Peroxidase-antiperoxidase Immunocytochemical Technique Research Articles

Neuron-specific enolase-like immunoreactivity in human Leydig cells

Using the peroxidase anti-peroxidase immunocytochemical technique, neuron-specific enolase (NSE)-like immunoreactivity (NSE-LI) was revealed in Leydig cells of adult human testes at the light microscopic level. Differences in the NSE staining intensity were observed between the individual Leydig cells, separate cell groups within a testis and between the testes of individual patients. Together with the already established substance P-like immunoreactivity (SP-LI), the results obtained provided further evidence for the possible neuroectodermal origin of human Leydig cells and their presumable relation to the APUD- or the Diffuse Neuroendocrine System (DNES).

Neuropeptide Y-immunoreactive intracardiac neurones, granule-containing cells and nerves associated with ganglia and blood vessels in rat and guinea-pig heart

Intrinsic neuropeptide Y-containing neurones in rat and guinea-pig hearts were studied at the ultrastructural level by the pre-embedding peroxidase-antiperoxidase immunocytochemical technique. Intracardiac neuronal cell bodies were often weakly or moderately immunostained, and the labelling was usually pronounced in the Golgi complex, multivesicular bodies, some cisterns of granular endoplasmic reticulum and large granular vesicles. Neuropeptide Y-immunoreactive nerve fibres were also observed in association with intracardiac neurones. A subpopulation of neuropeptide Y-immunoreactive granule-containing cells in the rat heart are described for the first time and were very heavily labelled; other granule-containing cells were non-immunoreactive, but were contacted by neuropeptide Y-containing nerves. Preterminal regions of nerve fibres that were located in nerve bundles were only weakly neuropeptide Y-immunoreactive, in contrast to the heavy labelling observed in varicosities that contained many synaptic vesicles. Many neuropeptide Y-immunoreactive nerve fibres were associated with the coronary vasculature and were particularly prominent in the walls of small arteries and arterioles where labelled nerve varicosities were present close to the smooth muscle cells. Immunoreactive nerves were also seen in the myocardium, usually near to capillaries. In axonal varicosities, the central core of large granular vesicles was immunolabelled, and electron-dense immunoreactive material outlined the membranes of small and large clear vesicles. The significance of neuropeptide Y-immunoreactive intracardiac neurones and granule-containing cells and the origin of associated labelled nerve fibres in the heart are discussed.

Serotonin‐containing projections to the mesencephalic trigeminal nucleus of the cat

It is well known that the mesencephalic trigeminal nucleus (MTN) neurons transmit somatosensory information from proprioceptors in the oral-facial region. Several mechanisms of sensory transduction in these specialized receptors have been proposed, but the neurotransmitters which are responsible for mediating proprioceptive information are still unknown. The current study concentrates on the distribution of one putative neurotransmitter system, serotonin (SER), in the cat MTN. A second objective was to clarify the location and sources of serotoninergic projections on the MTN neurons. To determine whether SER was localized in the MTN, the peroxidase-antiperoxidase (PAP) immunocytochemical technique was applied at light and electron microscopic levels in colchicine-treated animals. The origin of SER-containing fibers in the MTN was studied using a double-labeling method combining retrograde transport with wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) and SER immunocytochemistry. There were no SER-containing neurons in the MTN. The cell bodies of immunonegative MTN neurons were closely surrounded by fine SER-positive fibers and terminals. The labeled fibers were in most cases very thin and sometimes varicose. Ultrastructurally, direct synaptic contacts between SER-containing terminals and perikarya of MTN neurons of all sizes could be seen. The majority of SER-labeled structures were synaptic terminals in which the immunoreactive material was located within the small round clear as well as the small granular vesicles (diameter 50-80 nm) and a few large dense-cored vesicles (up to 150 nm). Retrograde tracing demonstrated that most of cells in the nuclei raphe dorsalis, pontis and magnus were WGA-HRP-labeled. These results indicated that MTN neurons received serotoninergic projections from the raphe nuclei of the brainstem. In light of these morphological data, it is concluded that the MTN of the cat is under the influence of SER-containing axons and this serotoninergic input may modulate MTN neuronal activity at the first synaptic relay.

