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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.

We have studied the distribution of microtubule-associated protein 2 (MAP2) in the Purkinje cell dendrites of rats whose cerebella were exposed to X-irradiation during the second postnatal week. The Purkinje cells of such animals have abnormally elongated apical primary processes that branch in the other molecular layer rather than close to the cell body as in normal tissue. The results show that in these distorted dendrites the MAP2 distribution is "shifted" distally relative to the normal pattern, in which MAP2 is distributed evenly throughout the dendritic tree. Tubulin and other microtubule-associated proteins, such as MAP1, are not affected and remain evenly distributed throughout the dendritic tree despite the anatomical distortion. We conclude that the distribution of MAP2 in Purkinje cells is not determined solely by its binding to tubulin. Other factors must be involved and these appear to be related to dendritic morphology and possibly to branching.

Axons that normally will encounter either CNS or PNS glia have been shown to contain a powerful mitogen for both Schwann cells and oligodendrocytes. The normally nonmyelinated, nonglial ensheathed cerebellar granule cells have been shown to possess a proliferative signal for Schwann cells, suggesting that a glial mitogen is common to all axons. To determine if a glial mitogen capable of stimulating both Schwann cells and oligodendrocytes is colocalized on all types of axons we have (1) cocultured granule cells with oligodendrocytes, (2) incubated oligodendrocytes with granule cell membranes, and (3) evaluated the ability of heparin extracts of granule cell membranes, splenic nerve microsomes, and axolemma-enriched fractions isolated from rat and bovine CNS to stimulate mitosis of cultured oligodendrocytes. Neither the intact granule cells nor the granule cell membrane fraction stimulated cultured oligodendrocytes to divide. However, heparin extracts of the granule cell membranes were significantly mitogenic to the cultured oligodendrocytes. Heparin extracts of splenic nerve microsomes were more mitogenic than the comparable extract obtained from bovine CNS axolemma-enriched fractions. These results suggest that the neuronal mitogen for oligodendroglia is colocalized with the neuronal mitogen for Schwann cells.

The distribution of dopamine in the brain of the teleost Gasterosteus aculeatus L. was demonstrated with the indirect peroxidase-antiperoxidase immunohistochemical method using highly specific antibodies against a dopamine-glutaraldehyde-thyroglobulin conjugate. Dopamine-immunoreactive (DAir) neuronal somata were observed in all main brain regions. In the forebrain, DAir neurons were located in a continuous cell column extending from the caudal part of the olfactory bulbs to the preoptic area. The neurons lie lateral to the dorsal (and caudally to the subcommissural) portion of the ventral telencephalic area, and ventromedial to the central nuclei of the dorsal area. In the diencephalon, cerebrospinal fluid-contacting neurons were located in the paraventricular organ and in the subependymal layers of the dorsal and caudal zones of the periventricular hypothalamus. Small DAir neurons were observed in the suprachiasmatic nucleus, in the parvocellular preoptic nucleus and in the ventromedial thalamic nucleus, while large perikarya were observed dorsolateral to the dorsal zone of the periventricular hypothalamus ('PVO-accompanying cells'), in the posterior tuberal nucleus and in the most rostral portion of the mammillary bodies. Numerous small DAir neurons were located in the periventricular pretectal nucleus. In the brainstem, DAir neurons were observed in the isthmus region, in the dorsal raphe nucleus and in the lateral parts of the nucleus of the solitary tract. DAir perikarya were also observed in the area postrema. Direct comparison with the distribution of tyrosine hydroxylase- and dopamine-beta-hydroxylase-immunoreactivity (THir and DBHir) gave the following results: THir neurons were found in all areas where DAir neurons were located, except for the paraventricular organ and the dorsal and caudal zones of the periventricular hypothalamus, which were devoid of THir. DBHir (putatively noradrenergic or adrenergic) neurons were observed in the lateral parts of the nucleus of the solitary tract, and in the isthmus region. The DBHir neurons in the isthmus region, which have previously been shown to be noradrenergic, appeared to be identical with the THir and DAir neurons of the same area. DAir axons were found in high numbers in most parts of the brain. Especially dense innervation was found in the ventrolateral and posterior parts of the dorsal telencephalic area, the region surrounding the lateral recesses of the third ventricle, the interpeduncular nucleus, the dorsal and median raphe nuclei (the rostral raphe nuclei), and in the nucleus of the solitary tract.(ABSTRACT TRUNCATED AT 400 WORDS)

Serial frontal sections of male and female mouse hypothalamus were immunostained with an antiserum to oxytocin, in order to study the topographical distribution of oxytocinergic perikarya and processes. Numbers of immunostained perikarya were counted in various hypothalamic regions. The oxytocin content of microdissected hypothalamic tissue samples was measured in radioimmunoassays. While the overall topographical distribution of oxytocin neurons in the classical magnocellular nuclei was similar in both genders, quantitative differences could be observed. The numbers of immunostained perikarya and the amounts of oxytocin found in females exceeded by far the numbers and amounts found in males. Male mice had fewer oxytocin-immunostained axons, projecting within the brain, than females. This was especially apparent in parts of the limbic system. Oxytocin-immunostained neurons in the perifornical region, the lateral hypothalamus and the ventral ansa lenticularis were mostly absent in males. It is possible that the observed sex differences in oxytocin immunoreactive brain architecture are due to the different hormonal conditions in males and females.

