Expression of Thy-1, H-2 and NS-4 cell surface antigens and tetanus toxin receptors in early postnatal and adult mouse cerebellum

The expression of several cell surface components (Thy-1, H-2 and NS-4 antigens and tetanus toxin receptors) was studied by indirect immunofluorescence in situ using histological sections and in vitro using freshly dissociated and cultured cells from mouse cerebellum. Thy-1 alloantigen is expressed in adult cerebellum predominantly in neuron-rich regions, i.e. molecular, Purkinje cell, and granular layers, however, it is not detectable at postnatal day 8. In cerebellar cultures of 6-day-old mice Thy-1 is absent from more than 99% of all cells when these are maintained as monolayers in vitro for up to 3 days. After 4 days in vitro some GFA protein-positive astrocytes and some fibronectin-positive fibroblast-like cells start to express Thy-1 antigen. After 14 days in vitro not all fibroblast-like cells and astrocytes are Thy-1 antigen-positive. Neurons with small cell bodies and oligodendrocytes never express Thy-1 at any stage examined. H-2 is not expressed sufficiently to be detectable in histological sections in early postnatal or adult cerebellum. In cerebellar cultures of 6-day-old mice H-2 becomes detectable on some fibroblast-like cells and some astrocytes after 7 days in culture. In histological sections of adult and early postnatal cerebellum NS-4 antigen and tetanus toxin receptors are expressed predominantly in neuron-rich regions. In the developing cerebellum both are expressed at higher levels on more mature granule cells. In cerebellar cultures NS-4 antigen and tetanus toxin receptors are expressed on neurons. Occasionally some astroglia can also show detectable levels of expression. NS-4 antigen is also present on some 04 antigen-positive oligodendrocytes, while tetanus toxin receptors are never detectable on these cells.     

Neurochemical and morphological consequences of axon terminal degeneration in cerebellar deep nuclei of mice with inherited purkinje cell degeneration

The concentrations of free amino acids and the activities of transmitter-related enzymes, glutamic acid decarboxylase (GAD), choline acetylase (ChAC) and GABA-transaminase (GABA-t) were measured in cerebellar cortex and deep cerebellar nuclei from the mouse mutant Purkinje cell degeneration (pcd) at various times before and after Purkinje cell loss. Axosomatic synapses on target cells in pcd deep nuclei were quantified by electron microscopy during and after degeneration. The concentration of GABA (nmol/mg wet weight), the Purkinje cell transmitter, was normal in pcd cerebellar cortex and deep nuclei before onset of Purkinje cell degeneration on postnatal day 15. Just after the major period of Purkinje cell loss in cerebellar cortex, GABA concentration was unchanged in the cortical layers but fell to 50% of normal values in the deep nuclei of pcd animals killed either by decapitation or by microwave irradiation. No other measured free amino acid decreased. There were no long-term increases following Purkinje cell degeneration in the concentration of any transmitter amino acids or related enzymes, GAD, ChAC or GABA-t, and thus no indication of axonal sprouting reactions. Progressive losses occurred in wet weight and protein and in activity of GABA-t in both the cerebellar cortex and the deep nuclei of pcd animals. Electron microscopic analysis indicated that Purkinje cell axon terminals contact 30% or more of the somatic surface of principal neurons of the lateral nucleus of the normal cerebellum, but only about 2% of the corresponding sites in the pcd cerebellum. Glial leaflets, rather than other synaptic terminals take their place. Axon terminals may degenerate earlier than Purkinje somata in the pcd disease.     

Immunohistochemical mapping of enkephalin containing cell bodies, fibers and nerve terminals in the brain stem of the rat

Enkephalin immunoreactive perikarya, fibers and nerve terminals, visualized by the indirect immunohistofluorescent method in colchicine-pretreated animals, are localized in many discrete regions of the rat brain stem. These specific immunohistofluorescent patterns are similar after staining with selective primary antisera directed against either methionine-enkephalin or leucine-enkephalin. Cell bodies are found in the substantia gelatinosa and interpolaris zones of the trigeminal nuclear complex, the nucleus of the solitary tract, in the vicinity of the nucleus raphé magnus, in the dorsal cochlear, medial vestibular, and paraolivary nuclei and, dorsal to this last region, in the parabrachial nuclei and the dorsal tegmental nucleus of Gudden, in the periaqueductal gray matter and interpeduncular nucleus and along the borders of the lateral lemniscus and medial geniculate. In some areas, such as the parabrachial region, nucleus of the solitary tract and substantia gelatinosa of the trigeminal nucleus, these perikarya are associated with densities of fibers and terminals. Other regions, such as the dorsal cochlear nucleus and the vicinity of the nucleus raphé magnus, contain cell bodies associated with low densities of processes and terminals. In still other nuclei, such as the nucleus of the facial nerve and the locus coeruleus, fiber and terminal densities without associated cell bodies are evident. Many of these enkephalin localizations can be rationalized on the basis of known actions of opiate drugs and the brain stem distribution of opiate receptors.     

