The nucleus raphe dorsalis of the rat and its projection upon the caudatoputamen. A combined cytoarchitectonic, immunohistochemical and retrograde transport study

The nucleus raphe dorsalis of the albino rat has been studied in the following three ways: (1) the cell mass was subjected to a detailed cytoarchitectonic analysis, based upon Nissl-stained material; (2) serotonin--as well as the noradrenaline--immunoreactive neurons present in the area of the nucleus raphe dorsalis were plotted; (3) following large injections of the fluorescent dye propidium iodide into the caudatoputamen complex, the cells in the nucleus raphe dorsalis projecting to this complex were labeled and subsequently stained with an antibody against serotonin. Cytoarchitectonic analysis showed that three cell types are present within the confines of the nucleus raphe dorsalis: small, medium and large. Moreover, differences in concentrations of cell bodies made it possible to subdivide the nucleus raphe dorsalis into four regions. Immunohistochemical analysis showed that the borders of the serotoninergic cell groups B6 and B7 of DAHLSTROM and FUXE do not coincide with those of the nucleus raphe dorsalis. Serotonin-immunoreactive perikarya in the nucleus raphe dorsalis were categorized as medium and large neurons; noradrenaline-immunoreactive neurons in the nucleus raphe dorsalis do all belong to the category--large neurons. With the combined use of immunofluorescence and fluorescent retrograde tracing, it was found that the projection from the nucleus raphe dorsalis to the caudatoputamen complex originates from serotoninergic as well as non-serotoninergic cells, both of which can be categorized as being medium-sized neurons. The data presented in this paper provides a guide for further studies of afferent and efferent connections of the nucleus raphe dorsalis and for electrophysiological experiments on its constituent neurons.     

Expression of porcine cholecystokinin cDNA in a murine neuroendocrine cell line. Proteolytic processing, sulfation, and regulated secretion of cholecystokinin peptides

The cDNA for porcine preprocholecystokinin (pre-pro-CCK) was engineered for expression in mammalian cells under the control of the Rous sarcoma virus-long terminal repeat promoter. This expression construct was transfected into the murine anterior pituitary cell line, AtT-20. A stable cell line (AtT-20/CCK) was derived that expresses CCK mRNA indistinguishable from the CCK mRNA found in pig brain or gut. The AtT-20/CCK cells carry out proteolytic processing and sulfation reactions to generate authentic sulfated CCK8 from pro-CCK. The cells also store and secrete CCK-immunoreactive peptides. This secretion can be stimulated with corticotropin releasing factor, the natural secretagogue for anterior pituitary cells. In contrast, monkey kidney epithelial cells (COS cells), which are transiently transfected to express CCK, predominantly secrete nonsulfated pro-CCK into the medium. These studies show that a murine neuroendocrine cell line contains the complete processing machinery required to generate authentic porcine CCK8. The processing events include simultaneous proteolytic processing at one and two basic amino acid sites and sulfation of tyrosine residues. The cell line thus duplicates exactly the processing patterns found to occur in pig brain cortex.     

Lung, heart, and kidney express high levels of mRNA for the vitamin K-dependent matrix Gla protein. Implications for the possible functions of matrix Gla protein and for the tissue distribution of the gamma-carboxylase

We have used cDNA probes for two small vitamin K-dependent bone matrix proteins, bone Gla protein (BGP) and matrix Gla protein (MGP), to evaluate the possibility that either of these proteins might be synthesized by the various soft tissues previously shown to have gamma-carboxylase activity. BGP mRNA was found in bone but not in any of the soft tissues tested, a result which reinforces the view that plasma BGP is a specific marker for bone metabolism. In contrast, MGP mRNA was found in all rat tissues examined. Lung and heart have 10-fold higher levels of MGP mRNA than bone, and kidney has a 5-fold higher level. Despite the high levels of MGP mRNA in heart and kidney, these tissues contain 40-500-fold lower concentrations of MGP protein than bone. Immunofluorescence was used to identify cells that contain MGP in kidney, lung, heart, and spleen. In each tissue, MGP was found in discrete tissue-specific cell types. In most of the soft tissues tested, MGP is the first well characterized substrate for the vitamin K-dependent carboxylase found to be synthesized. The exceptionally broad tissue distribution for MGP synthesis demonstrates that the function of MGP is not specific to connective tissues, and the low levels of MGP antigen in soft tissues with high MGP mRNA levels indicate that MGP is unlikely to act solely by virtue of its accumulation in an extracellular matrix.     