We describe a method for the selective labelling, isolation and electrophoretic analysis of cell-surface molecules and extracellular matrix components. Intact tissues are reacted with activated esters of biotin and the labelled surface molecules identified on Western blots with horseradish-peroxidase-coupled or 35S-labelled streptavidin. Alternatively, the biotinylated proteins can be purified from tissue homogenates by affinity chromatography on an avidin-agarose column. Evidence is presented to show that this method is indeed specific for membrane and matrix components. Its practical application to embryonic neural tissues is demonstrated.

Using an immunosuppression technique, the monoclonal antibody 2A1 has been generated specific for a 140 x 10(3) Mr cytoplasmic-membrane-associated protein as shown by subcellular fractionation and Western blot analysis. The antigen is initially confined to perikarya of postmitotic migratory ganglion cells of the embryonic chick retina as revealed by bromodeoxyuridine labeling. During the subsequent period of axon outgrowth, the antigen becomes restricted to ganglion cell axons but disappears during the innervation of the tectum opticum, suggesting a tectal inhibition of antigen expression in retinal axons. To analyse whether the tectum suppresses 2A1-antigen expression, optic nerves of chick embryos were severed to prevent tectal innervation. 2A1-immunoreactivity was determined in deflected axons in comparison to control axons. In addition, retinal axons were grown in vitro on a substratum consisting of alternating stripes of laminin and tectal membranes, in order to investigate whether retinal axons become devoid of the 2A1-antigen once they cross from laminin to tectal membranes. However, neither prevention of target innervation by optic nerve transection in vivo nor exposure of retinal axons to soluble or particulate tectal components in vitro modify 2A1-antigen regulation in ganglion cell axons, suggesting a retina inherent-control of gene expression. Antigen expression is essentially restricted to the period of axonal outgrowth and therefore the 2A1-protein is likely to be involved in processes essential for neurite extension, independent of the synaptic target.

Titrimetric, 45Ca dialysis, and autoradiographic methods were used to examine how axoplasmic proteins from the giant neuron of the marine annelid Myxicola infundibulum bind calcium. Following the autoradiographic method of Maruyama et al., the 150-160 kD neurofilament subunits were identified as prominent intracellular Ca-binding peptides. Using equilibrium dialysis, extracts of axoplasmic proteins (greater than 50% neurofilament subunits) were examined in 300 mM KCl at different concentrations of free Ca and Mg, and at different pH. Axoplasmic proteins showed a high affinity Ca binding site (K1/2 3-6 microM, capacity 3-7 mumole g-1 protein) at pH 6.8 or pH 7.5. Changing the Mg concentration from 0 to 5 mM had no effect on the Ca binding. Elevating the dialysis pH from 7.0 to 9.0 reduced the apparent number of binding sites for Ca. Using microelectrodes to record the free Ca, microtitrations of axoplasmic proteins were completed by adding small amounts of CaCl2 to 100 microliters volumes of protein solutions. In a medium containing ionic constituents closely resembling those of the Myxicola axon, a Ca binding capacity of 5.0 mumole g-1 protein and a K1/2 of approximately 1 microM were measured.

The localisation of endogenous glutamate in the dragonet retina was investigated by light microscopic postembedding silver-enhanced immunogold labeling after incubation with an anti-glutamate antiserum. Rod and cone inner segments and synaptic terminals, as well as the inner plexiform layer, are moderately labeled. Bipolar cells and ganglion cell bodies show strong labeling. In the dorsal inner plexiform layer, the levels with square-patterned bipolar synaptic boutons can be identified by their prominent glutamate-immunoreactivity. These results support the idea that the majority of the neurons that constitute the direct, centripetal pathways through the retina use glutamate as their neurotransmitter.

The effect of excitatory amino acid stimulation on the cytoskeleton of cultured spinal cord and cortical neurons was monitored with antibodies against microtubule-associated proteins tau and MAP2. In unstimulated cultures tau-1 immunoreactivity was restricted to axon-like processes. Stimulation with glutamate (0.1-1 mM) or N-methyl-D-aspartate (NMDA) (0.1 mM) resulted in a dramatic increase in the intensity of tau labelling in axons and the appearance of staining within a proportion of neuronal cell bodies and dendrites. Quisqualate or kainate stimulation resulted only in an increase in tau immunoreactivity within axons. The NMDA mediated events were calcium dependent and the effects of all excitatory amino acids could be blocked by specific antagonists. In contrast, following stimulation with excitatory amino acids, MAP2-immunoreactivity was associated with filaments which formed a complex network within the cell body. This suggests that the different excitatory amino acid receptor subtypes can have differential effects on the neuronal cytoskeleton.