Band 1 and voltage-sensitive Na channels are not codistributed in cultured rodent neuronal cells

The relationship of the mouse nervous system specific band 1 protein to the putative high molecular weight component of the Na+ channel was investigated using antibody to band 1. Morphologic differentiation of cultured neuroblastoma cells has been reported to increase the quantity of the putative Na+ channel high molecular weight component. Morphologically differentiated clone NB2a neuroblastoma cells have 2-3 times the amount of band 1 and 1.5 times the relative rate of synthesis of band 1 as undifferentiated cells. The anti-band 1 serum reacts with both adult mouse and rat brain but not 3 cultured rat neuronal lines known to have active Na+ channels. Thus either band 1 is not a component of the Na+ channel or individual cultured murine neuronal lines has distinct macromolecular Na+ channels.     

Neurotensin-containing cell bodies, fibers and nerve terminals in the brain stem of the rat: Immunohistochemical mapping

Neurotensin immunoreactive perikarya, fibers and nerve terminals, visualized by the indirect immunohistofluorescent method in colchicine-pretreated animals, are localized in many discrete regions of the rat brain stem. Cell body groups are found in the inner aspect of the substantia gelatinosa of the caudal trigeminal nuclear complex, the nucleus of the solitary tract, the parabrachial nuclei, the locus coeruleus, the dorsal raphé nucleus, the periaqueductal gray matter, and the ventral tegmental area of Tsai. These areas of cell body density are accompanied by concentrations of fibers and terminals, while the occasional positive perikaryon noted in the dorsal cochlear nucleus is accompanied by only sparse fluorescent fiber/terminal patterns. Other brain stem regions, such as the floor of the fourth ventricle and aspects of the caudal ventrolateral reticular formation, possess substantial numbers of fibers and terminals that are not accompanied by cell bodies. Many aspects of this distribution coincide with the brain stem distribution of the enkephalin pentapeptides, though significant differences in localization are also evident. Interactions of neurotensin with other neurotransmitter candidates are also suggested by its presence in areas enriched in norepinephrine, dopamine, serotonin, and substance P. Certain neurotensin localizations suggest an association of the peptide with functional brain systems preferentially involving these regions. In particular periaqueductal gray and substantia gelatinosa neurotensin synapses are plausible sites for the analgesia elicited after intercisternal injection of low doses of neurotensin.     

Biochemical differentiation of aggregating cell cultures of different fetal rat brain regions.

Rotation-mediated aggregating cell cultures of mechanically dissociated fetal rat brains divided into three (telencephalon, mesencephalon-diencephalon and rhombencephalon), or two (telencephalon and mesencephalon-diencephalon plus rhombencephalon) parts were examined for their biochemical differentiation by measuring the specific activities of choline acetyltransferase, acetylcholinesterase, glutamic acid decarboxylase, tyrosine 3-monooxygenase, aromatic L-amino acid decarboxylase, catechol methyltransferase and monoamine oxidase. The results showed that such parts yielded cultures that were relatively enriched for acetylcholine-synthesizing (telencephalon) or catecholamine-synthesizing (mesencephalon-diencephalon and mesencephalon-diencephalon plus rhombencephalon) enzymes. For cultures which were derived from two brain divisions, the sum of the total activity for each enzyme in the parts after 30 days equalled that in whole brain cultures derived from the same group of embryos, suggesting that development of these enzymes was unaffected by division of the brain in two. In experiments to determine the effects of culture conditions on this development, chronic administration of certain drugs was found to selectively influence the specific activity of certain neurotransmitter metabolizing enzymes. Thus, in cultures of whole brain, ascorbic acid (0.2 mM) decreased tyrosine 3-monooxygenase and aromatic L-amino acid decarboxylase while other enzymes were slightly increased; and in cultures of telencephalon and mesencephalon-diencephalon plus rhombencephalon, N6, O2'-dibutyryladenosine 3',5'-cyclic phosphate (0.2 mM) decreased the specific activities of choline acetyltransferase acetylcholinesterase, glutamic acid decarboxylase and monoamine oxidase. These results demonstrate the feasibility of growing these cultures for pharmacological studies in developmental neurobiology.     

Ligand binding studies in the mouse olfactory bulb: identification and characterization of a L-[3H]carnosine binding site.

