Identification and localization of ryanodine binding proteins in the avian central nervous system.

Ryanodine binding proteins of the CNS have been identified using monoclonal antibodies against avian skeletal muscle ryanodine binding proteins. These proteins were localized to intracellular membranes of the dendrites, perikarya, and axons of cerebellar Purkinje neurons using laser confocal microscopy and immunoelectron microscopy. Ryanodine binding proteins were not found in dendritic spines. Immunoprecipitation and [3H]epiryanodine binding experiments revealed that the cerebellar ryanodine binding proteins have a native molecular weight of approximately 2000 kd and are composed of two high molecular weight (approximately 500 kd) polypeptide subunits. A comparable protein having a single high molecular weight polypeptide subunit was observed in the remainder of the brain. If the ryanodine binding proteins in muscle and nerve are similar in function, then the neuronal proteins may participate in the release of calcium from intracellular stores that are mechanistically and spatially distinct from those gated by inositol trisphosphate receptors.

Rapid increase of prostaglandin F2-alpha in neurons after subarachnoid hemorrhage in rats: an immunohistochemical study.

The effects of subarachnoid hemorrhage (SAH) with various degrees of increase in intracranial pressure (ICP) on the staining of prostaglandin F2-alpha (PG F2alpha) were studied in rat brains. SAH was produced in 18 rats by injection of 0.18-0.20 ml of autologous arterial blood/100 g body weight into the cisterna magna. By changing the speed of injection, the ICP was transiently increased by 346 +/- 68 (mean +/- S.D.) mm Hg in eight rats (including three pretreated with indomethacin), by 200 +/- 42 mm Hg in five rats, and by 6 +/- 4 mm Hg in the other five. Three rats injected with the same volume of mock cerebrospinal fluid (CSF) with ICP increased by 217 +/- 67 mm Hg and five normal rats without injection served as controls. All animals were decapitated 15 min after injection. The cryosections were stained for PG F2 alpha using an indirect immunofluorescence method. Positive staining for PG F2 alpha was noted only in pial vessels in all normal and mock-CSF-injected rats. In SAH rats with ICP increased by 6 +/- 4 mm Hg, there was a positive reaction in hippocampal neurons and Purkinje cells as well as blood vessels. SAH rats with higher ICP showed stronger PG F2 alpha staining in the above areas, as well as in cerebellar granule cells. All rats pretreated with indomethacin showed a smaller increase in staining. The above results indicate that subarachnoid blood clots per se produce a rapid increase of PG F2 alpha in neurons and blood vessels of both cerebrum and cerebellum, and that this increase is augmented by intracranial hypertension.

An immunochemical analysis of the distribution of a brain-specific polypeptide, PEP-19.

Immunochemical and immunohistochemical techniques were used to map the tissue distribution and cellular localization of a rat brain-specific polypeptide, termed PEP-19. PEP-19 was found to be abundant in the cerebellum and olfactory bulbs but was present at much lower levels in other gross brain regions. It was undetectable in all nonneural tissues examined but was present in the cerebellum of several vertebrates, including rat, mouse, guinea pig, monkey, and human. Immunohistochemical analysis revealed that PEP-19 was localized to the soma, axon, and dendritic processes of rat cerebellar Purkinje cells with no demonstrable immunoreactivity in nonneuronal cell types. Furthermore, mutant mice showing degeneration of Purkinje cells exhibit markedly decreased levels of PEP-19. Because PEP-19 appears during the final stages of maturation of Purkinje cells, it may be utilized as a probe to monitor the development of these neurons in vivo.

Differential distribution of beta-tubulin isotypes in cerebellum.

We describe the structure and expression of a mammalian beta-tubulin isotype (M beta 6) that is weakly expressed in testis but is abundant in developing brain, with transcripts declining to lower levels in the adult brain. The expression of M beta 6 was undetectable in any other mouse tissue examined. A serum specific for this isotype was prepared using a cloned fusion protein as immunogen. M beta 6 is one of five known beta-tubulin isotypes expressed in brain, and using the anti-M beta 6 serum along with sera, anti-M beta 2, anti-M beta 3/4 and anti-M beta 5, previously characterized, we have examined the pattern of expression of beta-tubulin isotypes in rat cerebellum. The isotypes each have characteristic cell-type specific patterns of localization in cerebellum. M beta 2, M beta 3/4 and M beta 5 are present in both neuronal and non-neuronal cells, but in contrast M beta 6 was only detectable in neurons in tissue sections and in dissociated cerebellar cell culture. The majority of sequence differences among the beta-tubulin isotypes lie at the carboxy terminus, the region of beta-tubulin involved in MAP binding. In the case of M beta 2 and M beta 6, the patterns of expression are similar or identical to the patterns of expression of MAP3 and MAP1A respectively. These results suggest that beta-tubulin isotypes may contribute to the determination of the specific association of MAPs with microtubules of diverse function. However, the strict subcellular segregation of other MAPs in brain may be determined by other factors.