The early development of interneurons in the chick embryo spinal cord was studied using a monoclonal antibody against a neuron-specific beta-tubulin isoform. Early developing interneurons were divided into two cell groups on the basis of their location and the pattern of growth of their axons. One group is composed of cells that establish a primitive longitudinal pathway (PL-cells), whereas the other group contains cells constituting a circumferential pathway (C-cells). The onset of axonal development in both cell groups occurs at stage (st.) 15 (embryonic day, (E), 2) in the branchial segments, which is prior to axonogenesis of motoneurons. PL-cells develop in the region between the floor plate and the motoneuron nucleus. Their axons are the first neuronal processes ('pioneer axons') to arrive in the ventrolateral marginal zone and they project both rostrally and caudally to establish a primitive longitudinal association pathway at the ventrolateral surface of the neural tube. This pathway is formed before axons of C-cells arrive in the ventrolateral region. The first C-cells are initially located in the most dorsal portion of the neural tube, whereas later appearing C-cells are also located in both intermediate and ventral regions of the neural tube. The axons of C-cells project ventrally, without fasciculating, along the lateral border of the neural tube. Some of their axons enter the ipsilateral ventrolateral longitudinal pathway at st. 17. We often observed apparent contacts and interactions between preexisting axons of PL-cells and newly arriving axons of C-cells. The axons of commissural C-cells first enter the floor plate at st. 17 and cross the midline at st. 18. Axons of C cells begin to join the contralateral ventrolateral longitudinal pathway at st. 18+ to st. 19. In the floor plate region, contacts between growth cones and axons were often observed. However, axons in the floor plate at these stages were not fasciculated. These observations establish the timing and pattern of growth of axons from two specific populations of early developing interneurons in the chick spinal cord. Additionally, we have identified an early and apparently previously undescribed 'pioneer' pathway that constitutes the first longitudinal pathway in the chick spinal cord.

A procedure is described to isolate a fraction enriched in cerebellar granule cell neuritic membranes. Morphological markers that are specific for either the granule cell perikarya or neuritic membranes have been identified. Concanavalin A (Con A) has been shown to bind predominantly to the granule cell neurites whereas, the enzymes acetylcholinesterase (AChE) and 2',3',cyclic nucleotide-3'-phosphohydrolase (CNPase) are localized predominantly in the neuronal cell bodies. The membrane fraction enriched in Con A binding has been used to generate a monoclonal antibody which morphologically recognized the cerebellar granule cell neuritic membrane. Following fractionation of the granule cells, each marker was used to identify the cellular origin of the fractions. The neuritic markers Con A and the neuritic membrane antibody MR2 bound predominantly to membranes found in the 29.1% and 31.5% region of the sucrose gradient. The perikaryal markers, CNPase and AChE activity were most enriched in membrane fractions found at a sucrose concentration of 23% and 21%, respectively. Morphological examination of the neuritic enriched fraction shows that it contains predominantly membranous material with few subcellular organelles. The protein profiles of the cerebellar granule cell fractions are unique when compared with the protein profiles of other neuronal and non-neuronal fractions. The membrane fraction isolated from the cerebellar granule cells should prove useful in furthering our understanding of the axonal influence on glial development.

The confocal scanning laser microscope (CSLM) offers improved optical resolution and contrast, high photometric precision, and the ability to make optical sections. These benefits were explored for use in quantitative analysis of immunofluorescence-labeled axon terminals. Guidelines were obtained for adjustments of the CSLM parameters. In the present applications, bleaching of the fluorescence did not represent a serious obstacle to analysis with the CSLM. A method was developed to distinguish the background fluorescence from the specific fluorescence labeling. This procedure made way for the development of automated quantification of immunolabeled axon terminals. The automated procedures substantially reduced the man-hour expenditure for analysis and provided highly reproducible quantifications compared with manual methods. The increased resolution and contrast of the CSLM allowed measurements of the fluorescence signal strength of individual axon terminals. The CSLM also allowed detection of co-localized neurochemicals in axon terminals.

In five species of lungless salamanders, family Plethodontidae, which all show highly developed visual abilities, the ultrastructure of the optic nerve was investigated and the total number of retinal ganglion cell axons, the percentage of myelinated axons, and the volume densities of glia and axons were determined. More than 80% of all axons were smaller than 0.4 micron and only 2-3% were larger than 0.8 micron. In individual nerves the degree of myelination varied between 1 and 9% which is in the range reported for other amphibian species. The miniaturized and highly paedomorphic species Batrachoseps attenuatus was an exception because only very few or even no myelinated axons were present in the nerve, which is unique among gnathostome vertebrates. The five investigated species had total numbers of axons ranging from 26,000 in Batrachoseps attenuatus to about 50,000 in Plethodon jordani. These numbers are the lowest found among vertebrates with an elaborated visual system. The amount of glial material in the optic nerve varied between 25 and 50%, with larger nerves possessing more glia than smaller ones. Ultrastructural analysis revealed that the optic nerve of each species contained both astrocytes and oligodendrocytes, although often in immature form. In Batrachoseps attenuatus the glia showed features of both astrocytes and oligodendrocytes which reflect an undifferentiated state.

