Localization of calmodulin in rat cerebellum by immunoelectron microscopy.

Calmodulin, a multifunctional Ca(++)-binding protein, is present in all eucaryotic cells. We have investigated the distribution of this protein in the rat cerebellum by immunoelectron microscopy using a Fab-peroxidase conjugate technique. In Purkinje and granular cell bodies, calmodulin reaction product was found localized both on free ribosomes and on those attached to rough endoplasmic reticulum (RER) and the nuclear envelope. No calmoduline was observed in the cisternae of RER or the Golgi apparactus. Calmodulin did not appear to be concentrated in the soluble fraction of the cell under the conditions used. Rather, peroxidase reaction product could be seen associated with membranes of the Golgi apparatus the smooth endoplasmic reticulum (SER), and the plasma membrane of both cell bodies and neuronal processes. In the neuronal dendrites, calmodulin appeared to be concentrated on membranes of the SER, small vesicles, and mitochondria. Also, granular calmodulin was observed in the amorphous material. In the synaptic junction, a large amount of calmodulin was seen attached to the inner surface of the postsynaptic membrane, whereas very little was observed in the presynaptic membrane or vesicles. These observations suggest that calmodulin is synthesized on ribosomes and discharged into the cytosol, and that it then becomes associated with a variety of intracellular membranes. Calmodulin also seems to be transported via neuronal processes to the postsynaptic membrane. Calmodulin localization at the postsynaptic membrane suggests that this protein may mediate calcium effects at the synaptic junction and, thus, may play a role in the regulation of neurotransmission.

Antibodies to neurofilament, glial filament, and fibroblast intermediate filament proteins bind to different cell types of the nervous system.

Antisera were raised to the 210,000-dalton and the 49,000-dalton proteins of a fraction enriched in intermediate (10 nm) filaments from human brain. Proteins of the filament preparation were separated by SDS-polyacrylamide gel electrophoresis and used for immunization and subsequent analysis of the reactions of the sera by rocket immunoelectrophoresis. Anti-210,000-dalton serum precipitated proteins of molecular weights 210,000, 160,000, and 68,000, and, thus, reacted with all the neurofilament triplet components. Anti-49,000-dalton serum did not react with the triplet proteins but precipitated the 49,000-dalton protein. By immunofluorescence on tissue sections, anti-210,000-dalton serum bound to neuronal axons in sciatic nerve and cerebellum. In dissociated cell cultures, rat dorsal root ganglion cells and their processes bound the serum, whereas nonneuronal cells did not. Some cultured cerebellar neurons were also positive, whereas astrocytes were not. At the ultrastructural level, anti-210,000-dalton serum bound to intermediate filaments inside axonal processes. Anti-49,000-dalton serum bound to astrocytes in sections of the cerebellum, and cultured astrocytes had filaments that stained, whereas other cell types did not. In sciatic nerve sections, elements stained with this serum, but cultured cells from newborn sciatic nerve were negative. An antiserum against the 58,000-dalton protein of the cytoskeleton of NIL-8 fibroblasts strongly stained sciatic nerve sections, binding to Schwann cells but not to axons or to myelin. In cerebellar sections, astrocytes were positive, as were blood vessels and cells in the pia. In cell cultures, anti-58,000-dalton serum stained filaments inside Schwann cells, fibroblasts, and astrocytes, but neurons were negative. Cells in the cultures and tissue sections of the nervous system failed to react with antiserum to the 58,000-dalton protein of skin intermediate filaments. In these studies, astrocytes in vivo and in culture were the only cells which had antigens related to two classes of intermediate filaments.

Putative 51,000-Mr protein marker for postsynaptic densities is virtually absent in cerebellum.

