Substance P immunoreactivity in the anterior pituitary gland of the guinea pig.

The aim of this study was to demonstrate the presence of Substance P (SP) immunoreactive cells within the anterior pituitary gland of the adult guinea pig. By utilizing the PAP technique of Sternberger, immunoreactive SP-containing cells were visualized in all animals studied. These cells were most dense in the ventral portion of the gland. When adjacent tissue sections were incubated with anti-SP serum and anti-rat TSH serum, it was observed that most, if not all, the SP immunoreactive cells examined also exhibited TSH immunoreactivity. However, not all TSH immunoreactive cells contained SP immunoreactivity. Immunoabsorption controls indicated that the immunohistochemical staining reactions were specific for both SP and TSH. The results of this study indicate that immunoreactive SP is present in the guinea pig anterior pituitary and that this peptide is localized within TSH immunoreactive cells.

Substance P, somatostatin, and methionine enkephalin immunoreactive elements in the spinal cord of the domestic fowl, Gallus domesticus.

The occurrence and distribution of substance P (SP), somatostatin (SOM), and enkephalin (ENK) immunoreactive elements were examined in the spinal cord of the domestic fowl, Gallus domesticus. SP immunoreactive fibers and their varicosities were densest in laminae I and II, although they were also found within deeper regions of the dorsal horn. In contrast, the intermediate gray area, the area around the central canal, and the ventral horn, contained fewer SP immunoreactive fibers. The distribution of ENK immunoreactivity in the gray matter was similar to that described for SP although immunoreactive fibers were denser around the central canal and in the intermediate zone. Few SOM immunoreactive fibers were present in the dorsal horn, the area around the central canal, and the ventral horn. All three peptidergic immunoreactive elements were found in and around the nucleus of Terni, an autonomic area. Throughout the lumbosacral enlargement SP, SOM, and ENK immunoreactive varicosities were found adjacent to the lumbosacral sinus and in fibers traversing the glycogen body. In addition, at caudal lumbar and rostral sacral levels a plexus of SP and SOM immunoreactive fibers was observed to be in close relationship with presumed motoneurons.

Localization of serotonin immunoreactivity in the opossum cerebellum.

We have used the indirect antibody peroxidase-antiperoxidase technique to analyze the course of serotonin (5-hydroxytryptamine; 5HT) fibers to the deep cerebellar nuclei; the distribution of serotonin within the nuclei; the continued course of 5HT fibers to the cerebellar cortex; and the lobular and laminar distribution of this indoleamine in the cerebellar cortex. Only rarely are fibers found in either the restiform body or the brachium pontis. However, a distinct bundle of serotoninergic axons is present in the medial aspect of the brachium conjunctivum. Axons arise from this bundle and course dorsally into the neuropil of the deep cerebellar nuclei. The densest immunostaining is present in posterior and ventral regions of all four cerebellar nuclei. Within the nuclei large (24% of total) and small (76% of total) varicosities are present. The average distance between varicosities on individual axons is 3.85 micron (S.D. = 1.2). The innervation of the cerebellar cortex is derived primarily from fibers that course through the deep nuclei. At levels caudal to the deep nuclei a single midsagittal band courses into lobules VIII and IX. In the cerebellar cortex, serotoninergic axons and varicosities are present in all lobules; however, the fiber density is not uniform. The densest distribution is present in vermal lobule VIII and the dorsal folia of lobule IX. Within the granule cell layer of lobules VIII and IX, immunoreactive elements form a midsagittal band, and to a lesser degree, two parasagittal bands. Beaded serotoninergic fibers course through the deep and middle portion of the granule cell layer and give rise to a plexus at the border between the Purkinje cell and granule cell layers. Within this plexus axons extend long distances in the transverse and sagittal planes. Long beaded axons oriented in the transverse plane of the folia are also present in the deep molecular layer. A few radial serotoninergic fibers ascend to the pial surface and give rise to very short tangential branches. In all three cortical layers, both large (19% of total) and small (81% of total) varicosities are present. The average distance between varicosities on individual fibers is 5.3 micron (S.D. = 2.2).(ABSTRACT TRUNCATED AT 400 WORDS)

A comparison of the ultrastructure of substance P and enkephalin-immunoreactive elements in the nucleus of the dorsal lateral funiculus and laminae I and II of the rat spinal cord.