The adrenergic innervation of somatostatin synthesizing neurons located in the anterior region of the rat hypothalamic periventricular nucleus was studied by means of a light and electron microscopic immunocytochemical double labelling technique. This region which is the source of hypophysiotrophic somatostatin immunoreactive (IR) neurons also receives a dense plexus of adrenergic axons as determined by immunocytochemistry of phenylethanolamine-N-methyltransferase (PNMT), the marker enzyme for the central adrenergic system. The simultaneous detection of PNMT and somatostatin antigens in hypothalamic sections of colchicine pretreated animals revealed a congruency in the distribution of the labelled elements and also close juxtaposition of PNMT-IR axons to somatostatin producing neurons. At the ultrastructural level, axo-somatic and axo-dendritic synaptic connections were found between PNMT-containing axons and somatostatin expressing neurons. These morphological findings support the view that the central adrenergic system might influence the production and secretion of growth hormone in the pituitary gland by a direct monosynaptic interaction with somatostatin synthesizing neurons.

We have examined neuronal differentiation and the formation of axon tracts in the embryonic forebrain and midbrain of the zebrafish, between 1 and 2 days postfertilisation. Axons were visualised with three techniques; immunocytochemistry (using HNK-1 and antiacetylated tubulin antibodies) and horseradish peroxidase (HRP) labelling in whole-mounted brains, and transmission electron microscopy. Differentiation was monitored by histochemical staining for acetylcholinesterase (AChE). These independent methods demonstrated that a simple grid of tracts and commissures forms the initial axon scaffold of the brain. At 1 day, the olfactory nerve, four commissures, their associated tracts and three other non-commissural tracts are present. By 2 days, these tracts and commissures have all greatly enlarged and, in addition, the optic nerve and tract, and three new commissures and their associated tracts have been added. Small applications of HRP at various sites revealed the origins and projections of some of these earliest axons. Retrogradely labelled cell bodies originated from regions that were also positive for AChE activity. At 1 day, HRP-labelled axons were traced: (1) from the olfactory placode through the olfactory nerve to the dorsal telencephalon; (2) from the telencephalon into the tract of the anterior commissure and also to the postoptic region of the diencephalon; (3) from the hindbrain through the ventral midbrain and diencephalon to the postoptic commissure; (4) from the dorsal diencephalon (in or near the epiphysis) to the tract of the postoptic commissure; (5) from ventral and rostral midbrain through the posterior commissure. Three new projections were demonstrated at 2 days: (1) from the retina through the tract of the postoptic commissure to the tectum; (2) from the telencephalon to the contralateral diencephalon; and (3) from the telencephalon to the ventral flexure. These results show that at 1 day, the zebrafish brain is impressively simple, with a few small, well-separated tracts but by 2 days the brain is already considerably more complex. Most of the additional axons added onto pre-existent tracts rather than pioneered new ones supporting the notion that other axons play a crucial role in the guidance of early central nervous system (CNS) axons.

By means of immunochemical method the presence of vasopressin in the melanocytes of the man's brain arteries was found. The terminals of the cholinergic axons come to an end on the surface of these cells. It is suggested that the melanocytes take part in the regulation of the brain arteries mobility.

The electron microscopic investigation was performed to analyze somatostatin-contained nerve terminals in the median eminence of 21 days old malnourished rats' hypothalamus. In nerve terminals of malnourished animals in compared with controls ones there was found the increased density of granular vesicles (11.62 +/- 0.40 and 8.56 +/- 0.39 in 1 micron2, respectively) and decreased density of electron lucent vesicles with 120-160 nm diameter (1.66 +/- 0.18 and 3.43 +/- 0.26 in 1 micro2, respectively). The revealed increase in density of granular vesicles in axon terminals with positive immunohistochemical reaction to somatostatin in malnourished rats was explained by slow somatostatin release.

Axons in the duodenum, ileum and rectum of the domestic fowl were identified as catecholamine-containing (CA) on the basis of positive reactivity following chromaffin fixation for electron microscopy. CA-axons in association with blood vessels in all regions of the intestine and in non-vascular sites in the small intestine had a 'typical' adrenergic appearance, in that they contained many small granular vesicles (SGV) and variable numbers of large granular vesicles (LGV). In the rectum the non-vascular CA-axon profiles were atypical, in that there were many elongated LGV and few SGV, and the chromaffin reactivity was weak. The nerve profiles in the rectum were dramatically reduced following 6-hydroxydopamine and reserpine treatment and were absent in rectum cultured in the absence of extrinsic ganglia. It was concluded that the profiles, in spite of their low chromaffin reactivity, truely represent CA-axons. The possibility was raised that the atypical morphology and reduced chromaffin reactivity is due to the presence of adrenaline.

Antisera to two putative neurotransmitters, luteinizing hormone-releasing hormone (LHRH) and molluscan cardioexcitatory tetrapeptide (H-Phe-Met-Arg-Phe-NH2; FMRF-amide), bind specifically to neurites in the inner nuclear and inner plexiform layers of the goldfish retina. Retrograde labeling showed that intraocular axon terminals originate from the nervus terminalis, whose cell bodies are located in the olfactory nerves. Double immunocytochemical and retrograde labeling showed that some terminalis neurons project to the retina; others may project only within the brain. All terminalis neurons having proven retinal projections were both LHRH- and FMRF-amide-immunoreactive. The activity of retinal ganglion cells was recorded with microelectrodes in isolated superfused goldfish retinas. In ON- and OFF-center double-color-opponent cells, micromolar FMRF-amide and salmon brain gonadotropin-releasing factor ( [Trp7, Leu8] LHRH) caused increased spontaneous activity in the dark, loss of light-induced inhibition, and increased incidence of light-entrained pulsatile response. The nervus terminalis is therefore a putatively peptidergic retinopetal projection. Sex-related olfactory stimuli may act through it, thereby modulating the output of ganglion cells responsive to color contrast.

