R.M. Bowker

Noradrenergic projections to the spinal cord of the rat

Noradrenergic terminals were identified in the spinal cord of rats by immunocytochemical staining for dopamine-beta-hydroxylase. Although immunoreactive fibers and terminals were observed throughout the spinal grey matter, heavier accumulations of terminal labeling were observed in the marginal layer of the dorsal horn, in the ventral horn among motoneurons, and in the autonomic lateral cell columns of the thoracic and sacral spinal cord. Two specific retrograde transport techniques were employed to identify the origins of these noradrenergic terminations in the spinal cord. Cells of origin were observed in the locus coeruleus, the subcoeruleus, the medial and lateral parabrachial, and the Kölliker-Fuse nuclei, as well as adjacent to the superior olivary nucleus. These regions correspond to the A5-A7 cell groups of the pons. No spinally projecting noradrenergic cells were ever observed in the medulla. It was concluded that pontine noradrenergic cell groups are the sole source of noradrenergic terminals in the spinal cord.

Descending serotonergic, peptidergic and cholinergic pathways from the raphe nuclei: A multiple transmitter complex

The localization of serotonergic, various peptidergic and possibly cholinergic neurons in the medullary raphe nuclei that project to the lumbosacral spinal cord have been studied using a retrograde transport method combined with immunocytochemical and histochemical techniques. Spinally projecting neurons stained for serotonin-like, substance P-like, enkephalin-like and thyrotropin-releasing hormone-like immunoreactivity and for the histochemical marker acetylcholinesterase were all observed in each of the raphe nuclei of the medulla, as well as in the adjacent ventrolateral reticular formation. The similar distributions of the descending serotonergic and peptidergic neurons in the raphe nuclei as well as quantitative data on their relative numbers suggest that a large fraction of raphe-spinal neurons contain serotonin co-existing with one or more peptides in the same cell.

Origins of serotonergic projections to the spinal cord in rat: An immunocytochemical-retrograde transport study

The origins of the serotonergic projections to the spinal cord in the rat were determined by employing the retrograde cell marker HRP coupled with the unlabeled antibody, peroxidase-antiperoxidase immunocytochemical method of Sternberger. Large numbers of stained neurons (greater than 70%) in the medullary raphe nuclear complex were found to contain both HRP retrogradely transported from the spinal cord and positive 5-HT staining. These serotonergic cell groups, including the nucleus raphe obscurus, raphe pallidus, raphe magnus, and the ventral parts of the reticular formation, project to all spinal cord levels. In addition, some neurons contained HRP granules, but were unstained for 5-HT, suggesting that they may contain other non-serotonergic neurotransmitters. More rostrally in the midbrain reticular formation, many 5-HT neurons were found to have projections exclusively to the cervical spinal cord. These findings indicate that the descending serotonin inputs to the spinal cord originate not only from the serotonergic neurons located in the medullary raphe complex, but also from other new sources located in the central gray and reticular formation of the midbrain.

Serotonergic and peptidergic projections to the spinal cord demonstrated by a combined retrograde HRP histochemical and immunocytochemical staining method.

A simple technique is described for simultaneously visualizing the cells of origin of descending brain stem-spinal systems and immunocytochemically defined neurotransmitters. The combination of horseradish peroxidase (HRP) histochemistry, enhanced by the use of CoCl2, with the unlabeled antibody, peroxidase-antiperoxidase (PAP) immunocytochemical staining method, revealed double labeled cells containing black punctate HRP granules within a homogeneous brown cytoplasm stained positively for serotonin (5-HT), substance P or enkephalin-like immunoreactivity. The advantages of this method over other double labeling techniques are discussed.

Origins and terminations of descending noradrenergic projections to the spinal cord of monkey

This report describes the distribution of noradrenergic cells in the brainstem and the pattern of terminal varicosities in the spinal cord of monkey using the immunocytochemical localization of dopamine-beta-hydroxylase (DBH). Using two separate and equally reliable techniques, retrograde transport of the antibody to DBH and a double-labeling method, the cells of origin of noradrenergic fibers in the spinal cord have been identified. The results of these studies indicate that 79% of all noradrenergic cells with axons projecting to the spinal cord are located in the nucleus subcoeruleus and nucleus locus coeruleus. Other pontine noradrenergic cell groups contribute the remainder of the fibers to the cord. No medullary cells contribute to the noradrenergic innervation of the spinal cord.

