular formation and the cerebellum. The main differences come from the specific localization of their descending bulbospinal tracts inside the lateroventral funiculus of the spinal cervical cord.