The polyene antibiotic filipin reacts specifically with membrane cholesterol and produces distinctive membrane lesions. We treated frog cutaneous and sartorius muscles with 0.04% filipin in a glutaraldehyde solution with or without prefixation with glutaraldehyde. Freeze-fracture of these muscles revealed numerous 19 to 38-nm protuberances and depressions (filipin-sterol complexes) in most areas of muscle, axon, and Schwann cell membranes. In the presynaptic membrane, however, these filipin-sterol complexes were absent from active zones consisting of ridges bordered with double rows of particles. In the postsynaptic membrane, filipin-sterol complexes were also virtually absent from the areas occupied by aggregates of large particles representing acetylcholine receptors. These results suggest that the membrane regions of active zones and acetylcholine receptor aggregates have a low cholesterol content.

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.

High-voltage electron microscopy (HVEM) has been used to determine the distribution of cationic precipitates in myelinated axons resulting from the application of two cytochemical techniques: a direct osmium pyroantimonate treatment for precipitating Na+, Ca2+ and Mg2+; and a 5 mM Ca2+ inclusion procedure (Oschman & Wall) for imparting electron density to Ca2+ binding sites. Electron probe wavelength spectroscopy was then used on semi-thick tissue sections to identify the species of ions present in the following regions: Schwann cell paranodal loops, axoplasm at the node, compact myelin and extracellular matrix. With these combined procedures we were able to localize elevated concentrations of both Na+ and Ca2+ to cytoplasmic compartments of the Schwann cell paranodal loops, as well as to detect the presence of Ca2+ at elevated levels in compact myelin. The involvement of the Schwann cell paranodal loops in providing a source and/or sink for Na+ involved in impulse conduction is suggested by these results, and the significance of such a role is discussed. A role for Ca2+ in the formation and stabilization of myelin lamellae is also suggested.

Rat and mouse CNS neurofilament proteins (NFPs) were characterized and compared, in terms of electrophoretic properties on polyacrylamide gels and by peptide mapping, with one another and with other co-purifying lower-molecular-weight CNS proteins, including alpha and beta tubulin. NFPs were partially purified by modification of the axon flotation procedure of Norton and co-workers and were demyelinated with Triton X-100. On one-dimensional SDS polyacrylamide gels the molecular weights of the triad of NFPs from both rat and mouse were approximately 200,000, 140,000, and 70,000. Prominent lower-molecular-weight proteins (63,000-16,000) as well as minor amounts of tubulin and actin were observed after gel electrophoresis. On two-dimensional gels (isoelectric focusing followed by SDS gel electrophoresis) each of the NFPs appeared to be composed of more than one component and the corresponding NFPs from rat and mouse had similar isoelectric points. Gel electrophoresis peptide mapping using Staphylococcus aureus V8 protease indicated the following: (1) the triad of NFPs of different sizes have different peptide maps; (2) alpha and beta tubulin have nonidentical digestion products, which are dissimilar to those of the NFPs; (3) other proteins that co-purify by the axon flotation procedure also have nonidentical peptide maps; and(4) the corresponding NFPs from rat and mouse have similar peptide maps. The co-purifying proteins examined in detail (63,000-49,000) do not appear to be derived by proteolytic cleavage of NFPs and may represent other cytoskeletal constituents.

The effects of Leu-enkephalin, Met-enkephalin and morphine on the electrical activity of the internal anal sphincter were studied in anesthetized spinalized cats and in vitro on sphincteric muscle strips. All the effects of enkephalins and morphine were antagonized by naloxone (2 mg/kg, i.v. in vivo and 10(-6)M in vitro). In vivo, the enkephalins (0.01 mg/kg i.v.) and morphine (2 mg/kg, i.v.) decreased the amplitude of the excitatory responses evoked in the sphincter by stimulation of the hypogastric nerves. Opiates presumably act on the sympathetic nerve endings by reducing the release of noradrenaline. In vitro, the enkephalins (10(-6)M) and morphine (10(-6)M) had a similar inhibitory effect, indicating that opiates act, at least partly, at intramural level. In vivo, the enkephalins and morphine produced an inhibition of the spontaneous electrical activity of the internal anal sphincter. This inhibition occurs also in vitro; it is thus due to a peripheral effect of opiates acting either directly on the sphincteric smooth muscle cells, or through the nervous structures controlling sphincteric motility. In addition, the distribution of nerves containing enkephalin-like immunoreactivity, using whole mount preparations of cat internal anal sphincter, indicates that this area is supplied with a dense Leu- and Met-enkephalinergic innervation. Met- and Leu-enkephalin-like immunoreactive axons were detected within the circular and longitudinal muscles.

