Bibie M. Chronwall

A comparison of the anatomical distribution of substance P and substance P receptors in the rat central nervous system

A comparison of anatomical distributions of substance P (SP) and substance P receptors in the rat central nervous system was performed. SP was localized by microdissection and radioimmunoassay and SP fibers and cell bodies by immunohistochemistry. Receptors for 125I-Bolton Hunter labelled SP (125I-BH-SP) were characterized pharmacologically by a slice binding technique in sections that contained primarily striatum. The receptor was saturable and had an equilibrium dissociation constant (KD) of 0.30 nM and maximum number of binding sites (Bmax) of 37.8 fmol/mg protein. Pharmacological characterization using C terminal fragments and naturally occurring analogues of SP reflected characteristics of the receptor which had been shown previously in bioassays and biochemical assays. Comparison of distribution of SP fibers and cell bodies and SP receptors indicated that there is no consistent relationship between the amount of SP receptor and density of SP fibers or cell bodies in a given region of the brain.

Anatomy and physiology of the neuroendocrine arcuate nucleus

The arcuate nucleus surrounds the ventral part of the third ventricle and contains densely packed small neurons with 1-3 dendrites. At least fifteen transmitters and neuropeptides have been found in perikarya of arcuate neurons. Each transmitter and neuropeptide have a characteristic distribution. In many cases distributions overlap (for example, dopamine and somatostatin, dopamine and neurotensin, neuropeptide Y and somatostatin) and alpha-MSH and beta-endorphin seem to have identical distributions but there are also distinctive neuronal populations containing only one of the described transmitters or neuropeptides (neuropeptide Y and alpha-MSH). Studies show extensive colocalization of dopamine and neurotensin and sparse colocalization of dopamine and GABA, neuropeptide Y and FMRF-NH2 and neuropeptide Y and somatostatin. Colocalization does not seem to be the rule in the arcuate, however, it is possible that colocalization may vary with the physiological state or sex of the animal. It also should be noted that our techniques may not be sensitive enough. To study efferent projections as a possible organizing principle within the arcuate, retrograde fluorescent tracing was combined with transmitter and neuropeptide immunohistochemistry. Mainly NPY and alpha-MSH neurons were studied and both peptides are present in projections to the preoptic area as well as to the midbrain periaqueductal gray. Some arcuate neurons were found to have collateral axons to both these areas. The arcuate communicates primarily with the pituitary gland, hypothalamus, limbic system, midbrain periaqueductal gray and autonomic nuclei of the brain stem. In this way, the arcuate may be involved in integrating emotional, sensory, vegetative homeostatic and autonomic functions with endocrine functions.

Neuropeptide Y and peptide YY neuronal and endocrine systems

An extensive system of neuropeptide Y (NPY) containing neurons has recently been identified in the central and peripheral nervous system. In addition, NPY and a structurally related peptide, peptide YY (PYY), containing endocrine cells have been identified in the periphery. The NPY system is of particular interest as the peptide coexists with catecholamines in the central and sympathetic nervous system and adrenal medulla. Evidence has been presented which indicates that NPY may play important roles in regulating autonomic function.

Coexistence of neuropeptide Y and somatostatin in rat and human cortical and rat hypothalamic neurons.

The distribution and coexistence of neuropeptide Y (NPY) and somatostatin (SOM) were evaluated in rat and human cerebral cortex and in the rat hypothalamic arcuate nucleus (n) using double immunofluorescent staining in which primary antisera were raised in different species. The results of the study indicate extensive coexistence of NPY and SOM in both rat and human cortex but only occasional coexistence in the rat arcuate n.

Characterization and distribution of FMRFamide immunoreactivity in the rat central nervous system

FMRFamide immunoreactive material (irFMRFamide) was studied in rat brain and gastrointestinal tract. Highest irFMRFamide concentrations were found in tissues of the gastrointestinal tract and, in the brain, highest concentrations were found in the hippocampus, midbrain, brainstem and hypothalamus. High pressure liquid chromatographic characterization of irFMRFamide demonstrated that the immunoreactive material in brain, pancreas and duodenum was different from molluscan FMRFamide but it was also distinct from any known neuropeptide.

Histochemical localization of FMRFamide-like immunoreactivity in the rat brain

Molluscan cardioexcitatory neuropeptide or FMRFamide is present in the invertebrate central nervous system (CNS) and FMRFamide like peptide has been demonstrated in the mammalian CNS. In this study, the distribution of FMRFamide immunoreactivity was studied in rat brain using the indirect immunofluorescent method. The highest number of FMRFamide staining cell bodies was found in the nucleus (n) arcuatus. N. paraventricularis, n. hypothalamus, n. ventromedialis, n. dorsomedialis and n. tractus solitarii also contained high numbers. FMRFamide positive nerve fibers and terminals were widely distributed. The septal complex contained high densities, especially in n. interstitialis striae terminalis. N. paraventricularis hypothalami, n. paraventricularis, n. hypothalamicus, n. ventromedialis and n. dorsomedialis showed a high to very high degree of immunoreactivity. In myelencephalon, n. tractus solitarii had the densest innervation. Spinal cord had a dense band of FMRFamide positive fibers in lamina I and II of the dorsal horn. The present findings support a neurotransmitter role for a FMRFamide like peptide in the mammalian brain, possibly related to endocrine and autonomic regulation as well as pain modulation.