Cerebrum and cerebellum contain numerous asymmetric synapses characterized by the presence of a postsynaptic thickening prominently stained by phosphotungstic acid and other electron-dense stains suitable for electron microscopy. A 51,000-Mr protein, copurified in postsynaptic density-enriched fractions from cerebrum, is considered to be a well established marker for the postsynaptic density. On the basis of two criteria, our studies demonstrate that the 51,000-Mr protein marker for postsynaptic densities is virtually absent in cerebellum, First, it is present in negligible amounts in deoxycholate-insoluble fractions from cerebellum but abundant in parallel fractions from cerebrum. Secondly, the 51,000-Mr protein, which binds 125I-calmodulin after SDS PAGE is readily visualized in membrane samples from cerebrum but is virtually undetectable in cerebellar samples. It is apparent that these results require reexamination of the role of the 51,000-Mr protein in postsynaptic density structures.

An intermediate filament-associated protein, p50, recognized by monoclonal antibodies.

Monoclonal antibodies (mAB) were raised to be used as probes to identify cytoplasmic components associated with intermediate filaments (IF). Four hybridomas (B27, B76, B78, and B100) secreting mAB were generated by fusing mouse myeloma cells with the spleen cells of mice immunized intraperitoneally with Triton-high salt insoluble materials from BHK-21 cells. This insoluble material consists mostly of IF, a small number of microfilaments, and some polyribosomes. Biochemical studies show that the Triton-insoluble materials contain many proteins, including vimentin (decamin) and desmin. Immunofluorescence microscopy of BHK-21 cells stained with the four mAB showed that these mAB decorate the IF in a dotted pattern. Double staining with polyclonal antibody to vimentin confirmed the reactivity of the mAB with the IF. These mAB also stained the vimentin-containing filament system in a variety of other cells including epithelial cells (PTK1 and HeLa) and cells of astroglial origin. Histological studies showed that mAB-B100 stained many types of tissue including epidermis, smooth muscle, and subdermis pericytes, but not the white matter nor the gray matter of the cerebellum and spinal cord. Immunoelectron microscopy with colloidal gold has shown that the mAB-B100 decorated the IF in clusters or aggregates around proteinaceous materials associated with the filaments. Results of immunoprecipitation indicate that mAB-B100 reacted with a protein of 50,000 daltons. These findings suggest that the mAB-B100 we have developed recognizes one of the many components of what appears to be an integrated cytoskeletal structure connected with intermediate filaments.

Ultrastructural localization of glial fibrillary acidic protein in mouse cerebellum by immunoperoxidase labeling.

Glial fibrillary acidic protein was localized at the electron microscope level in the cerebellum of adult mice by indirect immunoperoxidase histology. In confirmation of previous studies at the light microscope level, the antigen was detectable in astrocytes and their processes, but not in neurons or their processes, or in oligodendroglia. Astrocytic processes were stained in white matter, in the granular layet surrounding synaptic glomerular complexes, and in the molecular layer in the form of radially oriented fibers and of sheaths surrounding Purkinje cell dendrites. Astrocytic endfeet impinging on meninges and perivascular membranes were also antigen positive. In astrocytic perikarya and processes, the immunohistochemical reaction product appears both as a diffuse cytoplasmic label and as elongated strands, which by their distribution and frequency could be considered glial filaments.

Immunocytochemical demonstration of vimentin in astrocytes and ependymal cells of developing and adult mouse nervous system.