By immunocytochemical and biochemical techniques, we observed the localization and expression of pp60c-src in nerve growth factor (NGF)-treated PC12 cells. Immunostaining of pp60c-src is detected in the neuronal soma and the tips of neurites (growth cones). Immunofluorescence in the neurites is less significant. High-resolution microscopy reveals that the location of pp60c-src in growth cone is in good agreement with the adhesive site of growth cone to the substratum. The pp60c-src kinase activity and the pp60c-src protein level increase 3.1- to 3.5-fold and 2.0-fold during differentiation of PC12 cells, respectively. The pp60c-src levels in the neurite fraction are also higher than those in the neuronal soma fraction. These results support the immunocytochemical finding that pp60c-src is localized in growth cones of differentiated PC12 cells. Furthermore, we discuss the possible role of pp60c-src in growth cone.

The proteins of axons prepared from myelinated axons and isolated as myelin-free entities were separated by sodium dodecyl sulfate polyacrylamide electrophoresis and found to consist of more than 10 different molecular weight species. The molecular weights range from 13 000 to over 200 000 with a prominent protein of molecular weight 47 000. The amino acid composition of the seven major proteins showed that the protein with a molecular weigt of 47 000 is distinct from all the other proteins analyzed. A group of three low molecular weight proteins have amino acid compositions which are similar to each other as do a group of three high molecular weight proteins although the two groups are distinctly different from each other and the major axonal protein. Histones, DNA, myelin basic protein and glycoprotein were absent from the proteins but neurotubule protein was present as indicated by cochicine binding activity in the axonal preparations. The cellular origin of these proteins and their relationship to other central nervous system proteins are discussed.

This report presents the ultrastructural nerve study of a patient with sensorimotor neuropathy by a shoe glue containing an association of n-hexane and methylethylketone. Giant axons, distended by microfilamentous proliferation typical of such cases of neuropathy, were found in significant amounts. An unusual histological feature of this case was the considerable distension by a marked accumulation of glycogen granules in certain unmyelinated axons. Such a lesion has never been described in neuropathy induced by industrial agents.

1. Axoplasm from Myxicola contains two major polypeptides associated with neurofilaments, together with actin, tubulin and many minor polypeptide components. 2. Some of the minor polypeptides with molecular weights between 140,000 and 50,000 purify with neurofilaments under a variety of conditions and they appear to represent an integral part of the filament structure. 3. Peptide fingerprinting shows that the two major neurofilament polypeptides are almost identical. The fingerprint patterns from these major polypeptides share features with those obtained from the minor components. 4. Peptide fingerprinting has enabled us to propose a scheme for the main sites at which papain cleaves the major neurofilament polypeptides. In addition fingerprinting indicates how the minor components are related to the major polypeptides. 5. It is suggested that many of the minor neurofilament polypeptides could arise by proteolysis in vivo.

The onset of hormone synthesis in LH-RH and somatostatin neurons of the brain was studied in rats using the unlabelled antibody-PAP immunohistochemical method. LH-RH appeared in detectable amounts first in neuronal perikarya in the preoptic region of fetuses sacrificed on the 17th day of gestation. Minute amounts of LH-RH could already be detected in some nerve fibers of the preoptic region of fetuses on the 18th gestational day. On the 19th day the vascular organ of the lamina terminalis (OVLT) and on the 20th day the median eminence (ME) already contained loose networks of nerve fibers and axon terminals accumulating LH-RH. Somatostatin perikarya and axon terminals with detectable amounts of the hormone appeared simultaneously on the first postnatal day in the anterior periventricular region and the median eminence, respectively. Somatostatin containing nerve fibers became detectable in OVLT first on the 2nd day of life.

Using an immunoperoxidase technique at the ultrastructural level, vasopressin (VP) and neurophysin (NP) were localized in the axons of the median eminence of normal and adrenalectomized rats. Whereas VP and NP were generally found in 150-nm granules in the internal zone of the normal rat median eminence, in the adrenalectomized rat, many granules of about 80 to 100 nm in diameter were found to contain NP and VP in the external zone of the median eminence.

Squid giant axons were injected with aequorin or arsenazo III and impaled with a Ca-sensing electrode. The light output of aequorin or the spectrophotometer output when measuring arsenazo was compared with the voltage output of the electrode when the squid axon was depolarized with high-K solutions, when the seawater was made Na-free, or when the axon was tetanized for several minutes. The results from these treatments were that the optical response rose (as much as 50-fold) with all treatments known to increase Ca entry, while the electrode remained unaffected by these treatments. If axons previously subjected to Ca load are treated with electron-transport poisons such as CN, it is known that [Ca]i rises after a time necessary to deplete ATP stores. In such axons one expects a rise of [Ca]i in axoplasm which does not necessarily have to be uniform although the source of such Ca is the mitochondria and these are uniformly distributed in axoplasm. Under conditions of CN application, the optical signals from aequorin or arsenazo and Ca electrode output do rise together when [Ca]i is high, but there is a region of [Ca]i concentration where aequorin light output or arsenazo absorbance rises while electrode output does not. Axons not loaded with Ca but injected with apyrase and vanadate have mitochondria that still retain some Ca and this can be released by CN in a truly uniform manner. The results show that such a release (which is small) can be readily measured with aequorin, but again the Ca electrode is insensitive to such [Ca]i change.