The ultrastructure of substance P (SP)- and enkephalin (ENK)-immunoreactive elements in the nucleus of the dorsal lateral funiculus (NDLF) and in laminae I and II of the spinal cord was examined in the rat using the peroxidate-antiperoxidase (PAP) technique. Electron-microscopic observations were made of a large number of immunolabelled terminals (n = 428; many followed in serial sections), axons, and immunoreactive cell bodies and dendrites which were occasionally encountered. Morphometric analysis was used to describe and compare the fine structural features of immunolabelled elements. Both SP- and ENK-immunoreactive terminals contained clear synaptic vesicles and dense-cored vesicles of similar size but the ENK-immunoreactive profiles contained significantly more dense-cored vesicles than SP-immunolabelled profiles. Both SP- and ENK-immunoreactive profiles in the dorsal laminae of the dorsal horn contacted mainly smaller dendritic elements. Only rarely were axo-axonic interactions noted. The NDLF contains widely scattered cell bodies dispersed within a neuropil which is rich in synaptic complexes and is interdigitated between fascicles of myelinated and unmyelinated axons. Numerous SP- and ENK-immunoreactive profiles were observed in the NDLF, many of which made asymmetric synaptic contacts with NDLF neurons. Although both the dorsal gray and NDLF contain large numbers of SP- and ENK-immunoreactive elements which are similar in morphology in both regions, the NDLF can be distinguished from laminae I and II by a number of criteria, including the nature of the neuropil, principle sources of SP innervation, and the termination patterns of ascending projections.

Immunohistochemical localization of substance-P, somatostatin, and methionine-enkephalin in the spinal cord and dorsal root ganglia of the North American opossum, Didelphis virginiana.

The occurrence and distribution of substance-P (SP), somatostatin (SOM), and enkephalin (ENK) were studied in the North American opossum, a generalized marsupial. Substance-P immunoreactivity was present in the tract of Lissauer as well as within lamina I, the outer part of lamina II, the lateral portion of laminae III through VII, and lamina X at all spinal levels. Although present, it was very spare in the ventral horn. Substance-P was also localized within autonomic areas of the thoracolumbar cord. ENK immunoreactivity was present in laminae I-III and laminae VII-X, as well as within autonomic areas. There was more ENK immunoreactivity in the ventral horn than SP. The densest aggregates of ENK, however, were found within the outer part of lamina II. In contrast, SOM could not be localized within the spinal cord of the opossum. (See NOTE ADDED IN PROOF).

Organization of midbrain catecholamine-containing nuclei and their projections to the striatum in the North American opossum, Didelphis virginiana.

Presumptive catecholamine (CA) neurons in the opossum midbrain were identified by tyrosine hydroxylase immunohistochemistry. In the midline, small to moderate number of CA cells were present in the rostral third of the nucleus raphe dorsalis and throughout the nucleus linearis. Ventrolaterally, such cells were observed in the deep tegmental reticular formation, in all subnuclei of the ventral tegmental area, and in the three subdivisions of the substantia nigra. The CA cells in these areas conform to the dopamine cell groups, A8, A9, and A10 as described in the rat. In several areas there appeared to be no separation between the CA neurons belonging to cytoarchitecturally different nuclei. In order to determine which CA neurons gave rise to striatal projections, the neostriatum was injected with True Blue (TB), and sections through the midbrain were processed for tyrosine hydroxylase (TH) and visualized by immunofluorescence. Neurons containing both TB and TH were observed in each of the CA cell groups mentioned above. The distribution of these cells confirmed organizational features that may be unique to the opossum's substantia nigra. In addition, different patterns of labeling resulted from caudate versus putamen injections, suggesting a rudimentary medial to lateral topography in the organization of nigrostriatal projections. Although our results suggest that the organization of midbrain CA neurons in the opossum is similar to that in placental mammals, it is clear that differences exist.