Forebrain projections from cholecystokininlike-immunoreactive neurons in the rat midbrain.

The purpose of the present study was to analyze the distribution of cholecystokininlike-immunoreactive (CCK-I) neurons within the rat ventral mesencephalon which project to several forebrain areas. The peroxidase-antiperoxidase immunocytochemical technique was used to examine the anatomical localization of CCK-I within the ventral midbrain and in the following forebrain regions: caudate-putamen, nucleus accumbens, olfactory tubercle, bed nucleus of the stria terminalis, septum, amygdala, and prefrontal, anterior cingulate, and piriform cortices. CCK-I perikarya were distributed throughout the substantia nigra, ventral tegmental area, and several midline raphe nuclei to a greater extent than previously reported, particularly in the substantia nigra pars compacta. Terminallike immunoreactivity for CCK was observed in all of the above forebrain sites. In addition, infrequent CCK-I cell bodies were localized in the caudate-putamen, nucleus accumbens, olfactory tubercle, septum, and bed nucleus of the stria terminalis. To analyze forebrain projections of the ventral midbrain CCK-I neurons, indirect immunofluorescence was combined with fluorescence retrograde tracing. CCK-I neurons of the substantia nigra and/or ventral tegmental area were found to project, to varying extents, to all of the above CCK-I forebrain terminal fields. The nucleus accumbens, olfactory tubercle, and septal and prefrontal cortical projections arose primarily from CCK-I perikarya in the ventral tegmental area whereas the projections to the caudate-putamen and anterior cingulate cortex arose predominantly from immunoreactive neurons in the substantia nigra pars compacta. The amygdala received innervation mainly from CCK-I cell bodies located in the substantia nigra pars lateralis. CCK-I afferents to the bed nucleus of the stria terminalis and piriform cortex originated from perikarya distributed approximately equally across the ventral tegmental area and substantia nigra pars compacta. The general topography of CCK-I forebrain innervation observed in this study is similar to that previously reported for the ascending dopaminergic projections from ventral mesencephalic neurons. CCK-I neurons of the midline raphe nuclei were found to provide relatively minor afferents to the caudate-putamen, bed nucleus of the stria terminalis, septum, and prefrontal cortex and more substantial projections to the amygdala. The results of this study demonstrate that CCK-I neurons of the ventral midbrain supply a much broader innervation of forebrain regions than previously appreciated.(ABSTRACT TRUNCATED AT 400 WORDS)

Distribution of enkephalin-immunoreactive nerve fibres and terminals in the region of the nucleus basalis magnocellularis of the rat: a light and electron microscopic study.

This investigation was carried out on the distribution of enkephalin-containing nerve fibres and terminals in the region of the nucleus basalis magnocellularis (NBM) of the rat. At the light microscope (LM) level, enkephalin-immunoreactive sites and endogenous choline acetyltransferase (ChAT) were demonstrated by employing the two-colour immunoperoxidase staining technique, using highly specific monoclonal antibodies against enkephalin and ChAT. A pharmacohistochemical procedure to reveal acetylcholinesterase (AChE)-synthesizing neurons combined with the peroxidase-antiperoxidase (PAP) immunocytochemical technique to detect endogenous enkephalins, provided ultrastructural data on the relationships of neuronal elements containing AChE and enkephalins in the region of the NBM. At the LM level, cholinergic neurons of the NBM were surrounded by a dense network of enkephalin-immunoreactive nerve fibres. Electron microscopic (EM) observations of histochemically characterized structures, that were first identified in the LM, revealed that intensely AChE-stained structures in the region of the NBM received sparse synaptic inputs from enkephalin immunoreactive terminals. Synaptic inputs of immunoreactive terminals onto intensely AChE-stained neuron cell bodies were not detected. Synaptic contacts onto proximal AChE-positive dendrites were sparse, but the density increased on more distal regions of the dendrites. All immunoreactive boutons studied established symmetrical synaptic contacts with AChE-positive post-synaptic structures. The pattern of the synaptic input to these cells differs strikingly from that onto typical globus pallidus neurons. The perikarya and dendrites of the latter neurons were characteristically ensheathed in immunoreactive synaptic boutons. Results are consistent with the view that enkephalin-like substances in the rat might be synaptic transmitters or neuromodulators in the area of the NBM and that cholinergic neurons of the NBM (Ch4) are integrated into the circuitry of the basal ganglia. Enkephalins may play an important role regulating the extrinsic cholinergic innervation of the neocortex.