The properties of a new fluorescence histochemical method for arylethylamines based on reaction with a mixture of 4% formaldehyde and 0.5% glutaraldehyde in aqueous solution are described. At room temperature the aldehyde mixture produced a well-localized fluorescence reaction in tissues, which, when examined microscopically in aqueous solution, was sufficiently intense for fine terminal noradrenergic axons to be seen. If the tissue was subsequently dried, the fluorescence intensity increased. At the same time as inducing the fluorophores, the aldehyde mixture fixed the tissue to a standard well suited for electron microscopy. It thus proved possible to locate amine containing cells in the fluorescence microscope and subsequently examine their ultrastructure. In aqueous models, the aldehyde mixture formed fluorescent products with adrenaline, noradrenaline, dopamine, dopa, 5-hydroxytryptamine and 5-hydroxytryptophan, but not with histamine or octopamine. The fluorescence induced in the aldehyde mixture remained stable if the tissue was subsequently transferred to saline or distilled water and when it was dehydrated in ethanol and cleared with xylene, benzene, chloroform or acetone.

A bright yellow-green specific fluorescence is induced by formaldehyde histochemistry for monoamines in the secretory nerve trunks of the Octopus vulgaris posterior salivary duct, and in their ramification in the gland tubules. In contrast, the motor nerve trunks of the duct contain few fluorescent elements. The muscular and connective coat of the duct is provided with fluorescent globular and varicose structures, of various sizes and colours, which become numerous in the duct branches. At least some of these peripheral structures belong to varicose monoamine nerve fibres. In the gland, on the contrary, the muscle cells surrounding the tubules are not supplied with fluorescent nerve fibres.

Cytoskeletal proteins (e.g., tubulin, actin, and neurofilament proteins) in the squid giant axon are separable into KF-soluble and -insoluble forms. The KF-insoluble cytoskeletal components appear to constitute the major proteins in the subaxolemmal fibrous network on the inner surface of the axon. These cytoskeletal proteins and the subaxolemmal network are both highly soluble in KI solutions. Whereas giant axons tolerate prolonged perfusions in KF solutions with no loss of excitable properties, a relatively short perfusion with KI solution completely eliminates the excitability of the axon. The loss of this excitability correlates with the simultaneous dissolution of the subaxolemmal network of cytoskeletal proteins and the release of its proteins into the perfusate. These data support the hypothesis that cytoskeletal proteins associated with the inner surface of the axolemma are involved in the regulation of axonal excitability.

Oxytocin- and vasopressin-immunoreactive nerve fibers, apparently originating from a dorsal subunit of the paraventricular nucleus, were demonstrated in the pineal gland of the hedgehog. The majority of these fibers (pinealopetal projections) is intimately related to the capillaries of the pineal organ, whereas only a few elements are scattered throughout the pineal parenchyma. The number of peptidergic elements observed in the central portion of the pineal organ exceeds that of fibers located at the periphery. In relation to the functional state of the animals, the amount of immunoreactive material in these pinealopetal nerve fibers exhibits conspicuous variations. In hibernating hedgehogs (group 1), these nerve fibers were considerably richer in oxytocin than in non-hibernating or arousing winter animals (group 2 and 3). In contrast, only weak immunoreactivity for vasopressin was found in intrapineal nerve fibers of hibernating hedgehogs (group 1), whereas the fibers of arousing or non-hibernating hedgehogs (group 2 and 3) contained slightly larger amounts of vasopressin. In the pineal organ of animals sacrificed during the summer period (group 4), no immunoreactivity for both neuropeptides was found. The functional significance of the connection between the hypothalamic paraventricular nucleus and the pineal organ is discussed with special reference to the vascular terminals of the pinealopetal peptidergic nerve fibers.

A study was made of the distribution of Substance P-immunoreactive fibers in chick cornea and uvea in whole mount preparation, using the indirect immunofluorescence technique. The development of these fibers during embryogenesis was also investigated. SP-fibers were present in all chick eye structures, in the various prenatal and postnatal stages examined. Their distribution was comparable with that observed by other workers in mammals. Transformation of the iris musculature from smooth to striated, during development, is not accompanied by significant changes in SP-ergic innervation.

The innervation of the gut of the venerid bivalve mollusc, Chione stutchburyi, has been examined by fluorescence histochemistry, electron microscopy and autoradiography. Specific green and yellow varicose fluorescent fibres indicate the presence of dopaminergic and serotonergic axons, respectively. Three different types of axons can be distinguished by the morphological characteristics of their vesicles. Type I axons contain predominantly small granular vesicles (average diameter 65 nm), Type II axons possess large granular vesicles (average diameter 100 nm) and Type III axons contain large opaque vesicles (average diameter 150 nm). The granular vesicles in both Types I and II axons react positively to dichromate, and their granularity is reduced by reserpine indicating that they are monoaminergic. Only Type I axons accumulate tritiated dopamine and are selectively damaged by 6-hydroxydopamine. It is concluded that Type I axons are dopaminergic. Type II axons are serotonergic: they alone take up tritiated 5-hydroxytryptamine, and 5,7-dihydroxytryptamine selectively causes degenerative changes in these axons. Type III axons contain an unidentified neurotransmitter substance. The large opaque vesicles of these axons do not react to dichromate and are unaffected by reserpine, 6-hydroxydopamine or 5,7-dihydroxytryptamine.