Origins and terminations of bulbospinal axons that contain serotonin and either enkephalin or substance-P in the North American opossum.

We have shown previously that some enkephalin, substance-P, and serotoninergic neurons in the medullary raphe and adjacent reticular formation project to the spinal cord in the opossum. In the present study we have combined the retrograde transport of True Blue and immunofluorescence histochemistry to determine whether methionine enkephalin or substance-P containing bulbospinal neurons are serotoninergic. Furthermore, we have used the same immunofluorescence protocol to determine whether spinal axons contain the same substances. Neurons that immunostained for both enkephalin and serotonin were observed in many brainstem nuclei. However, those that projected to the spinal cord were limited to the nuclei raphe magnus and obscurus, and the ventral part of nucleus reticularis gigantocellularis, pars ventralis. Neurons that immunostained for both substance P and serotonin were fewer in number, but some of the ones in the above nuclei and within the nucleus raphe pallidus, projected to the spinal cord. Spinal axons exhibiting both enkephalin- and serotonin-like immunoreactivity were observed in the superficial laminae of the dorsal horn, lamina X, and the intermediolateral cell column, whereas those showing both substance-P and serotonin-like immunoreactivity were seen primarily in lamina X, the intermediolateral cell column, and the ventral horn. Some of the axons in the ventral horn were in close apposition to presumed motoneurons. Comparison of the above results with those obtained from previous studies of bulbospinal projections has allowed us to infer the origins of axons that innervate different spinal targets.

Development of catecholaminergic projections to the spinal cord in the North American opossum, Didelphis virginiana.

The intent of our study was to determine when catecholaminergic axons grow into each of their adult targets in the spinal cord of the North American opossum (Didelphis virginiana) and to identify the origin of catecholaminergic axons in the lumbosacral cord at different stages of development. Tyrosine hydroxylase-like immunoreactive axons, presumed to be catecholaminergic, were demonstrated at different stages of development by the indirect antibody peroxidase-antiperoxidase technique of Sternberger. The neurons giving rise to such axons in the lumbosacral cord were identified by using the retrograde transport of Fast Blue and immunofluorescence for tyrosine hydroxylase-like immunoreactive neurons. At birth, 12-13 days after conception, tyrosine hydroxylase-like immunoreactive axons are present in the marginal zone throughout the length of the spinal cord. Such axons are particularly numerous in the dorsolateral marginal zone, the region containing most of them in adult animals. By postnatal day 3, a few immunoreactive axons are present in the intermediate (mantle) zone of the spinal cord; and by postnatal day 8, they are most concentrated in the presumptive intermediolateral cell column. Laminae I and II of the dorsal horn are not innervated by such axons until approximately postnatal day 15. By postnatal day 44, the distribution of tyrosine hydroxylase-like immunoreactive axons in the spinal cord resembles that in adult animals, although some areas may be hyperinnervated. At birth, tyrosine hydroxylase-like immunoreactive cell bodies are present in all of the brainstem areas providing catecholaminergic projections to the spinal cord in adult animals (Pindzola et al.: Brain Behav. Evol. 32:281-292, '88); and by at least postnatal day 5, lumbosacral injections of Fast Blue retrogradely label tyrosine hydroxylase-like immunoreactive neurons in all such areas. Retrogradely labeled immunoreactive neurons were also found in areas that do not contain them in adult animals. Such areas include the dorsal part of the nucleus coeruleus and certain areas of the reticular formation. During development, spinally projecting tyrosine hydroxylase-like immunoreactive neurons are numerous medial to the nucleus ventralis lemnisci lateralis (the paralemniscal region), whereas only a few are present in the same location in adult animals. Our results suggest that catecholaminergic axons grow into the spinal cord prenatally, that they innervate their adult targets postnatally and over an extended time period, and that during some stages of development they originate from areas that do not supply them in the adult animal.