The occurrence of vimentin, a specific intermediate filament protein, has been studied by immunoflourescence microscopy in tissue of adult and embryonic brain as well as in cell cultures from nervous tissue. By double imminofluorescence labeling, the distribution of vimentin has been compared with that of subunit proteins of other types of intermediate filaments (glial fibrillary acidic [GFA] protein, neurofilament protein, prekeratin) and other cell-type specific markers (fibronectin, tetanus toxin receptor, 04 antigen). In adult brain tissue, vimentin is found not only in fibroblasts and cells of larger blood vessels but also in ependymal cells and astrocytes. In embryonic brain tissue, vimentin is detectable as early as embryonic day 11, the earliest stage tested, and is located in radial fibers spanning the neural tube, in ventricular cells, and in blood vessels. At all stages tested, oligodendrocytes and neurons do not express detectable amounts of vimentin. In primary cultures of early postnatal mouse cerebellum, a coincident location of vimentin and GFA protein is seen in astrocytes, and both types of filament proteins are included in the perinuclear aggregates formed upon exposure of the cells to colcemid. In cerebellar cell cultures of embryonic-day-13 mice, vimentin is seen in various cell types of epithelioid or fibroblastlike morphology but is absent from cells expressing tetanus toxin receptors. Among these embryonic, vimentin-positive cells, a certain cell type reacting neither with tetanus toxin nor with antibodies to fibronectin or GFA protein has been tentatively identified as precursor to more mature astrocytes. The results show that, in the neuroectoderm, vimentin is a specific marker for astrocytes and ependymal cells. It is expressed in the mouse in astrocytes and glial precursors well before the onset of GFA protein expression and might therefore serve as an early marker of glial differentiation. Our results show that vimentin and GFA protein coexist in one cell type not only in primary cultures in vitro but also in the intact tissue in situ.

Posttranslational modifications of alpha-tubulin: acetylated and detyrosinated forms in axons of rat cerebellum.

The distribution of acetylated alpha-tubulin in rat cerebellum was examined and compared with that of total alpha-tubulin and tyrosinated alpha-tubulin. From immunoperoxidase-stained vibratome sections of rat cerebellum it was found that acetylated alpha-tubulin, detectable with monoclonal 6-11B-1, was preferentially enriched in axons compared with dendrites. Parallel fiber axons, in particular, were labeled with 6-11B-1 yet unstained by an antibody recognizing tyrosinated alpha-tubulin, indicating that parallel fibers contain alpha-tubulin that is acetylated and detyrosinated. Axonal microtubules are known to be highly stable and the distribution of acetylated alpha-tubulin in other classes of stable microtubules suggests that acetylation and possibly detyrosination may play a role in the maintenance of stable populations of microtubules.

Immunoelectron microscopic localization of the neural cell adhesion molecules L1 and N-CAM during postnatal development of the mouse cerebellum.

The cellular and subcellular localization of the neural cell adhesion molecules L1 and N-CAM was studied by pre- and postembedding immunoelectron microscopic labeling procedures in the developing mouse cerebellar cortex. The salient features of the study are: L1 displays a previously unrecognized restricted expression by particular neuronal cell types (i.e., it is expressed by granule cells but not by stellate and basket cells) and by particular subcellular compartments (i.e., it is expressed on axons but not on dendrites or cell bodies of Purkinje cells). L1 is always expressed on fasciculating axons and on postmitotic, premigratory, and migrating granule cells at sites of neuron-neuron contact, but never at contact sites between neuron and glia, thus strengthening the view that L1 is not involved in granule cell migration as a neuron-glia adhesion molecule. While N-CAM antibodies reacting with the three major components of N-CAM (180, 140, and 120 kD) show a rather uniform labeling of all cell types, antibodies to the 180-kD component (N-CAM180) stain only the postmigratory granule cell bodies supporting the notion that N-CAM180, the N-CAM component with the longest cytoplasmic domain, is not expressed before stable cell contacts are formed. Furthermore, N-CAM180 is only transiently expressed on Purkinje cell dendrites. N-CAM is present in synapses on both pre- and post-synaptic membranes. L1 is expressed only preterminally and not in the subsynaptic membranes. These observations indicate an exquisite degree of fine tuning in adhesion molecule expression during neural development and suggest a rich combinatorial repertoire in the specification of cell surface contacts.

Light and electron microscopic studies on the localization of hyaluronic acid in developing rat cerebellum.