Immunocytochemical and morphometric study of prolactin cells during amphibian morphogenesis

The peroxidase-antiperoxidase (PAP) immunocytochemical technique and rabbit antihuman prolactin (PRL) antiserum were used to localize and identify PRL-producing cells in the pars distalis of Bufo calamita, Hyla meridionalis, Alytes cisternasii, Pelobates cultripes, and Rana perezi tadpoles at different stages of development as well as in 1-year-old postmetamorphic animals. This cell type was located throughout the gland in P. cultripes and R. perezi and in the caudal two-thirds in B. calamita, H. meridionalis, and A. cisternasii in premetamorphic animals. These distribution patterns do not show changes throughout development. Morphometry was used to evaluate the changes observed in pars distalis volume and stereological parameters of PRL immunoreactive cells during development. Pars distalis volume increased during the larval growth period and decreased throughout the metamorphic climax. PRL volume density and cellular area showed different patterns in the different species, although the correlation between these parameters suggests a period of great proliferative rate followed by changes in cellular size. The changes observed in PRL total volume suggest the existence of two phases in amphibian development: (i) a period of PRL storage during pre- and prometamorphosis and (ii) a period of release at the metamorphic climax.

Enkephalin-like immunoreactivity in the nucleus submedius of the cat and rat thalamus.

In the present study, we employed the peroxidase-antiperoxidase immunocytochemical technique to study the presence and distribution of enkephalin in the nucleus submedius of both cats and rats at the light- and electron-microscopic levels. The enkephalin-like immunoreactive (ENK-LI) fibers were present in a concentrated, albeit limited, manner in the nucleus submedius of both species. These fibers were located close to the dorsal and caudal edge of the nucleus, and were confined to a small area that never exceeded 350 microns in the rostrocaudal or 250 microns in the dorsoventral direction. Mediolaterally, however, the fibers extended some 700 microns. No ENK-LI cell bodies were seen in the nucleus submedius, even in colchicine-treated animals. At the electron-microscopic level, the ENK-LI terminals were seen to synapse on dendrites. These data indicate a previously unsuspected role of enkephalin in synaptic transmission processes within the nucleus submedius, and provide additional support for the role of this nucleus in the processing of nociceptive information at medial thalamic levels.

Development of the ultrastructural features of somatostatin-immunoreactive neurons in the rat visual cortex.

The peroxidase-antiperoxidase immunocytochemical technique has been used to examine the development of the ultrastructural features of somatostatin (SRIF)-immunoreactive neurons in the visual cortex of the rat between embryonic day 17 and postnatal day 32. In the adult, stained neurons are distributed in layers II through VI and characterized by an abundance of cytoplasm containing a plethora of organelles, most conspicuous of which are cisternae of granular endoplasmic reticulum organized in parallel arrays. In embryonic tissue, SRIF-positive cells are present in the subplate and in the border between the cortical plate and marginal zone. These cells possess scanty cytoplasm containing a few organelles; synapses onto immunoreactive perikarya and dendrites are evident at this stage. At birth and in early postnatal life, labelled cells are confined to the subplate region. Already at this age a number of cells display signs of ultrastructural features which characterize them in adult life. At the end of the first postnatal week, SRIF-immunoreactive neurons span a considerable spectrum of maturity. At one extreme are a few cells with little cytoplasm surrounding a large nucleus and at the other are the majority of labelled neurons showing abundant cytoplasm including prominent arrays of granular endoplasmic reticulum. Labelled cells first appear in the more superficial layers at the beginning of the second postnatal week and attain a distribution similar to that observed in adult animals at the end of this week. At this time their ultrastructural features closely resemble those of their adult counterparts, and differences in cytoplasmic maturity between superficial and deep labelled cells are not evident. This suggests that the SRIF-producing neurons in the superficial layers begin to express this peptide after they complete their migration and have acquired their morphological features. Maturation proceeds during the third postnatal week; at this stage most cells acquire their mature nuclear and cytoplasmic features and an adult complement of synapses. However, a number of SRIF-immunoreactive cells contain a particularly prominent accumulation of cytoplasmic organelles and appear hypertrophic.