The distribution and synaptic organization of serotoninergic elements in the inferior olivary complex of the opossum.

Immunohistochemistry and high-resolution autoradiography were used to analyze the distribution and synaptic organization of serotonin (5HT) - containing elements in the inferior olivary complex of the opossum. Immunoreactive beaded varicosities are present throughout the olivary complex. The densest 5HT immunostaining is present in subnucleus b of the caudal medial accessory nucleus. The rostral principal olive is sparsely populated with immunoreactive elements. Fine beaded fibers arborize throughout the neuropil of all the olivary nuclei except in subnucleus b of the caudal medial accessory nucleus where they also circumscribe neuronal cell bodies. In addition, a distinct population of large beaded fibers are occasionally encountered in the neuropil of the medial accessory nucleus. Ultrastructurally, labeled profiles that correspond in size to the smaller beads (less than 1 micron) contain tubulovesicular elements, large dense-cored vesicles, and clear vesicles. In contrast, larger profiles (greater than 2 microns) are characterized by numerous clear synaptic vesicles. Synaptic junctions were encountered in only 2% of the labeled elements. The majority of the labeled profiles were in juxtaposition to small-diameter dendrites (less than 2 microns) except in the caudal medial accessory nucleus, where they also were found in apposition to olivary cell bodies. Our results, when compared with other accounts, indicate that rather than major differences in the nuclear distribution of serotonin between species, there are differences in the density of serotoninergic elements in specific nuclei of the mammalian inferior olive. Based on the size of the labeled profiles and the distinct vesicle populations, our data suggest there are at least two populations of 5HT varicosities that are in juxtaposition to olivary neurons. Further, boutons containing 5HT primarily interact with the distal dendrites of olivary neurons except in the caudal medial accessory nucleus where cell bodies are in apposition to 5HT varicosities.

Brainstem origin of serotonin- and enkephalin-immunoreactive afferents to the opossum's cerebellum.

Previous studies have described the distribution of serotonin- and enkephalin-immunoreactive elements in the posterior lobe vermis of the opossum's cerebellum. In the present study we have used a double labeling paradigm which combines the retrograde transport of horseradish peroxidase (HRP) with serotonin and enkephalin immunohistochemistry to determine the brainstem origin of serotoninergic and enkephalinergic neurons that project to the opossum's cerebellar cortex. Subsequent to HRP injections into the posterior lobe vermis, widespread areas of the medulla and pons were found to contain retrogradely labeled neurons. Serotonin-immunoreactive somata are present primarily in the raphe nuclei and the adjacent reticular formation. Enkephalinergic neurons were numerous in the raphe nuclei, medial accessory olive, gigantocellular reticular formation, locus coeruleus, and the nucleus of the trapezoid body. However, serotoninergic neurons that project to the cerebellum were located only in the medullary pyramids and the reticular formation adjacent to the raphe. Double-labeled enkephalinergic neurons were located 1) within the medullary pyramids, 2) throughout the extent of the caudal medial accessory olive, 3) in the rostral subnucleus a of the medial accessory olive, 4) in the nucleus reticularis gigantocellularis pars ventralis, 5) in the nucleus reticularis lateralis, and 6) in the nucleus reticularis ventralis lateral to the inferior olivary complex. These results indicate that although neurons containing serotonin and enkephalin immunoreactivity may be present in some of the same pontine and medullary nuclei, those serotoninergic and enkephalinergic neurons that project to the cerebellum are present primarily in restricted and spatially separate regions of the caudal medulla.