Binding sites for the dipeptide L-carnosine (beta-alanyl-L-histidine) have been detected in membranes prepared from mouse olfactory bulbs. The binding of L-[3H]-carnosine was saturable, reversible and stereospecific and had a Kd of about 770 nM. The stereospecific binding of L-carnosine represented about 30% of the total binding at pH 6.8, and decreased markedly with increasing pH. Binding was stimulated by calcium, unaffected by zinc, magnesium or manganese and inhibited by sodium and potassium. Carnosine binding was sensitive to trypsin and phospholipases A and C, but not to neuraminidase. Nystatin and filipin, which interact with membrane lipids, also interferred with binding. Some peptide analogues of carnosine were potent inhibitors of binding, but a variety of drugs serving as potent inhibitors in other binding systems had no effect on carnosine binding. Carnosine binding to mouse olfactory bulb membranes was 15-fold higher than that seen in membranes prepared from cerebral hemispheres, 5-fold higher than that seen in membranes prepared from cerebral hemispheres, 5-fold higher than in cerebellum membranes and 3-fold higher than in membranes from spinal medulla and the olfactory tubercle-lateral olfactory tract area. Binding sites for 6 other radiolabeled receptor ligands were also detected in bulb membranes. Peripheral deafferentation of the olfactory bulbs by intranasal irrigation with ZnSO4 led to a loss greater than 90% of the L-[3H]carnosine binding in 4--5 days with much smaller losses in binding of the other 6 ligands over a 180-day observation period. This initial loss of carnosine binding after denervation was due to a loss of binding site stereo-specificity followed by a loss of binding sites. The characteristics of the carnosine binding site in olfactory bulb fulfil 6 of the 7 criteria considered relevant for a functional receptor.     

Divalent cation dependent phosphorylation of proteins in squid giant axon

In vitro and in situ (after intracellular infusion) incubation of axoplasm from the squid giant axon with [gamma-32P]ATP produces a phosphorylation of primarily two proteins (of mol.wt. 200,000 and greater than 400,000). The phosphorylation of these proteins is stimulated by Mg2+, inhibited by Ca2+, and unaffected by 10(-7) to 10(-5) M cyclic nucleotides. The 200 kdalton and greater than 400 kdalton phosphorylated peaks appear to be neurofilament proteins, and phosphorylation of these peaks in situ is decreased by electrical stimulation of the axon.     

Rapid loss of nicotine-cholinergic receptor binding activity in the deafferented avian optic lobe

The levels of alpha-bungarotoxin (alpha-BuTX) sensitive receptor sites were investigated in the optic lobe after optic deafferentation in the neonatal and adult chicken. Within two days a 30% loss of alpha-BuTX binding sites per optic lobe is observed in the neonatal chick after enucleation. The results are similar with the adult chicken in experiments where the receptor binding activity is measured in the optic lobe and in the optic tectum after enucleation. The possibility that acetylcholine is a neurotransmitter in the vertebrate retinotectal pathway is discussed.     

Amino acid neurotransmitter candidates in rat cerebellum: Selective effects of kainic acid lesions

Kainic acid injections directly into the cerebellum destroy Purkinje, stellate, basket and Golgi II cells selectively with much less damage to granule cells. We have utilized such kainic acid lesions to evaluate the disposition of amino acid transmitter candidates in different neuronal populations of the cerebellum. Kainic acid lesions produce a 65-70% decrease in high affinity [3H]GABA uptake into synaptosomal fractions and a similar decrease in glutamic acid decarboxylase with a 50% reduction in endogenous GABA. Synaptosomal accumulation of [3H]glutamate and [3H]-aspartate is reduced 25-30% following such lesions while no decline in uptake of numerous other amino acids is observed. No significant changes are found in endogenous levels of glycine and serine are elevated following such lesions. These findings are consistent with the possibility that glutamate is the transmitter of granule cells and that GABA is the transmitter of the other cell types in the cerebellum.     

Spectrophotometry of the CSF (CSF-SPE) and computer tomography (CT) in traumatic head injuries

Combined examinations with quantitative CSF spectrophotometry (CSF-SPE) and computer tomography (CT) were performed on 53 patients with traumatic head injuries. In cerebral concussion the results were mainly normal in both examinations. In cerebral contusion bleeding patterns were found by CSF-SPE in all subjects, with a special bleeding pattern (S2 pattern) occurring in 86%. CT showed findings described as typical for contusion in 8 of 14 examined patients, the remaining CT scans showing questionable or normal signs. In extra- and intracerebral haematomas, all patients had bleeding patterns on the CSF-SPE. A special bleeding component (H factor) was found in about 72%. The H component was not observed during the first 3 to 4 days after the trauma. All but one patient examined later than the 4th day had an H component with or without an S-pattern. CT demonstrated a haematoma in 14 of 18 verified haematoma patients, while 4 subjects with subdural haematoma (e.g. one third of this patient group) had questionable CT findings.The combined examinations with CT and CSF-SPE, being complementary to each other, are of great value in the differential diagnosis of traumatic head injuries.     