Persistence of immunoreactive TRH and GnRH in long-term primary anterior pituitary cultures

Intact anterior pituitary tissue and primary anterior pituitary cultures were stained with 1:30,000 anti-TRH and 1:10,000 anti-GnRH using the peroxidase antiperoxidase immunocytochemical technique. Stains applied to serial ultrathin sections of intact pituitaries showed that TRH immunoreactivity could be localized in secretory granules of thyrotropes, gonadotropes and corticotropes whereas GnRH immunoreactivity was found only in gonadotropes and corticotropes. Long-term primary pituitary cultures were studied to remove the anterior pituitary cells from hypothalamic influences. In these cell populations both TRH and GnRH immunoreactivity persisted. In addition, quantification of the stained cells at the light microscopic level demonstrated that the volume fraction of TRH and GnRH immunoreactive cells remained constant up to 3 weeks of culture. Studies of serial ultrathin sections through cells from these cultures showed TRH or GnRH localized in secretory granules of cells that contained LH and ACTH, but not TSH. Both liquid and solid phase immunoabsorption specificity controls were used to validate the immunocytochemical stains. These studies suggest that the pituitary TRH and GnRH immunoreactivities may not be completely of hypothalamic origin, but may also be endogenous to a subpopulation of unique multihormonal pituitary cells.

Synapses between GABA-immunoreactive axonal and dendritic elements in monkey substantia nigra

γ-Aminobutyric acid (GABA)-positive elements in the monkey substantia nigra pars reticulata (SNr) were identified using the peroxidase-antiperoxidase immunocytochemical technique with a direct GABA antibody. Light-microscopic examination revealed fine-caliber immunostained fibers piercing the cerebral peduncle and entering the SNr, where they formed a dense plexus. Ultrastructural observations included boutons with densely packed ovoid synaptic vesicles forming synapses with labeled and unlabeled dendritic elements. Most terminals contained small dark mitochondria, although some endings held larger, paler and fewer of these organelles. The synapses observed between GABA-immunoreactive profiles may represent the morphologic basis for disinhibition of pars compacta neurons reported to follow neostriatal or pallidal stimulation.

Distribution of cholecystokinin-like immunoreactivity in the rat main olfactory bulb.

The anatomical localization of cholecystokinin-like immunoreactivity (CCK-I) within the rat main olfactory bulb was analyzed by using the peroxidase-antiperoxidase immunocytochemical technique. Neurons or neuronal processes containing CCK-I were localized within all laminae of the olfactory bulb except the olfactory nerve fiber layer. A large population of CCK-I neurons, with morphology, size, and distribution corresponding to that of the middle and external tufted cells, was observed within a zone extending from the deep periglomerular region through the superficial one-half to one-third of the external plexiform layer. A smaller number of immunoreactive perikarya were found in the deep external plexiform layer, the glomerular layer, and rarely within the inner plexiform layer. These CCK-I neurons appeared to correspond to internal tufted cells, periglomerular cells, and deep short-axon cells, respectively. Dense CCK-I staining of fibers and terminals was present within the internal plexiform layer and, less densely, within the neuropil of the granule cell layer. In addition, terminal-like CCK-I was localized within layer 1A of the anterior olfactory nucleus, the olfactory tubercle, and the most rostral piriform cortex. This observation provides corroboration for the identification of the principal CCK-I neuron in the rat olfactory bulb as the centrally projecting middle tufted cell. The present results, demonstrating the localization of CCK-I to both local circuit and projection neurons of the olfactory bulb and to terminal-like puncta in the internal plexiform and granule cell layers, suggest that CCK may be significantly involved in olfactory processing at several levels.