Characterization of preprocholecystokinin products in the porcine cerebral cortex. Evidence of different processing pathways

Using gel, ion-exchange, and reverse-phase chromatography monitored by radioimmunoassays specific for five sequences of preprocholecystokinin (prepro-CCK), its processing products were measured in neutral and acid extracts of porcine cerebral cortex before and after incubation with trypsin, carboxypeptidase B, and arylsulfatase. Three categories of peptides were found: biologically active peptides, i.e. peptides with the alpha-amidated COOH terminus Trp-Met-Asp-Phe-NH2, comprising large CCKs, i.e. peptides larger than CCK-58 and peptides eluting like CCK-58, CCK-33, and CCK-22; CCK-octapeptides in sulfated and traces of nonsulfated forms; and small CCKs, i.e. traces of CCK-7, large amounts of CCK-5, and modest concentrations of CCK-4 (the structures of CCK-5 and -4 were confirmed by sequence analysis); four NH2-terminal fragments, of which the two predominant ones correspond to the desnonapeptide fragments of CCK-58 and CCK-33; and COOH-terminal extended peptides corresponding to glycine-extended CCK-58, CCK-33, and CCK-8 in small but significant amounts. Thus, in addition to CCK-8 the porcine cerebral cortex synthesizes larger and smaller active CCK peptides in quantities of an order similar to those of CCK-8. The occurrence of these together with the NH2-terminal fragments and glycine-extended peptides can be explained only by the existence of different processing pathways for preproCCK. Consequently, the results suggest that cerebral CCK neurons are heterogeneous and comprise at least three populations with different biosynthetic machineries.     

Postnatal Development of Cholecystokinin-Like Immunoreactivity and Its mRNA Level in Rat Brain Regions

Developmental changes of preprocholecystokinin mRNA (CCK mRNA) and cholecystokinin-like immunoreactivity (CCK-LI) were examined in rat brain regions (frontal cortex, colliculi, hippocampus, striatum, and cerebellum) using RNA dot blot assays with cholecystokinin (CCK) cDNA and radioimmunoassay, respectively. The CCK-LI levels in all regions examined were very low at birth. Excluding the cerebellum, the levels in these regions increased postnatally and reached adult values at 28 days of age. In contrast to CCK-LI, CCK mRNA levels changed dramatically during development. A considerable amount of CCK mRNA was detected in the frontal cortex and hippocampus at birth. The changes in the level of CCK mRNA in the frontal cortex and colliculi paralleled those of CCK-LI, including a rapid increase from 7 to 14 days of age. The synthesis of CCK mRNA preceded the appearance of CCK-LI. CCK mRNA levels in the hippocampus and striatum exhibited a transient increase, with a peak at 14 days of age. In the adult brain, the CCK mRNA levels were high in the frontal cortex, moderate in the hippocampus and colliculi, and low in the striatum. The cerebellum contained only a negligible amount of CCK mRNA during development. The relatively high level of CCK-LI compared with the low level of CCK mRNA in the striatum supports the idea that most of the striatal CCK-LI is supplied from extrastriatal regions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intracellular localization of messenger RNAs for cytoskeletal proteins

We have analyzed intracellular distributions of mRNAs for the cytoskeletal proteins actin, vimentin, and tubulin by in situ hybridization. Although polyadenylated RNA was homogeneously distributed throughout the cell, actin mRNA demonstrated a nonhomogeneous distribution in 95% of randomly selected chicken embryonic myoblasts and fibroblasts, as detected by isotopic and nonisotopic techniques. Actin mRNA concentrations were highest at cell extremities, generally in lamellipodia, where grain densities were up to 16-fold higher than in areas near the nucleus. Vimentin mRNA, unlike actin mRNA, was distributed near the nucleus. Tubulin mRNA appeared most concentrated in the peripheral cytoplasm. These results demonstrate that cytoplasmic mRNAs are localized in specific, nonrandom cellular patterns and that localized concentrations of specific proteins may result from corresponding localization of their respective mRNAs. Hence, actin mRNA distribution may result in increased concentration of actin filaments in lamellipodia of motile cells.     

Accumulation of nonamidated preprogastrin and preprocholecystokinin products in porcine pituitary corticotrophs. Evidence of post-translational control of cell differentiation

Using gel and ion-exchange chromatography monitored by radioimmunoassays specific for sequences essential in the processing of preprogastrin and preprocholecystokinin, the products were characterized in extracts of porcine pituitary lobes before and after incubation with trypsin, carboxypeptidase B, and arylsulfatase. The intermediate and neural lobes contained only fully activated (i.e. alpha-amidated) preprogastrin products (component I, gastrin-34, and gastrin-17). In contrast, the anterior lobe contained, in addition to traces of alpha-amidated gastrin (2 pmol/g), hundredfold higher amounts of a nonamidated progastrin (189 pmol/g; Mr approximately 7000) and two nonamidated procholecystokinin fragments (75 pmol/g; Mr approximately 7000 and 5000). These results show that hormone genes, in spite of translation of their mRNA, are not necessarily expressed in functional peptides in cells outside the principal production regions. Hence, the study indicates that differentiation of endocrine cells may be controlled at the post-translational level.     