Organization of Descending Serotonergic Projections to the Spinal Cord

Publisher Summary This chapter discusses the organization of descending serotonergic projections to the spinal cord. The indolamine serotonin is believed to have important neurotransmitter and/or neuroregulatory roles in the descending pathways influencing various somatosensory, motor, and autonomic functions within the spinal cord. The autonomic preganglionic neurons of the thoracic spinal cord, as well as the motoneurons of the ventral horn, are also under the influence of descending serotonergic cell groups. Cells in these various motoneuron pools can be inhibited and/or excited by iontophoretic application of serotonin or by electrical stimulation of the descending serotonergic pathways. In recent years, the introduction of immunocytochemistry to neurobiology has provided a very sensitive method for studying the organization of descending serotonergic pathways. In contrast to the previously employed histofluorescence methods with which rapid photodecom-position of the yellow fluorescing cells and terminals was a constant problem, the peroxidase-antiperoxidase (PAP) immunochemical method results in a relatively permanent marker for 5-HT neurons and terminals.

Serotonergic projections to the caudal brain stem: a double label study using horseradish peroxidase and serotonin immunocytochemistry

Cells of origin of serotonergic and non-serotonergic projections to the caudal brain stem in the primate were examined using a double label technique. Following HRP injections into medullary raphe nuclei and the adjacent reticular formation double labeled cells were found in the dorsal raphe nucleus, the central superior nucleus and the ventrolateral tegmentum. Retrogradely labeled cells that did not stain for serotonin-like immunoreactivity were found primarily in the periaqueductal gray (PAG) and the mesencephalic and pontine reticular formation. The results are discussed in relation to the descending pathway(s) mediating the effects of PAG stimulation.

Transmitters of the raphe-spinal complex: Immunocytochemical studies

The localization of serotonergic and various peptidergic neurons in the medullary raphe nuclei that project to the lumbosacral spinal cord have been studied using a retrograde transport method combined with immunocytochemistry. Spinally projecting neurons stained for serotonin-like, substance P-like, enkephalin-like and thyrotropin-releasing hormone-like immunoreactivity were all observed in the raphe nuclei of the medulla, as well as in the adjacent ventrolateral reticular formation. The distribution of the descending serotonergic and peptidergic neurons in the raphe nuclei as well as quantitative data on their relative numbers suggest that a large fraction of raphe-spinal neurons contain serotonin co-existing with one or more peptides in the same cell.

A Combined Retrograde Transport and Immunocytochemical Staining Method for Demonstrating the Origins of Serotonergic Projections

Principle of Technique In recent years considerable interest has been directed toward defining projections of neurons containing specific neurotransmitten substances. As a result, numerous techniques have been developed that use fluorescent retrograde cell markers (2,6,10,1 1,19) on horseradish peroxidase (HRP) (3,4,13,14,17) combined with the fluorescence histochemistry (2, 1 7,19), immunohistofluonescence (6,10), or the unlabeled antibody, peroxidase-antiperoxidase (PAP) method (4,5,14) to identify the projections of neurotransmitter-defined neurons. While these techniques have contributed to the advancement of our basic understanding of the anatomical organization of certain pathways, they are often laborious and inconvenient or the existence of the different cell markers is relatively short-lived. A technique (4) has been developed in our laboratory that combines the retrograde transport of horseradish peroxidase (HRP) or of wheat germ agglutinin conjugated to HRP (WGA-HRP) with the PAP method of Sternberger. This technique can be employed to determine the cells ofonigin ofserotonergic pathways as well as pathways containing other putative neurotransmitter/modulator substances. In the present article, these methods are applied to determining the origin of descending serotonergic pathways to the spinal cord (4,5), but the techniques can be readily adapted to studies ofother systems (15,20). Instruments

The relationship of the medullary catecholamine containing neurones to the vagal motor nuclei

We have re-examined in the rat the nuclear localization of the medullary catecholamine-containing cell groups (A1 and A2) and their relation to the vagal motor nuclei using a double labeling method. The vagal nuclei were defined by the retrograde transport of horseradish peroxidase applied to the cervical vagus, and noradrenergic and adrenergic neurons were stained with the peroxidase-antiperoxidase immunocytochemical method using an antibody to dopamine beta-hydrolase. The method allows visualization of both labels within single neurons. The neurons of the A2 group are primarily distributed in both the nucleus of the solitary tract and the dorsal motor nucleus of the vagus in a complex interrelationship that depends on the rostrocaudal level. Caudal to the obex, cells of the dorsal motor nucleus of the vagus are scattered among cells immunoreactive for dopamine beta-hydroxylase in the area considered to be the commissural subnucleus of the nucleus of the solitary tract. At levels near and slightly rostral to the obex, the dopamine beta-hydroxylase-positive cells are largely confined to nucleus of the solitary tract. However, the rostral third of the A2 group lies predominantly within dorsal motor nucleus, as defined by horseradish peroxidase labeled cells, with only a few cells in the nucleus of the solitary tract. A subset of the dopamine beta-hydroxylase positive cells within the rostral dorsal motor nucleus of the vagus are also vagal efferents. Our results suggest that a second population of dopamine beta-hydroxylase positive vagal efferents may exist ventrolaterally where neurons of the AI cell group intermingle with those of nucleus ambiguus.