Raymond H. Ho

The distribution and origin of serotonin immunoreactivity in the rat cerebellum

The distribution of serotonin immunoreactivity in the rat cerebellum was studied using the indirect antibody peroxidase-antiperoxidase (PAP) technique of Sternberger. Furthermore, the origin of these chemically defined cerebellar afferents was studied using a procedure which combines the retrograde transport of horseradish peroxidase (HRP) with the PAP technique. Serotoninergic fibers and varicosities distribute throughout the cerebellar cortex. However, within the cortex there are density variations in the distribution of this indoleamine to the granule cell and molecular layers as well as differences in the spatial orientation of labeled elements, especially in the latter lamina. Serotonin-positive fibers are also present in the Purkinje cell layer. Some of the fibers pass from this layer into the overlying molecular layer while others form a plexus around the somata of Purkinje cells. Subsequent to injections of HRP into the vermis and immediately adjacent portions of the cerebellar cortex, several reticular and raphe nuclei (n.) were found to project to the cerebellum including the paramedian reticular nucleus, n. raphe pallidus, n. raphe obscurus, n. raphe magnus, n. reticularis gigantocellularis, n. reticularis paragigantocellularis, n. pontis oralis, n. reticularis tegmenti pontis and n. centralis superioris. Double-labeling experiments, however, reveal that the neurons giving rise to serotoninergic afferents to the cerebellum are located almost exclusively in the n. reticularis gigantocellularis, the n. reticularis paragigantocellularis and the n. pontis oralis. In conclusion, the findings of the present study further support the view that the cerebellar cortex is not uniform in its histological structure. Although serotoninergic elements are distributed throughout the cerebellar cortex, there are lobular variations in the laminar distribution of this indoleamine. These data suggest that serotonin may be exerting its physiological effect on different populations of cortical neurons in different lobules. Thus this putative neurotransmitter may play different roles in the circuitry of the cerebellum in disparate regions of the cortex. Further, the data obtained in the double-label experiments demonstrate a fairly restricted origin for serotoninergic afferents in the medullary and pontine reticular formation. Moreover, the majority are not located in the raphe nuclei.

Substance P immunoreactivity in the median eminence of the North American opossum and domestic fowl

Substance P (SP) was first detected in extracts of equine brain and intestine by von Euler and Gaddum in 1931 z3. Since then, this undecapeptide has been isolated, purified, biochemically characterized, and synthesized1, zl. The availability of pure synthetic SP in conjunction with recent advances in producing antibodies to small peptides allowed immunohistochemical identification of the peptides immunoreactivity* within specific regions of the nervous system. Extensive immunohistochemical mapping of SP in the rat nervous system has been accomplished 4,16. Although these studies revealed an abundance of SP-containing neuronal elements in the hypothalamus, relatively few of them were found within the median eminence (ME). Corroborating these morphological findings, radioimmunoassay studies have shown low concentrations of SP immunoreactivity in the rat ME 18. Recently, Hfkfel t et al. have reported a very dense plexus of SP varicosities and terminals in the external layer of the ME in the monkey and human t2. The location of these terminals adjacent to the primary portal vessels suggests that the peptide is released into the portal vessels and subsequently transported to the anterior pituitary gland. This concept fits well with the classical Portal Vessel-Chemotransmitter hypothesis of Harris 10. On the other hand, the presence of these SP elements in the M E may also suggest a SP-mediated indirect control of anterior pituitary function via possible axo-axonic interactions within the ME 8. A similar SP plexus was not observed in the rat ME 12. The comparative studies we report herein corroborate the relative lack of SP within the ME of the rat, but they also reveal that a SP plexus is present in the ME of at least two non-primate species, namely, the North American opossum, Didelphis virginiana (a marsupial) and the white leghorn cockerel, Gallus domesticus (a bird).

Stimulatory Role of Substance P on Gonadotropin Release in Ovariectomized Rats

Substance P (SP) has been shown to be present in the hypothalamus and anterior pituitary. To evaluate a possible physiological role of endogenous SP in the control of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) release, specific antiserum against SP (anti-SP) was injected intraventricularly (3 microliters into the third ventricle) or intravenously (50 or 200 microliters) into conscious, ovariectomized (OVX) rats. Third ventricular injection of the antiserum induced a significant decrease in both plasma LH and FSH levels when compared to values in control animals injected with normal rabbit serum (p less than 0.01 and p less than 0.025, respectively). The effect was observed within 10 mi and levels remained suppressed for 60 min. In contrast, intravenous injection of large doses of anti-SP had no effect on the release of both hormones. In order to confirm the stimulatory effect of SP itself, synthetic SP was injected intravenously and intraventricularly into estrogen-primed (E-primed), OVX rats. Synthetic SP dramatically stimulated LH release, but not FSH release when injected either intravenously or intraventricularly at doses of 10 and 50 micrograms (p less than 0.001, p less than 0.005 vs. control, respectively). To investigate any direct action of SP on gonadotropin release from the anterior pituitary gland, synthetic SP was incubated with dispersed anterior pituitary cells harvested from E-primed OVX rats. SP did not affect the release of gonadotropins in vitro. These results indicate that endogenous hypothalamic SP exerts a tonic stimulatory hypothalamic control of basal gonadotropin release in OVX rats.