The hyaluronic acid-binding region was prepared by trypsin digestion of chondroitin sulfate proteoglycan aggregate from the Swarm rat chondrosarcoma, and biotinylated in the presence of hyaluronic acid and link protein. After isolation by gel filtration and HPLC in 4 M guanidine HCl, the biotinylated hyaluronic acid-binding region was used, in conjunction with avidin-peroxidase, as a specific probe for the light and electron microscopic localization of hyaluronic acid in developing and mature rat cerebellum. At 1 w postnatal, there is strong staining of extracellular hyaluronic acid in the presumptive white matter, in the internal granule cell layer, and as a dense band at the base of the molecular layer, surrounding the parallel fibers. This staining moves progressively towards the pial surface during the second postnatal week, and extracellular staining remains predominant through postnatal week three. In adult brain, there is no significant extracellular staining of hyaluronic acid, which is most apparent in the granule cell cytoplasm, and intra-axonally in parallel fibers and some myelinated axons. The white matter is also unstained in adult brain, and no staining was seen in Purkinje cell bodies or dendrites at any age. The localization of hyaluronic acid and its developmental changes are very similar to that previously found in immunocytochemical studies of the chondroitin sulfate proteoglycan in nervous tissue (Aquino, D. A., R. U. Margolis, and R. K. Margolis. 1984. J. Cell Biol. 99:1117-1129; Aquino, D. A., R. U. Margolis, and R. K. Margolis. J. Cell Biol. 99:1130-1139), and to recent results from studies using monoclonal antibodies to the hyaluronic acid-binding region and link protein. The presence of brain hyaluronic acid in the form of aggregates with chondroitin sulfate proteoglycans would be consistent with their similar localizations and coordinate developmental changes.

Thyrotropin-releasing hormone: regional distribution in rat brain.

A sensitive and specific radioimmnunoassay has been used to measure the distribution of thyrotropin-releasing hormone (TRH) in rat brain. All areas of brain tested, except cerebellum, contained readily measurable amounts of TRH. The hypothalamus contained only 31.2 percent of the total brain content of TRH. These results support recent suggestions of central actions for TRH in addition to its hypophysiotropic functions.

Norepinephrine and dopamine: assay by mass fragmentography in the picomole range.

Gas chromatography-mass spectrometry makes possible the simultaneous measurement of norepinephrine and dopamine in concentrations of 0.1-milligram tissue samples. Specificity of the assay is confirmed both by the retention time of the compound and by the mass to charge ratio of the fragments recorded. The sensitivity is of the order of 0.5 picomole, and linearity of the response is maintained up to at least 200 picomoles.

Diazepam-binding inhibitor: a neuropeptide located in selected neuronal populations of rat brain.

An endogenous polypeptide of rat brain has been identified that is capable of displacing 1,4-benzodiazepines and the esters of the 3-carboxylic acid derivatives of beta-carbolines from their specific synaptic binding sites. This polypeptide was termed diazepam-binding inhibitor (DBI). Previous studies have shown that DBI injected intraventricularly in rodents elicits "proconflict" responses and antagonizes the "anticonflict" action of benzodiazepines. An antiserum to this peptide, directed toward an immunodeterminant near its amino terminus, makes it possible to detect, measure, and study the neuronal location of this peptide in rat brain. In the rat cerebral cortex, DBI immunoreactivity is located in neurons that are not GABAergic (GABA, gamma-aminobutyric acid); in the cerebellum and hippocampus, however, it might be present also in GABAergic neurons.

A nonpeptide morphine-like compound: immunocytochemical localization in the mouse brain.

A nonpeptide morphine-like compound (MLC) which cross reacts with morphine-specific antibodies has been localized with the use of immunocytochemistry. This morphine-like compound is found in neuronal perikarya or processes (or both) in nuclei related to vestibular, cerebellar, and raphe systems.

External imaging of cerebral muscarinic acetylcholine receptors.