Immunohistochemical localization of substance P, somatostatin, enkephalin, and serotonin in the spinal cord of the northern leopard frog, Rana pipiens.

Using the indirect antibody peroxidase-antiperoxidase method of Sternberger, we localized substance P (SP), somatostatin (SOM), enkephalin (ENK), and serotonin (5HT, 5-hydroxytryptamine) in the spinal cord of Rana pipiens. This is the first study to demonstrate all four substances in adjacent sections of frog spinal cord. The distribution patterns of ENK, SP, SOM, and 5HT in our study differ from that described for laminae I and II in amniotes. A high density of ENK, SP, and SOM fibers is present in a band ventral to the dorsal terminal field of cutaneous primary afferent fibers and slightly overlapping the ventral terminal field of muscle primary afferent fibers. However, a high density of 5HT fibers is present in the dorsal terminal field.

Raphespinal projections in the North American opossum: evidence for connectional heterogeneity.

Retrograde transport studies revealed that the nuclei pallidus, obscurus, and magnus raphae as well as the adjacent reticular formation innervate the spinal cord in the opposum. HRP-lesion experiments showed that a relatively large number of neurons within the nucleus obscurus raphae and closely adjacent areas of the nucleus reticularis gigantocellularis project through the ventrolateral white matter and that many cells within the nucleus magnus raphae, the nucleus reticularis gigantocellularis pars ventralis, and the nucleus reticularis pontis pars ventralis contribute axons to the dorsal half of the lateral funiculi. Neurons within the rostral pole of the nucleus magnus raphae and the adjacent nucleus reticularis pontis pars ventralis may project exclusively through the latter route. Each of the above-mentioned raphe and reticular nuclei contain nonindolaminergic as well as indolaminergic neurons (Crutcher and Humbertson, '78). When True-Blue was injected into the spinal cord and the brain processed for monoamine histofluorescence evidence for True-Blue was found in neurons of both types. Injections of 3H-leucine centered within the nuclei pallidus and obscurus raphae and/or the closely adjacent nucleus reticularis gigantocellularis labeled axons within autonomic nuclei and laminae IV-X. Labeled axons were particularly numerous within the intermediolateral cell column and within laminae IX and X. Injections of the caudoventral part of the nucleus magnus raphae or the adjacent nucleus reticularis gigantocellularis pars ventrialis labeled axons in the same areas as well as within laminae I-III. When the injection was placed within the rostral part of the nucleus magnus raphae or the adjacent nucleus reticularis pontis pars ventralis axons were labeled within laminae I-III and external zones of laminae IV-VII, but not within lamina IX. The immunohistofluorescence method revealed evidence for indolaminergic axons in each of the spinal areas labeled by injections of 3H-leucine into the raphe and adjacent reticular formation. They were particularly abundant within the intermediolateral cell column and within laminae IX and X. These data indicate that raphe spinal systems are chemically and connectionally heterogeneous.

Differential potencies of cocaine and its metabolites, cocaethylene and benzoylecgonine, in suppressing the functional expression of somatostatin and neuropeptide Y producing neurons in cultures of fetal cortical cells.

Using aggregate cultures derived from 17-day-old fetal rat cortex, we addressed the question: Does cocaine alter the functional expression of neuropeptide Y (NPY) and somatostatin (SRIF) neurons and, if so, are cocaethylene (CE) and benzoylecgonine (BZE) as active as cocaine? NPY/SRIF production in response to brain-derived neurotrophic factor (BDNF) or phorbol-12-myristate-13-acetate (PMA) was used as a functional criterion. A 5-day exposure to cocaine did not affect basal or stimulated (BDNF or PMA) production of NPY but it markedly suppressed BDNF- or PMA-stimulated production of SRIF. Exposure to CE led to a drastic suppression of basal as well as stimulated (BDNF or PMA) production of both NPY and SRIF. These effects of cocaine and CE were concentration dependent (1-100 microM). BZE did not alter any of these functional parameters. Next, we evaluated the fate of cocaine, CE, and BZE in the culture medium. Cocaine was converted to BZE, whereas BZE was not converted to cocaine. CE was converted to cocaine and BZE, with substantial amounts of cocaine and CE remaining in the medium after 72 hr (approximately 20% each). In summary, cocaine, CE, and BZE exhibited differential potencies in suppressing the expression of cultured NPY and SRIF neurons: CE was more potent than cocaine and BZE was inactive. SRIF neurons were more susceptible than NPY neurons to the effects of cocaine. The higher potency of CE may be due to a property of the compound and/or to CE serving as a source for a slow, continuous formation of cocaine by the brain cells themselves.