Immunocytochemical localization of human epidermal growth factor/urogastrone in several human tissues.

Epidermal growth factor (EGF) stimulates the growth of many tissues and inhibits stimulated gastric acid secretion. Its primary tissue of origin in man is still unknown. We used polyclonal anti-human EGF sera in the peroxidase-antiperoxidase immunocytochemical staining technique to identify immunoreactive human EGF (ihEGF) in tissue sections from 29 subjects ranging from fetuses to 63 years in age. In addition to acinar cells in the submandibular salivary glands and cells of Brunner's duodenal glands, previously reported to contain ihEGF, we found ihEGF in most anterior pituitary glycopeptide hormone-secreting cells, in gastric and pyloric gland cells of the stomach, and in bone marrow cells that resembled mononuclear phagocytes in subjects of all ages. The eccrine sweat glands in the skin of adults also contained ihEGF. Cells containing ihEGF were found singly or in clusters in the trachea of the fetus only. No fetal pancreatic islet cells stained, but occasional cells in neonates and a majority of islet cells in older subjects contained ihEGF; there was no constant association with insulin, glucagon, or somatostatin. Only the lactating breast contained ihEGF. In adults, outer adrenomedullary cells contained ihEGF. Intense immunostaining was observed in the renal medulla, apparently limited to the extracellular area between the renal tubules, and increased with age; the cortex was devoid of ihEGF. No ihEGF was detected in posterior pituitary gland, thyroid gland, heart, lung, or liver at any age. An adult prostate contained ihEGF only in an area of local injury, and some primordial follicles from the ovary of a newborn appeared to contain ihEGF. Thus, many tissues appear to synthesize hEGF, which may exert exocrine, endocrine, or paracrine functions in different tissues and at different ages.

Distribution of substance P neurons in the epithalamus of the rat: an immunohistochemical investigation.

Substance P (SP) immunoreactivity was studied in the epithalamus of intact and superior cervical ganglionectomized female rats by using the peroxidase-antiperoxidase immunocytochemical technique. Cells containing immunoreactive SP were located in the medial habenula, and SP fibers were observed in the lateral habenula, habenular commissure, pineal stalk, and the pineal organ. Bilateral removal of the superior cervical ganglia did not affect the immunocytochemical staining of SP-containing cells and fibers 15-30 days later. It is suggested that habenular neurons containing SP send projections into the pineal organ of the rat.

Distribution of immunoreactive substance P neurons in the hypothalamus and pituitary of the rhesus monkey.

The distribution of immunoreactive substance P (IR-SP) neurons was examined in the hypothalamus and pituitary gland of the rhesus monkey by using the peroxidase-antiperoxidase immunocytochemical technique. Immunoreactive SP cell bodies were observed in the arcuate nucleus, in the region lateral to the arcuate nucleus, and in the median eminence (ME). Immunoreactive SP cells were also seen in the periventricular area of the dorsal tuberal region. A rich network of SP fibers was concentrated in the arcuate region, and the fiber stain was particularly dense in the external zone of the median eminence and in the external layer of the infundibular stalk. Also, substance P fibers were seen in the internal layer of the pituitary stalk and in the neural lobe of the pituitary gland. Outside the hypothalamus a dense network of IR-SP fibers was observed in the globus pallidus.

Ultrastructural immunocytochemical localization of fibronectin in the developing rat lung.

In a previous study changes in the macrodistribution of fibronectin during rat-lung development were examined. Using the peroxidase-antiperoxidase immunocytochemical technique, we have demonstrated the presence of fibronectin in embryonic, neonatal, and adult rat lung at the ultrastructural level. In the embryo, fibronectin is found both in an intra- and extracellular association with isolated pneumoblasts, and in a periodic distribution along the basal lamina. The neonate displays fibronectin in an intracellular association with early type-I cells and on their basal and luminal surfaces, but not in association with type-II cells. Neonatal basal lamina is diffusely labeled by anti-fibronectin antiserum. Fibronectin in adult tissue is found both intracellularly and on the basal and luminal surfaces of type-I cells but not in type-II cells. The basal lamina and interstitial connective tissue are slightly or non-reactive. These observations confirm and extend our initial suggestion that fibronectin is involved in rat-lung development.