Partial characterization of immunoreactive substance P in the rat pituitary gland

Two distinct carboxy-terminus-directed anti-substance P (SP) sera (R-1C and R-6G) were used to characterize immunoreactive SP (I-SP) in acetic acid extracts of anterior pituitary (AP) and posterior pituitary (PP) glands of adult male rats. The tissue concentrations of I-SP measured by R-1C and R-6G were comparable. The contents of I-SP were 600-1150 pg/AP and 25-52 pg/PP. I-luteinizing hormone releasing hormone and I-somatostatin (I-SOM) were undetectable in AP extracts, but PP extracts contained the equivalents of 325-785 pg I-SOM/gland. Serial dilutions of AP and PP extracts produced displacement curves with both SP antisera that were parallel to the respective synthetic SP standard and hypothalamic extract displacement curves. Gel filtrations of AP and PP extracts on a Sephadex G-25 column produced I-SP peaks eluting in the same fractions as synthetic SP and hypothalamic I-SP. However, the AP I-SP profile also revealed a side peak migrating between the void volume and the major I-SP peak. Neither immunoreactive species in the AP extract were eliminated when eluted with 6.0 M guanidine HCl, a strong denaturing agent. In vitro incubation of paired anterior hemipituitaries for 30 min in the presence of a 56 mM K+ concentration resulted in a significant (p less than .0001), 25-fold increase in the release of I-SP into the incubation medium above the mean control value. Radiofrequency lesions placed in the median eminence-arcuate region of male rats caused a significant (p less than .001) reduction of I-SP in both the AP and PP. These reductions were inversely related to the plasma prolactin values. The elevation in plasma prolactin was taken as an index of completeness of lesions. We conclude that: 1) the rat pituitary contains I-SP as assessed by its immunologic and chromatographic behavior, 2) K+ depolarization is a potent stimulator of the release of AP I-SP in vitro, 3) the ME-arcuate region is important for the maintenance of pituitary I-SP levels in the rat.

Anatomical evidence for enkephalin immunoreactive climbing fibres in the cerebellar cortex of the opossum

SummaryEnkephalin immunoreactivity is present in the cerebellum of the adult opossum within axonal arbors that resemble mature climbing fibres. In the developing cerebellum, enkephalinergic axons form pericellular nests around the perikarya of Purkinje cells in a manner which resembles developing climbing fibres seen in Golgi impregnations. Serial electron micrographs of adult climbing fibres reveal elongate enkephalin immunoreactive profiles that contain synaptic vesicles and make contact with the thorns and shafts of Purkinje cell dendrites. These results suggest that a peptide, enkephalin or an enkephalin-like substance may mediate synaptic interactions between certain populations of climbing fibres and Purkinje cells in the cerebellum of the opossum. Enkephalin immunoreactive axonal arbors, present in the molecular layer, are localized in restricted areas of vermal lobules II–VIII and X. The intermediate cortex and hemispheres are devoid of enkephalinergic climbing fibres except in restricted areas of the paramedian lobule, paraflocculus and the flocculus. In an attempt to establish the origin of enkephalin axons in the cerebellum, a double labelling technique that combines retrograde labelling of cells with horseradish peroxidase and enkephalin immunohistochemistry has been employed. Enkephalin immunoreactive neurons within specific portions of the medial accessory olive are retrogradely labelled in this paradigm. The presence of enkephalin immunoreactivity in selected climbing fibres provides evidence for chemical heterogeneity within one of the major afferent systems to the cerebellum previously thought to be uniform in its transmitter content.

Widespread distribution of substance P-and somatostatin-immunoreactive elements in the spinal cord of the neonatal rat

SummaryThe distribution of substance P (SP)- and somatostatin (SOM)-immunoreactive elements in the spinal cord of the neonatal rat was examined. With few exceptions, the distribution of SP-immunoreactive elements is similar to that described for the adult. A major difference is the obvious presence of SP-immunoreactive fibers in all funiculi of neonatal cords. In addition, an obvious small bundle of longitudinal SP immunoreactive fibers is seen in the base of the dorsal horn at rostral cervical levels. Unlike that of the adult, the neonatal spinal cord shows a widespread distribution of SOM-immunoreactivity. SOM-immunoreactive fibers are present in all funiculi. SOM-immunoreactive perikarya of various shapes and sizes are widely dispersed throughout the gray matter. The cell density is increased in the superficial laminae of the dorsal horn, in a region ventral-lateral to the central canal and in the ventral horn. SOM-immunoreactive varicosities are present in moderate amounts in the superficial laminae of the dorsal horn but are extremely sparse in other regions of the gray matter. A few SOM-immunoreactive fibers course longitudinally at the base of the dorsal horn at rostral levels of the cord. These fibers are found in the same region occupied by the longitudinal SP-immunoreactive fibers referred to above.