Localization of immunoreactive epidermal growth factor receptors in human nervous system.

Epidermal growth factor is a well-defined peptide which stimulates cell growth and elicits cell responses in a variety of tissues by binding to specific receptors, EGF-R. A specific antiserum against the EGF receptor, which has previously been used to characterize EGF-R in human skin, fibroblasts, and smooth muscle, was used to survey the distribution of EGF-R in human nervous system. Portions of formalin-fixed, paraffin-embedded autopsy specimens were examined by use of immunohistochemical staining (PAP technique) with EGF-R antiserum. Many types of nerve cells, e.g., cerebral cortical pyramidal cells, hippocampal pyramidal cells, Purkinje cells, anterior horn cells, and dorsal root ganglion neurons, contained immunoreactive EGF-R. However, immunoreactive EGF-R were not detected in astrocytes, oligodendrogliocytes, and other small neurons such as granule cells. Intense immunostaining for EGF-R was also detected in ependymal cells from choroidal and extrachoroidal locations. Although immunoreactive EGF-R is widely distributed in human nervous system, the functional role of EGF and its receptor in the nervous system remains unknown.

Purification and characterization of PCPP-260: a Purkinje cell-enriched cyclic AMP-regulated membrane phosphoprotein of Mr 260,000.

PCPP-260 (Purkinje cell phosphoprotein of Mr 260,000), a substrate for cAMP-dependent protein kinase, appears to be an integral membrane protein highly enriched in Purkinje cells of the mammalian cerebellum (Walaas et al.: J. Neurosci., 3:291-301, 1983; Walaas et al.: J. Neurosci., 6:954-961, 1986). PCPP-260 has now been purified from a crude particulate fraction of bovine cerebellum, using the ionic detergent N-lauryl sarcosine (NLS) as solubilizing agent, and monitoring the purification by silver stain and autoradiography of 32P-phosphorylated samples, after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Concanavalin A was found to bind to PCPP-260, suggesting that it is a glycoprotein. PCPP-260 was therefore extracted, retained on a column of concanavalin A-agarose, and eluted by alpha-methyl mannoside. Further chromatography on Sephacryl S-400 yielded a preparation that was purified approximately 250-fold relative to the initial particulate fraction and that was at least 95% pure. The protein was estimated to represent approximately 0.4% of total membrane protein in the cerebellum. Peptide mapping and phosphoamino acid analysis following phosphorylation of the protein by cAMP-dependent protein kinase showed one major tryptic phosphopeptide containing phosphoserine. A similar, less prominent protein was also found in membranes from other brain regions but could not be detected in liver membranes. The availability of highly purified PCPP-260 should facilitate the investigation of its possible functional roles in the nervous system.

The polypeptide PEP-19 is a marker for Purkinje neurons in cerebellar cortex and cartwheel neurons in the dorsal cochlear nucleus.

This light and electron microscopic immunocytochemical study shows that the polypeptide PEP-19, a presumptive calcium binding protein specific to the nervous system, represents an excellent marker for cerebellar Purkinje cells and dorsal cochlear nucleus (DCoN) cartwheel cells. The polypeptide clearly reveals the entire populations of both types of neurons, including their complete dendritic and axonal arborizations. Other PEP-19 containing neurons in the two regions display weak immunoreactivity restricted to the cell body or to cell body and principal dendrites. Electron microscopic localization of PEP-19-like immunoreactivity reveals similarities between this polypeptide, parvalbumin, and a 28K vitamin D-dependent calcium binding protein. However, calmodulin, which is expressed in both Purkinje and granule cells, may differ from PEP-19. Similarities between the organization of the cerebellar cortex and the DCoN superficial layers have been known for some time, with several types of neurons in one system having their presumed homologue in the other. These data provide further support for the proposed structural and functional homology between Purkinje and cartwheel neurons, and establishes PEP-19 as a useful marker for examining degeneration of these two neuronal populations in murine cerebellar mutants.

beta-Endorphin-like immunoreactivity in the brain of the lizard, Lacerta muralis.

Using immunocytochemical methods, a beta-endorphin-like immunoreactive substance was identified in the brain of the lizard Lacerta muralis. beta-Endorphin-like neurons were observed in the dorsal posterior part of the paraventricular nucleus and in the caudal region of the nucleus ventromedialis hypothalami. beta-Endorphin-like immunoreactive fibers were also detected in the median eminence. Another cell group displaying beta-endorphin-like immunoreactivity was found in both subdivisions of the oculomotor nucleus and in the periaqueductal gray of the mesencephalon. In addition, a beta-endorphin-like immunoreaction was observed in the perikarya of the Purkinje cells and in their axonal processes. These patterns of immunoreactivity were completely abolished when a specific antiserum was absorbed with its corresponding antigen or with beta-lipotropin. These control tests suggest that the immunoreaction might correspond to a beta-endorphin- or lipotropin-like reaction. The results are discussed in relation to the possibility that a beta-endorphin-like peptide may be involved in hypophysial regulation or neuromodulator activity in the brain of the lizard L. muralis.