Anatomical evidence for interactions between somatostatin neurites in lamina II of the rat spinal cord.

Light microscopic analysis of adult and 10-14-day-old rat spinal cords suggested that somatostatin-immunoreactive (SOM-I) fibers apposed SOM-I cell bodies in lamina (L) II. Electron microscopic analysis of these relationships at both ages showed the presence of direct appositions between SOM-I fibers and SOM-I cells. However, synapse formation between SOM-I fibers and cells was observed only in the young rat. Similarly, synapses between SOM-I fibers and SOM negative cell bodies were only found in the young animal. Adjacent SOM-I perikarya directly contacted each other, but, again, membrane specializations were evident only in the young rat. Within L I of the adult dorsal horn, a SOM-I fiber directly apposed an unlabeled cell body. Despite analysis of serial sections through the apposition, no synaptic contacts were observed.

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.

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.

Evidence for a synergistic effect of the HIV-1 envelope protein gp120 and brain-derived neurotrophic factor (BDNF) leading to enhanced expression of somatostatin neurons in aggregate cultures derived from the human fetal cortex.

Changes in the expression of somatostatin (SRIF) have been observed in the brains of HIV encephalitis. Since gp120 is thought to play a major role in AIDS-associated abnormalities in the brain, we addressed the question: Does gp120 alter the functional expression of human fetal SRIF neurons in culture and if so, is this effect fetal-age dependent? Aggregate cultures, obtained from cortices of nine fetuses (14.9-20.7 weeks), were exposed for 7 days to BDNF or BDNF+gp120; BDNF induced production of SRIF during the subsequent 24-48 h was assessed. Similar effects of BDNF and gp120 were observed in the 9 brain-cultures. A 7-day exposure to BDNF alone led to a significant increase in SRIF production (p=0.014), whereas exposure to gp120 alone did not. Co-exposure to BDNF and gp120 led to an increase in BDNF-induced SRIF production which was significantly greater than that after BDNF alone (p=0.006). These effects were BDNF- and gp120-dose dependent and they were not accompanied by changes in DNA content of the aggregates nor in lactate dehydrogenase activity in the medium; indicating that gp120 did not lead to a major loss of cell integrity. These results are consistent with a synergistic effect of BDNF and gp120 leading to enhanced functional expression of the signalling pathway(s) mediating BDNF induction of SRIF production; an effect expressed by fetal brains throughout the 2nd trimester of gestation. Thus, this culture system can serve as a model to study the mechanism(s) underlying the early interactions between gp120 BDNF in the developing human brain.

Immunocytochemical analysis of somatostatin in the hypothalamus of obese and non-obese Zucker rats.