Biochemical and histochemical characterization of ranatensin immunoreactive peptides in rat brain: lack of coexistence with bombesin/GRP.

An antibody to des-pyroglutamyl ranatensin (RT 2-11) has been prepared and has been used to histochemically and biochemically identify ranatensin-like immunoreactivity (irRT) in the rat brain. The most most prominent stained cell group was situated in the dorsal tegmental pons. Areas of immunoreactive fibers were found in the ventral hippocampus, septal area and the hypothalamus. A similar distribution was found by radioimmunoassay data. The distribution of irRT was compared to that of bombesin-like immunoreactivity (irBN). The two peptides have different though partly overlapping distributions. Gel filtration of brain extracts show that the molecular size of irRT is similar to that of synthetic RT. However, HPLC characterization of irRT indicated that the immunoreactive material is different from synthetic RT and also different from irBN and irGRP. These results indicate the presence of two separate peptide systems, one RT-like, the other BN-like, in the mammalian CNS.

Presence and distribution of immunoreactive and bioactive FMRFamide-like peptides in the nervous system of the horseshoe crab, limulus polyphemus

FMRFamide immunoreactivity was detected in all regions of the Limulus nervous system, including the brain (6.5 +/- 0.6 pg FMRFamide/mg), cardiac ganglion (2.06 +/- 0.67 pg FMRFamide/mg), and ventral nerve cord (5.8 +/- 0.7 pg FMRFamide/mg). The distribution of immunoreactive FMRFamide (irFMRFamide) was mapped by immunofluorescence and the distribution corresponded to regional RIA data. A good proportion of the CNS and cardiac ganglion neuropile contained irFMRFamide, and fluorescent cell bodies were observed in several areas. High performance liquid chromatography (HPLC) was employed to separate and characterize the FMRFamide-like peptides from extracts of Limulus brains. HPLC fractions were analyzed using coincidental radioimmunoassay and bioassay (the radula protractor muscle of Busycon contrarium). There appear to be at least three FMRFamide-like peptides in the Limulus brain, including one similar to clam FMRFamide. FMRFamide acts on Limulus heart in a biphasic manner at relatively high concentrations (10(-5)M), but has no effect on the activity of the isolated ventral nerve cord. These data suggest that in Limulus FMRFamide-like peptides are acting as neurotransmitters, or neuromodulators.

Glial and nonglial neoplasms evaluated on frozen section by double immunofluorescence for fibronectin and glial fibrillary acidic protein

SummaryAntisera against fibronectin (FN) and glial fibrillary acidic protein (GFAP) were used combined on frozen sections of surgical biopsies to distinguish between astrocytoma and hemangioblastoma, meningioma, or sarcoma. The nonglial neoplasms contained FN in their parenchyma, whereas the gliomas contained GFAP. The immunofluorescent staining procedure takes 10 min. The immunoperoxidase staining for the permanent files takes 30 min. The stains have been used in this institute for more than 1 year, and 26 tumors have been evaluated.

Regulation of heterogeneity in D2 dopamine receptor gene expression among individual melanotropes in the rat pituitary intermediate lobe.

The regulation of D2 dopamine receptor binding, immunoreactivity, and mRNA was studied in melanotropes of the intermediate lobe of the rat pituitary. D2 dopamine receptor-binding experiments showed that chronic haloperidol treatment (2 mg/kg for 14 days) significantly increased the Bmax but not Kd of [3H]spiperone binding, whereas bromocriptine treatment (2 mg/kg for 14 days) produced no change in either parameter. Immunohistochemical experiments revealed that all melanotropes contained D2 receptor immunoreactivity but the D2long receptor isoform was intensely expressed by comparatively few melanotropes scattered among cells that contained substantially lower staining intensities. Chronic haloperidol treatment increased the overall intensity of total D2 receptor staining and concurrently increased both the number and staining intensity of melanotropes immunoreactive for the D2long isoform. Bromocriptine treatment produced no change in overall staining intensity; however, the number of melanotropes staining for the D2long isoform increased significantly. Nonradioactive in situ hybridization histochemistry further revealed that individual melanotropes contained differing amounts of both total D2 receptor and D2long mRNAs; the heterogeneity in D2long receptor expression was particularly striking. Following haloperidol treatment, D2 receptor mRNA levels increased in all melanotropes. Following bromocriptine treatment most melanotropes contained very low levels although many melanotropes retained substantial amounts of D2 receptor mRNA. The parallel increase in D2 receptor-binding densities, immunoreactivity, and mRNA levels following chronic antagonist treatment emulates the classic paradigm by which a cell increases its receptor population. Chronic agonist treatment did not follow the inverse paradigm and revealed heterogeneous regulation of a discrete subpopulation of melanotropes.