Induction of differentiation increases Met5-enkephalin and Leu5- enkephalin content in NG108-15 hybrid cells: an immunocytochemical and biochemical analysis

Regulation of cellular content of the endogenous opioid peptides Met5-enkephalin and Leu5-enkephalin was investigated in neuroblastoma X glioma hybrid cells NG108-15 grown in both serum-supplemented and serum-free defined media. Untreated cells and cells induced to differentiate were stained using anti-Met5-enkephalin and anti-Leu5-enkephalin with the peroxidase-antiperoxidase immunocytochemical technique at the light microscopic level. In untreated NG108-15 cells grown in serum-supplemented medium, intense enkephalin-like immunoreactivity was localized in cell bodies and short processes of a select population of cells. The volume fraction of stained untreated cells remained constant throughout the time period investigated. When cells were induced to differentiate with N6,O2'-dibutyryl adenosine 3':5'-cyclic monophosphate (dBcAMP) or 8-bromo cyclic adenosine monophosphate (1.0 mM) treatment for 5 days, staining was found throughout the cytoplasm of perikarya and the extensive processes which were expressed, and the volume fraction of stained cells increased over 2-fold. Receptor-mediated stimulation of adenylate cyclase by prostaglandin E1 (10 microM) for 5 days produced results similar to those with dBcAMP. Pure cultures of differentiated cells with intense staining were obtained by further treatment of cultures, grown in the presence or absence of dBcAMP, with arabinosylcytosine (araC). Untreated, dBcAMP-treated and araC-treated NG108-15 cells grown in defined medium expressed staining patterns and volume fractions of stained cells similar to those grown in serum-supplemented medium; sodium butyrate (1.0 mM), however, increased the volume fraction of stained cells grown in defined medium over 3-fold, whereas it had little effect on staining of cells grown with serum. The presence of both Met5- and Leu5-enkephalin-like activities in NG108-15 cells was confirmed in acid extracts of cells by radioreceptor assay after separation by reverse phase high pressure liquid chromatography. Induction of differentiation in NG108-15 cells by dBcAMP treatment increased the cellular concentration of both enkephalins to over 2 times the levels found in untreated cells. The biochemical analysis for Met5-enkephalin- and Leu5-enkephalin-like activity compared well with the immunocytochemical data indicating that the enkephalin content is correlated with the state of differentiation of NG108-15 cells.

Leucine-enkephalin-containing neuron system in the facial nucleus of the rat with special reference to its fine structure

The light and electron microscopic localization of leucine-enkephalin-containing terminals in the facial nucleus of the rat were investigated by means of the peroxidase-antiperoxidase (PAP) immunocytochemical technique. By light microscopy, leucine-enkephalin-like immunoreactive (LEI) terminals were unevenly distributed in the facial nucleus. The greatest accumulation of the terminals was seen in the medial part of the nucleus. Electron microscopic examination of LEI-terminals in the medial part of the nucleus revealed that the predominant type of synaptic contacts of LEI-terminals in this area were axo-dendritic contacts (about 75%). These dendrites which made synapses with LEI-terminals were relatively large and rich in cytoplasmic organella, suggesting that they belonged to the proximal segment of the dendrite. A small number of LEI-terminals was found to make synaptic contact with neuronal perikarya (5%). These perikarya were very large and had nuclei with less chromatin particles. These findings suggest that LEI-terminals make contact with neurons which exist in the facial nucleus. The rest of the LEI-terminals (20%) were in apposition to the non-labelled axon terminals which contain small, clear and round vesicles.