Neuroleptic and dopamine receptors: autoradiographic localization of [3H]spiperone in rat brain.

Rats were administered [3H]spiperone (SP: spiroperidol) by tail vein injection and 2 h later the brain was processed for light microscopic autoradiography. High densities of autoradiographic grains were found in all areas known to have a dopaminergic innervation, including the olfactory tubercles, nucleus accumbens, nucleus caudate-putamen, lateral septum, zona incerta, nucleus subthalamicus, arcuate nucleus, nucleus of the central amygdala, areas in the ventral tegmentum and the claustrum. There were also increased autoradiographic grain densities in other areas such as the midbrain and the frontal cortex indicating that binding occurred to other neurotransmitter receptors besides dopamine receptors. These studies delineate with a high resolution at an anatomical level the major binding sites for neuroleptic drugs in the forebrain. They suggest which areas of the brain are the most involved in neuroleptic drug action and they add further evidence that important regions are those receiving a dense dopaminergic innervation.     

Distribution of angiotensin II receptors in rat brain.

Angiotensin II binding activity of rat brain particles was examined using [125I]-angiotensin II (0.1-0.3 nM) in the presence and absence of excess unlabelled angiotensin II. Certain features of the binding suggested that physiological receptors were involved. The binding activity was temperature dependent and was increased 3-fold by the addition of 0.5 M EDTA. The binding appeared specific as judged by inhibition with angiotensin II agonists and antagonists. The "specific" binding was saturable, two-thirds reversible and occurred with high affinity. The equilibrium dissociation constant (Kd) of the "specific" binding was 0.9 nM. Subcellular fractionation studies indicated that over 90% of the binding was associated with particulate matter and was concentrated in the crude microsomal fraction. Binding was localized to the midbrain, thalamus, septum, hypothalamus and medulla; Very low levels of binding were found in the cortex, hippocampus and striatum; The lateral septum had the highest binding activity of all the tissues examined. Subdivision of the medulla showed that the highest binding activity was associated with the area postrema and medullary regions ventral to this organ.     

Acetylcholine receptors in cultured human muscle cells

Primary cultures prepared from human muscle biopsies were examined for the presence and distribution of acetylcholine receptors, as measured by the binding of ¹²⁵iodine-labelled α-bungarotoxin. The toxin bound to the human muscle cultures with a similar time dependence and specificity as found in muscle cultures from other species. The amount of toxin bound was lower than that obtained for neonatal mouse muscle under similar conditions.The distribution of receptors was similar in cultures derived from the muscles of patients with a variety of neuromuscular disorders. The toxin was located along the myotubes in a fairly even distribution; however, variations in labelling along a single myotube were observed, as well as variations between different myotubes in the same culture. Occasionally, the toxin also bound to other cells, which may have been monouncleated.Cultures prepared from patients with Duchenne muscular dystrophy produced multi-layered cell clusters, instead of the usual monolayer of cells. Within these clusters, only the myotubes bound α-bungarotoxin.     

Bilateral infarction of the anterior cingulate gyri and of the fornices: Report of a case

A 70-year-old woman had complex behavioural changes of sudden onset. The symptoms consisted of indifference, docility and inappropriate urination, but predominantly in a lack of attention. She was unable to maintain the attention necessary to perform a goal-directed activity and she was distractable by any stimulus, such as a sound, an object, or a word, which might induce behaviour irrelevant to the preconceived activity. She also exhibited a confabulatory-amnestic syndrome.Neuropathological examination of the brain revealed infarcts in the territories of both anterior cerebral arteries. The rostral part of the anterior cingulate gyrus (Acg), small areas of the adjacent medial prefrontal cortex, and the underlying white matter were destroyed bilaterally. Infarction involved the deep territory of the left anterior cerebral artery, with a bilateral lesion of the fornices.This cingulate damage was more restricted than the Acg lesions reported in some cases of akinetic-mutism, which extended more caudally, but was presumably larger than the lesions created in psychosurgery. The impairment of attention was analyzed according to the possible roles of the cingulate and of the fornix lesions as causing a dysfunction between the frontal lobes and the hippocampal formations.