Use of a biotinylated probe and in situ hybridization for light and electron microscopic localization of Po mRNA in myelin-forming Schwann cells

A biotinylated Po glycoprotein cDNA was hybridized in situ to aldehyde-fixed vibratome sections and to aldehyde-fixed thin sections of Lowicryl-embedded trigeminal ganglia of 15 day old rats. Alkaline phosphatase and peroxidase detectors were used for light microscopic (LM) studies and peroxidase or colloidal gold were employed for electron microscopic (EM) detection. In both LM and EM sections, probe was found in cytoplasmic areas of myelin-forming Schwann cells that were enriched in granular endoplasmic reticulum, demonstrating that these regions contain Po mRNA. Interestingly, Po mRNA tended to cluster in regions close to the developing myelin sheath. Relatively simple methods are here described for EM detection of mRNA with reasonable tissue preservation and high resolution. These methods may be useful for developmental and disease-related studies of specific mRNAs in mammalian tissues.     

The Effects of Kainic Acid Injections on Guanylate Cyclase Activity in the Rat Caudatoputamen, Nucleus Accumbens and Septum

The activity of soluble and particulate guanylate cyclase (EC 4.6.1.2) has been compared with the distribution of neurotransmitter candidates in three rat forebrain nuclei, and the effects of local kainic acid injections into these nuclei have been tested. Soluble guanylate cyclase was highly concentrated in both the caudatoputamen and the nucleus accumbens, with lower activity found in the septum. This distribution coincided with markers for acetylcholine and monoamines, but not with markers for γ-aminobutyrate (GABA) or glutamate neurons. In contrast, particulate guanylate cyclase was equally active in all regions. Local injections of kainic acid, which destroyed cholinergic and GABA neurons in the caudatoputamen and in the nucleus accumbens, caused a rapid (70–90%) decrease in the soluble guanylate cyclase and a slower 50-60% fall in the particulate guanylate cyclase in these nuclei. In the septum, where kainate destroyed GABA cells but not cholinergic neurons, the guanylate cyclase activity was unchanged after the lesion. Thus, both the soluble and particulate guanylate cyclases appear to be concentrated in local neurons in the caudatoputamen and nucleus accumbens. In the septum, however, most of the guanylate cyclase activity is located outside kainate-sensitive neurons.     

Tremor following intracerebral carbachol injection. 1.Carbachol sensitivity of various brain structures]

Tremor produced by intracerebral injection of carbachol. I. Susceptibility of different brain areas to carbachol Microinjections of carbachol into the lateral ventricle of rats caused tremor depending on dose. Intensity and duration of motor effects after injection of carbachol (30 mug/3 mul bilateral) into different brain areas were found to depend on localization: strongest tremor was elicited by injections into the nucleus caudatoputamen and cortex cerebri, moderate tremor by administration into the substantia nigra reticularis, globus pallidus and thalamic brain regions. Target areas of mean sensitivity were demonstrated in more rostral and caudal parts of the formatio reticularis. The injection of carbachol into the nucleus ruber, nucleus linearis and substantia nigra compacta brought about lowest tremor values. Ablation of the site of injection from the remaining brain abolished tremor induced by carbachol contrary to the tremor induced by oxotremorine.     

Translational active mRNPs from rabbit reticulocytes are qualitatively different from free mRNA in their translatability in cell-free system

The translatability of polyribosomal and free mRNPs from rabbit reticulocytes and their mRNA was compared. Both classes of mRNPs turned out to be active in rabbit reticulocyte lysates. Considerable differences between mRNPs and mRNA have been revealed. The most striking feature of mRNPs was that high concentrations of mRNPs do not inhibit protein biosynthesis, whereas high concentrations of mRNA strongly inhibit this process. This inhibition is specific for mRNA and does not occur at the addition of the same amount of rRNA from E. coli. The features of mRNP translation are not the result of addition of the supplementary translation factors within particles. The specific function of mRNP proteins in the process of translation is under discussion.     