Avian pancreatic polypeptide-like immunoreactivity in ground squirrel hypothalamus

The distribution of avian pancreatic polypeptide-like immunoreactivity (APP-LI) within the hypothalamus of the golden-mantled ground squirrel ( Spermophilus lateralis) was analyzed using the indirect peroxidase-antiperoxidase immunocytochemical technique. Avian pancreatic polypeptide-LI is widely distributed within the hypothalamus of the golden-mantled ground squirrel and in general, the distribution of APP-LI containing neuronal fibers in this species is qualitatively similar to the distribution found in the laboratory rat. This similarity in APP-LI distribution in two rodents from different taxonomic Families suggests that the distribution of APP-LI containing neurons is probably very conservative within the Order Rodentia.

Intestinal vitamin D-induced calcium-binding protein: time-course of immunocytological localization following 1,25-dihydroxyvitamin D3.

The vitamin D-induced calcium-binding protein (CaBP) was localized in histological sections of chick duodenum using the peroxidase-antiperoxidase immunocytochemical technique. The time-course of appearance of CaBP in rachitic chicks was investigated from 0 to 120 hr after stimulation by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). CaBP was not routinely detected at 0 hr after 1,25(OH)2D3 administration. CaBP was first noted in some, but not all, of the samples taken 2 hr following 1,25(OH)2D3 and was detected in all 2 1/2 hr samples. The number of CaBP-containing absorptive cells and the apparent CaBP concentration both increased to a maximum at about 16-24 hr. At later times, as CaBP free cells migrated up the villi, the CaBP-containing cells decreased in number, but even at 120 hr post 1,25(OH)2D3 dose there were significant numbers of CaBP-containing cells present. The relationships between time-course of CaBP location on intestinal villi, enterocyte migration rates, and the time-course of 1,25(OH)2D3 stimulated intestinal calcium transport are discussed.

Fine structural studies on a type of somatostatin-immunoreactive neuron and its synaptic connections in the rat neostriatum: a correlated light and electron microscopic study.

Somatostatin-immunoreactive neurons in the rat neostriatum were studied by correlated light and electron microscopy using the peroxidase-antiperoxidase immunocytochemical technique. Immunoreactivity was localized in neuronal perikarya and processes. The perikarya were of spindle or fusiform shape (average length 16.9 microns) and were found in all parts of the neostriatum. From each neuron there arose two to four straight immunoreactive dendritelike processes, which could frequently be traced as far as about 130 microns from their perikaryon. Immunoreactive varicose axonlike processes were occasionally found, some of which were proximal axons of identified immunoreactive cells. Nine of the light microscopically identified neurons showing somatostatin-immunoreactivity were studied in the electron microscope; two of them had proximal axons with varicosities. Each neuron had an oval or elongated nucleus, which was always indented. These morphological features correspond well to those of certain "medium-size aspiny" neurons classified by Golgi studies. Although the immunoreactive endproduct was diffusely located throughout the neuron, it was characteristically located in the saccules and large granules (diameter 133 nm) of the Golgi apparatus, and large immunoreactive vesicles of similar size to those in the Golgi apparatus frequently occurred in all parts of axon. Very little synaptic input was found on the perikarya and dendrites of somatostatin-immunoreactive neurons. The perikarya and proximal dendrites received both symmetrical and asymmetrical synaptic input, while the distal dendrites usually received boutons that formed asymmetrical contacts. The somatostatin-immunoreactive boutons contained pleomorphic electron-lucent vesicles (diameter 39.3 nm) and a few large immunoreactive granular vesicles; these boutons always formed symmetrical synapses. Their postsynaptic targets were dendritic shafts, spines, and unclassified dendritic profiles. On the other hand, the varicosities of identified proximal axons of somatostatin-positive neurons did not form typical synapses, since they lacked clusters of small vesicles, but some of them were in direct apposition (via membrane specializations) to unlabelled perikarya or dendrites. It is concluded that somatostatin is a useful marker for a particular type of neuron in the neostriatum. The presence of somatostatin immunoreactivity in synaptic boutons is consistent with the view that somatostatin could be a neurotransmitter in the neostriatum.