Cell bodies of origin of serotonin-immunoreactive afferents to the inferior olivary complex of the rat.

Previous studies have used immunohistochemistry to localize serotonin to distinct olivary nuclei in several mammalian species. However, the location of the cell bodies of origin for the serotoninergic projection to the inferior olive in any of these species was unknown. In the present study, a paradigm which combines transport of horseradish peroxidase (HRP) and serotonin immunohistochemistry (PAP) was used to identify the cell bodies of origin of this afferent system to the inferior olivary complex of the rat. Cells which contain both retrogradely transported HRP and brown cytoplasmic staining indicating that they are serotoninergic cells that project to the inferior olivary complex are found exclusively in an area dorsal to the rostrolateral dorsal accessory olive within the nucleus reticularis paragigantocellularis. Neurons within this nucleus were also found to be a source of serotoninergic afferents to the cerebellum and spinal cord of the rat. This raises the possibility that individual serotonin-immunoreactive neurons within this nucleus may project to all 3 areas. Future studies will be designed to address this possibility. No double-labeled cells were observed within any of the raphe nuclei.

Substance P and serotonin immunoreactivity in the rat inferior olive

The present study analyzes in detail the distribution of substance P and serotonin immunoreactivity in the inferior olivary complex of the rat using the indirect antibody peroxidase-antiperoxidase technique. The entire dorsal accessory olive contains an abundance of substance P fibers and varicosities. In addition, the dorsal cap of Kooy, nucleus Beta, parts b and c of the medial accessory olive, and the dorsomedial cell column contain numerous substance P immunoreactive elements. Fibers and varicosities positive for serotonin are present throughout the entire dorsal accessory olive. In addition, the rostral medial accessory olive contains an abundance of serotonin immunostaining. Scattered serotonin immunoreactive elements are present in the principal olive in particular in the area immediately subjacent to the lateral aspect of the dorsal accessory olive. Only scant serotonin immunolabeling is present in the dorsal cap of Kooy, nucleus Beta and the dorsomedial cell column. Speculations on possible functional roles of these two putative neurotransmitters are presented.

Reticular and Raphe Projections to the Spinal Cord of the North American Opossum. Evidence for Connectional Heterogeneity

Publisher Summary The chapter reviews the organization of reticular and raphe projections to the opossums spinal cord and describes possible neurotransmitters or modulators employed by some of them. The opossums central nervous system is relatively generalized in comparison with that of most mammals. Corticospinal tracts exist, but they are limited and can only be traced to laminae III–VI of the cervical cord. In contrast, axons from the red nucleus and vestibular complex, as well as from several reticular and raphe nuclei, project the length of the spinal cord. Reticular and raphe neurons projecting to different levels of the spinal cord are identified by the horseradish peroxidase (HRP) technique. In a study described in the chapter, HRP was injected into the desired level of the cord where it was incorporated by axons and transported retrogradely to the parent cell bodies. The presence of HRP in reticular and raphe cells was revealed by several histochemical techniques.

An immunohistochemical study of serotonin development in the opossum cerebellum

SummaryIn the present study we have used the indirect antibody peroxidase anti-peroxidase technique to analyze the development of serotonin in the cerebellum of pouch young opossums ranging in age from birth (postnatal day (PD) 1) — to PD 47. The pathways by which serotoninergic axons enter the cerebellum appear to change during development. Between PD 1 and PD 11 varicose serotoninergic fibers course though the continuity between the tectum and the dorsal medial aspect of the cerebellar plate and distribute primarily to the intermediate zone. By PD 11 serotoninergic fibers enter the cerebellum via a connecting band between the cerebellum and the dorsal lateral aspect of the rostral medulla. Fibers entering the cerebellum via this later route course around the lateral recess of the fourth ventricle and initially distribute to ventral and lateral areas of the cerebellum. At later developmental ages (PD 14–PD 47) serotoninergic fibers are present in: 1) the cellular zone of migration between the Purkinje cell layer and deep nuclei; 2) the Pukinje cell layer; 3) the internal granule cell layer; and 4) the deep cerebellar nuclei. The external granule cell and the molecular layers rarely contain serotoninergic fibers.The present study has shown that serotonin is present in the cerebellar anlage on PD 1 (within 13 days of conception). This is prior to the arrival of other major afferent systems (King et al. 1982; Bishop et al. 1983; Martin et al. 1983; Morgan et al. 1983). Further, serotoninergic axons reach the cerebellum via different routes at different stages of development. The presence of serotonin in the intermediate zone early in development supports the hypothesis that it may influence neuronal migration and differentiation (Lander and Krebs 1976). Axons containing serotonin and arriving later in development may function in synaptic transmission, a role proposed for this indoleamine in the adult (Strahlendorf et al. 1979).