Studies on the distribution of PHI in mammals

We have developed a sensitive and specific radioimmunoassay to PHI and investigated its distribution in four mammalian species (man, cat, guinea-pig and rat). PHI was present in high concentrations, not only in intestine but also in brain, respiratory tract, urogenital tract and other peripheral tissues. Its distribution was similar to that of VIP and in each tissue examined there was always a significant correlation between the concentrations of these two peptides. In a survey of endocrine tumours, PHI was found to be produced only in those tumours that also produced VIP. In addition PHI was only elevated in the plasma of patients that also had high plasma VIP concentrations. This parallel distribution and release was found to be due to the co-synthesis of VIP and PHI in the same pro-hormone peptide. However, the variable ratio of VIP/PHI in different anatomical areas suggest that in these areas there is a different post-translational enzyme processing of the precursor protein.     

Spatial and temporal expression of a maize lipid transfer protein gene.

We studied the temporal and spatial pattern of lipid transfer protein (LTP) gene expression, as well as the localization of this protein, in maize. Using an LTP gene, we observed an accumulation of LTP mRNA in embryos and endosperms during seed maturation. LTP gene expression was also investigated in young seedlings. After germination, the level of LTP mRNA in the coleoptile increased, with a maximum at 7 days, whereas LTP mRNA levels were low in the scutellum and negligible in roots. The high levels of LTP mRNA found in coleoptiles and embryos were confirmed by in situ hybridization. Moreover, LTP gene expression appeared to be localized in the external cellular layers and around the leaf veins. Using immunogold methods, we also observed that LTP was distributed heterogeneously in the different cells of coleoptiles and leaves. The highest concentrations of LTP were found in the outer epidermis of the coleoptiles as well as the leaf veins. Together, our observations indicate that LTP gene expression is not only organ specific and time specific but also cell specific.     

Distribution of corticotropin-releasing factor in ovine brain determined by radioimmunoassay

We have developed and used a sensitive and specific radioimmunoassay to demonstrate the presence of CRF-like immunoreactivity in extra-hypothalamic areas of ovine brain. Synthetic CRF displaced antibody bound tracer at an ED50 value of 200 pg and there was no cross-reactivity with LHRH, TRH, ACTH, beta-endorphin and several other peptides. Displacement of bound 125I-CRF by brain extracts exhibited curves parallel to synthetic CRF standards. Highest concentrations (1 ng/mg tissue) of CRF-like immunoreactivity were found in the median eminence but surprisingly, high concentrations of CRF-like immunoreactivity were found in frontal, parietal, occipital and particularly temporal areas of cerebral cortex. Much lower concentrations were found in other brain areas including the basal ganglia, limbic system and brain stem.     

Cellular observations and hormonal correlates of feedback control of luteinizing hormone secretion by testosterone in long-term castrated male rhesus monkeys

Testosterone at physiological levels cannot exert negative feedback action on LH secretion in long-term castrated male monkeys. The cellular basis of this refractoriness is unknown. To study it, we compared two groups of male rhesus macaques: one group (group 1, n = 4) was castrated and immediately treated with testosterone for 30 days; the second group (group 2, n = 4) was castrated and treated with testosterone for 9 days beginning 21 days after castration. Feedback control of LH by testosterone in group 1 was normal, whereas insensitivity to its action was found in group 2. Using the endpoints of concentrations of aromatase activity (P450(AROM) messenger RNA [mRNA]) and androgen receptor mRNA in the medial preoptic anterior hypothalamus and in the medial basal hypothalamus, we found that aromatase activity in both of these tissues was significantly lower, P: < 0.01, in group 2 compared with group 1 males. P450(AROM) mRNA and androgen receptor mRNA did not differ, however. Our data suggest that the cellular basis of testosterone insensitivity after long-term castration may reside in the reduced capacity of specific brain areas to aromatize testosterone. Because P450(AROM) mRNA did not change in group 2 males, we hypothesize that an estrogen-dependent neural deficit, not involving the regulation of the P450(AROM) mRNA, occurs in long-term castrated monkeys.     

Differences in cathepsin B mRNA levels in rat tissues suggest specialized functions

The tissue distribution of mRNAs encoding two lysosomal proteases, cathepsin B and cathepsin D, was examined using cloned cDNAs to probe Northern and dot blots of RNAs extracted from various rat tissues. Cathepsin B mRNA showed a wide range of variation in expression in the tissues analyzed with the highest concentrations found in spleen and kidney, while the cathepsin D mRNA levels were relatively uniform in these same tissues. Significant quantities of cathepsin B mRNA were detected in total RNA from isolated islets of Langerhans but was not detectable in equivalent amounts of RNA from whole pancreas. The wide variations in tissue levels of cathepsin B mRNA suggest that tissue specific controls may regulate its expression and are compatible with the participation of this protease in specialized cellular functions other than intralysosomal protein degradation.