Levels of growth hormone (GH) are reduced in the genetically obese Zucker rat, fa/fa, in comparison to lean littermates. In normal rats, GH release is regulated by stimulatory and inhibitory factors of hypothalamic origin. The present experiment focuses on hypothalamic somatostatin (SOM; growth hormone release inhibiting factor) in order to determine if abnormal hypothalamic SOM may be a correlate of depressed GH secretion in fa/fa rats. We compared immunocytochemical localization of hypothalamic SOM between 5 obese (fa/fa) Zucker rats and 5 non-obese littermates. Brain sections from pairs of animals were processed simultaneously. The distribution of SOM immunoreactive cell bodies in the hypothalamus agreed with previous reports. SOM-containing neurons in the periventricular area were counted and analyzed at 4 hypothalamic levels: (1) anterior to the suprachiasmatic nucleus (SCN); (2) through SCN; (3) between SCN and the ventromedial hypothalamic nucleus (VMH); and (4) through VMH. The greatest number of SOM-immunoreactive cell bodies was observed at levels (2) and (3). The numbers of SOM-containing cells did not differ significantly between obese and lean animals. No apparent difference in density of fiber staining was observed in the median eminence.

Enhanced survival of apparent presynaptic elements on polylysine-coated beads by inhibition of non-neuronal cell proliferation.

Increased survival of presynaptic-like neuronal profiles was found in cell cultures of rat cerebellum when the non-neuronal cell numbers were reduced with an antimitotic drug. In both treated and untreated cell cultures, neurites grew onto the polylysine-coated surface of sepharose beads and formed a swelling. The neuronal swelling contained an accumulation of synaptic vesicles and a membrane density at the site of contact with the bead and was called an apparent presynaptic element. The apparent presynaptic elements in untreated cultures increased in number from the time the beads were added to the culture to 7 days incubation and then showed a decrease to one half the 7-day value at 14 days incubation. A 75% reduction in cell division of non-neuronal cells was seen in cultures exposed to a 5 X 10(-6)M cytosine arabinoside (Ara-C) for 2 days. Adding polylysine-coated beads to cultures treated with Ara-C showed at 14 days incubation a 7-fold increase in the number of apparent presynaptic elements as compared to untreated cultures. Additional experiments examined the numbers of neurites on the beads and found only small differences between treated and untreated cultures. A decrease, however, was shown in the number of glial fibrillary acidic protein staining astrocytes on the surface of the beads in treated cultures. The reduction of astrocytes by Ara-C appeared to enhance the survival of apparent presynaptic elements but did not enhance the growth of neurites. These results suggest that proliferating non-neuronal cells at a site of injury in the central nervous system may inhibit the formation of synaptic contacts and the growth of neurites through the site of injury.

The early development of major projections to the dorsal striatum in the North American opossum.

We have employed immunocytochemical and axonal transport techniques to study the development of major projections to the dorsal striatum of the North American opossum. The opossum is born in a very immature state, 12-13 days after conception, and climbs into an external pouch where it remains attached to a nipple for several months. Its immaturity at birth and its protracted postnatal development make the opossum a good model for developmental studies. Although tyrosine hydroxylase-like immunoreactive (TH-LI), presumably dopaminergic, neurons were present in the ventral mesencephalon at birth (the presumptive substantia nigra and ventral tegmental area), there was no evidence for TH-LI axons in the striatal anlage. By postnatal day (PD)6, a few immunostained axons were found within the putamen. The subsequent growth of TH-LI axons into the striatum followed general caudal to rostral and ventrolateral to dorsomedial gradients and, at any age, they were most numerous in the areas exhibiting the greatest cytodifferentiation. By estimated (E)PD45, TH-LI axons were present in most, if not all, areas of the striatum. Serotoninergic (5-HT)-LI axons were found lateral to the presumptive striatum at birth but not within it. By PD7, however, a few 5-HT-LI axons could be identified in the putamen. The growth of 5-HT-LI axons into the striatum generally followed the same gradients described for TH-LI axons although at all ages their density was much less. Using the orthograde transport of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP), evidence was obtained for the existence of thalamostriatal projections by PD5 and for corticostriatal projections by PD10. Crossed corticostriatal projections were present by EPD23. Our results suggest that the development of major projections to the striatum occurs postnatally in the opossum, rather than prenatally as in placental animals. The timetable for striatal innervation is discussed in light of the developmental sequences established for other motor circuits.