Occurrence of beta-endorphin-like immunoreactivity in the brain of the teleost, Boops boops.

By immunocytochemical methods the present study describes beta-endorphin-like immunoreactive substance in the brain of Boops boops. Beta-Endorphin-like neurons and fibers were detected in both dorsal and ventral components of the preoptic nucleus and in the nucleus lateralis tuberis. This localization has been discussed in relation to the presence, in the same area, of a well-defined neurosecretory system involved in hypophysial regulation. A beta-endorphin-like immunoreaction was also detected in the Purkinje cells and in processes within the cerebellum molecular layer. Although this last finding remains enigmatic it may suggest a neuromodulatory activity for cerebellum beta-endorphin-like substance. No immunoreaction was observed when the specific antiserum was absorbed with corresponding antigen and with beta-LPH. These tests led to the conclusion that the immunostaining reaction might correspond to a beta-endorphin- or lipotropin-like reaction. Further, the present results show the phylogenetic antiquity of a beta-endorphinergic or lipotropinergic system in the brain, with a stable evolutionary history as a hypophysial regulatory factor or neuromodulatory agent.

Immunocytochemical localization of cyclic GMP: light and electron microscope evidence for involvement of neuroglia.

Guanosine 3',5'-cyclic monophosphate (cGMP) immunoreactivity in the rat's cerebellum was studied with light and electron microscopy by the indirect fluorescence method and the peroxidase-antiperoxidase method. Labeled cells included neuroglial cells in the cerebellar cortex, white matter, and deep nuclei; some stellate and basket cells in the cortex; and some large neurons in the deep nuclei. No evidence was found for sagittal microzonation in the cGMP distribution. In the labeled cells, cGMP immunoreactive sites were localized to surface membranes, organelles, and the cytoplasmic matrix. Specificity was indicated by the same pattern of labeling after treatment with cGMP immunoglobulin that had been adsorbed with adenosine 3',5'-cyclic monophosphate (cAMP) and by the failure to label after treatment with normal rabbit sera or with cGMP immunoglobulin that had been adsorbed with 1 mM cGMP. Cerebella treated with cAMP antisera, however, showed immunoreactivity in Purkinje cells, granule cells, and Golgi cells in addition to neuroglia in cortex and deep nuclei. Sequential norepinephrine and glutamate superfusions generally intensified cGMP immunoreactivity, not only in neuroglial cells but also in the background. Under these conditions some Purkinje cells and some granule cells were also labeled. Increased cGMP immunoreactivity was also obtained by treatment with harmaline, gamma-aminobutyric acid and aminooxyacetic acid, muscimol, gamma-aminobutyric acid, or apomorphine in order of decreasing effectiveness. Serotonin and colchicine produced no detectable increase of cGMP immunoreactivity above normal, and diazepam and sodium pentobarbital decreased it. In these experiments, diethyl ether was preferable to sodium pentobarbital for anesthesia on account of the depressive action of the latter on cGMP immunoreactivity. Thus, drugs that increase cerebellar activity enhance cGMP levels, whereas those that decrease cerebellar activity decrease cGMP levels. However, it is not clear whether these fluctuations in cGMP levels are a direct consequence of neurotransmitter function or are sequelae to other related events. The present study suggests that some neurons and many neuroglial cells are the major sites of cGMP in the cerebellum.

Cytophotometric identification of tetraploid Purkinje cells in young and aged rats.

DNA content of cerebellar basket and Purkinje cells of four month, one year, and two year old albino rats was determined by Feulgen cytophotometry. DNA content per nucleus remained essentially constant during aging although there was a slight shift to lower Feulgen-DNA values occurring in the one year age group. Purkinje cells of all three age groups were found to contain tetraploid amounts of DNA as compared to diploid basket cells.

Are all the neuronal nuclei polyploid?

The DNA analysis of rat brain nuclei by two independent cytochemical methods, namely microfluorometry and UV-absorption, brings completely different results to those published previously by many investigators. The neuronal nuclei possess twice as much DNA as the glial nuclei.

Adenosine 3',5'-monophosphate is localized in cerebellar neurons: immunofluorescence evidence.

Adenosine 3',5'-monophosphate is localized in specific cerebellar neurons, as shown by fluorescence immunocytochemistry with a specific rabbit immunoglobulin. Positive staining is exhibited by Purkinje neurons and granule cells. The increase in concentration of cyclic adenosine monophosphate in the cerebellum, which is known to follow decapitation, is represented by greatly increased fluorescence of Purkinje neurons only. These immunofluorescence data provide the first evidence for localization of cyclic adenosine monophosphate in specific neurons and may permit further exploration into the role of this cyclic nucleotide in neuronal function.

Tetraploid DNA content of Purkinje neurons of human cerebellar cortex.

Microspectrophotometric analysis of single cells in Feulgen preparations revealed tetraploid amounts of DNA in Purkinje neurons of human cerebellar cortex.