A radioiodinated ligand that binds to muscarinic acetylcholine receptors was shown to distribute in the brain by a receptor-mediated process. With single-photon-emission imaging techniques, radioactivity was detected in the cerebrum but not in the cerebellum, whereas with a flow-limited radiotracer, radioactivity was detected in cerebrum and cerebellum. Single-photon-emission computed tomography showed good definition of the caudate putamen and cortex in man.

Nerve growth factor gene expression in the developing rat brain.

The regulation of nerve growth factor (NGF) protein and NGF messenger RNA (mRNA) in the developing rat brain has been studied to assess the hypothesis that NGF supports the differentiation of cholinergic neurons in the basal forebrain. In the adult, the major targets of these neurons, the hippocampus and neocortex, contain the highest concentrations of NGF mRNA, but comparatively low ratios of NGF protein to its mRNA. In contrast, a high concentration of NGF protein and a low concentration of NGF mRNA were seen in the basal forebrain, consistent with retrograde transport of NGF protein into this region from the neocortex and hippocampus. In these two target regions NGF and NGF mRNA were barely detectable at birth, their concentrations increased to a peak at day 21, and then NGF mRNA, but not NGF protein, declined threefold by day 35. NGF accumulation in the basal forebrain paralleled that in the target regions and preceded an increase in choline acetyltransferase, suggesting that the differentiation of cholinergic projection neurons is indeed regulated by retrogradely transported NGF. In addition, high ratios of NGF protein to NGF mRNA, comparable to that in the basal forebrain, were seen in the olfactory bulb and cerebellum, suggesting that NGF may be transported into these regions by unidentified neurons.

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.

Cyclic adenosine monophosphate in brain areas: microwave irradiation as a means of tissue fixation.

Amounts of cyclic adenosine monophosphate in discrete regions of the brain were estimated after exposure of rats to microwave irradiation. Amounts were highest in the cerebellum and brainstem, intermediate in the hypothalamus and midbrain, and lowest in the hippocampus and cortex. Decapitation increased the concentration of cyclic adenosine monophosphate in all brain areas, although the increase in the cerebellum was three to four times greater than that in other areas. Microwave irradiation may provide a means of rapidly fixing brain tissue in situ while permitting easy dissection of the brain. In this way artifacts produced by decapitation can be eliminated, and concentrations of heat-stable compounds in the brain can be estimated under conditions which more closely approximate those in vivo.

Intrastrain difference in serotonin and norepinephrine in discrete areas of rat brain.

Determination of levels of serotonin and norepinephrine in various brain areas of male Sprague-Dawley rats obtained from four different breeder-suppliers showed considerably different basal levels among the various groups, as well as differences in response to monoamine oxidase inhibitors.

A novel mRNA of the A4 amyloid precursor gene coding for a possibly secreted protein.

The gene, encoding the A4 peptide found in the amyloid core of senile plaques isolated from the cerebral cortex of patients with Alzheimer's disease, produces at least three precursors that resemble cell surface receptors. A clone isolated from a human brain complementary DNA library contained the structural sequence for an A4 amyloid peptide precursor with a serine protease inhibitor domain in which 208 amino acids at the carboxyl terminal are replaced by 20 amino acids derived from nucleotide sequences with homology to the Alu repeat family. This protein devoid of the transmembrane domain most likely represents a secreted form of the A4 amyloid peptide precursor.

( 3 H)lysergic acid diethylamide: cellular autoradiographic localization in rat brain.

Intravenous administration of [(3)H]lysergic acid diethylamide(LSD) to rats resulted in accumulation of the drug in the brain within 15 minutes. Autoradiographic methods were used to differentiate free and bound [(3)H]LSD in brain tissue. Free [(3)H]LSD was generally distributed in the pituitary and pineal glands, cerebellum, hippocampus,and choroid plexus. Bound [(3)H]LSD was localized in neurons of the cortex, caudate nucleus, midbrain, and medulla,as well as in choroid plexus epithelium.