The auditory and vestibular sense-organs of the rabbit were examined for the presence of substance P(SP) by an immunohistochemical technique using a monoclonal SP-antibody. Between 30 and 40 per cent of the cells of the vestibular ganglia and about 50 per cent of the spiral ganglion cells innervating the extreme basal part of the cochlea showed SP-like immunoreactivity. The neural elements in the vestibular sensory epithelia, notably the calyx-formed nerve terminals of type I sensory cells of the maculae, also showed strong SP-like immunoreactivity. The findings suggest the possibility of a modulator or transmitter role for SP in the inner ear function.

Immunocytochemistry with antibodies against cytoskeletal proteins has been used to search for molecular differences in the spinal cord from patients with motor neuron disease (MND) of amyotrophic lateral sclerosis type and normal spinal cord. Monoclonal antibodies which recognize phosphorylated neurofilament epitopes diffusely labelled a proportion of normal and MND anterior horn cells, but did not permit differentiation between normal and MND tissue. However, in some MND and control anterior horn cells, dense 'floccular' accumulations were labelled by antibodies recognizing phosphorylated neurofilament epitopes. These accumulations of phosphorylated neurofilaments suggest abnormalities of cytoskeletal regulation, but were neither a common nor a specific feature of MND. Axonal spheroids, which were as common in normal as in MND tissue, were labelled by all antineurofilament antibodies. Normal-appearing axons, but not spheroids, in MND and control tissue were identified by an antiactin antibody, indicating that actin may be absent from the cytoplasmic domain which gives rise to spheroids. In summary, we have not found specific posttranslational changes of cytoskeletal proteins in MND and, in particular, phosphorylated neurofilament epitopes are common to both MND and control anterior horn cells.

Immunocytochemical analysis of the distribution of dynorphin B terminals in the sacral spinal cord of the cat revealed a pattern of staining very similar to that produced with antisera directed against the primary afferent derived, putative neurotransmitter, vasoactive intestinal polypeptide. Labeled axons and terminals were concentrated in lamina I and V and there was dense fiber staining in the tract of Lissauer. Of particular interest was the presence of immunoreactive axons in attached dorsal rootlets. To specifically focus on the possibility that some of the sacral primary afferent fibers are dynorphin-immunoreactive, we first tried to increase perikaryal labeling in the sacral dorsal root ganglia by topical treatment with colchicine. This did not produce immunoreactive labeling of cell bodies in the ganglia. Unilateral multiple dorsal rhizotomy (L5 to coccygeal 1), however, significantly decreased the staining of dynorphin-immunoreactive axons and terminals in the tract of Lissauer and in the dorsal horn of sacral segments ipsilateral to the deafferentation. No changes were detected in the lumbar cord. Finally, radioimmunoassay of caudal lumbar and sacral dorsal root ganglia was performed. Measurable immunoreactivity was found in all ganglia assayed, but, consistent with the histochemical analysis, sacral ganglia contained the highest concentration of immunoreactive dynorphin B. These data indicate that a significant component of the sacral spinal cord dynorphin terminal immunoreactivity derives from primary afferent fibers.(ABSTRACT TRUNCATED AT 250 WORDS)

The superior cervical ganglia (SCG), celiac superior mesenteric ganglia (CMG), and splanchnic nerve of unoperated guinea pigs, as well as both proximal and distal stumps of a previously transected branch of the postganglionic plexus of the CMG, were immunostained for somatostatin (SS). In addition, the PAP technique was adapted for fine-structural visualization of SS. A greater proportion of cells were labeled for SS in the CMG than in the SCG. PAP molecules were present in one type of intraganglionic axons. Only two labeled axons were found in the splanchnic nerve. Neither proximal nor the distal stump of the transected CMG postganglionic nerve contained labeled axons. The present results support the hypothesis that the intraganglionic axons labeled for SS arise from SS-containing intraganglionic neurons.

Following immobilization stress the supraoptic nucleus exhibits an increased number of coarse, heavily immunostained fibers in the basal glia labyrinth. Ultrastructural immunocytochemistry demonstrates a labeling of the rough endoplasmic reticulum in the neurosecretory perikarya and granule-free, immunoreactive material in the axons adjacent to the basal glia labyrinth. Furthermore, a labeling of the intercellular clefts of the neuropil is demonstrable in the supraoptic nucleus. These results lead to the hypothesis that 1) vasopressin is synthesized and released in two forms, in a granule-bound form and in a granule-free, probably more soluble form, and that 2) the latter might be released already in the nuclear area into the intercellular clefts from where it may reach its target cells via the cerebrospinal fluid of the subarachnoid space.

In the preceding paper (Salzer et al., 1980, J. Cell Biol. 84:753--766), evidence was presented that a neurite membrane fraction could be used to stimulate Schwann cell proliferation in culture. In this study, we present evidence that the mitogenic signal by which intact neurites or neurite membranes stimulate Schwann cell proliferation is located at the neurite surface. This conclusion is based on the following observations: (a) stimulation of Schwann cell proliferation by neurons requires direct contact between neurites and Schwann cells, separation of the two cells by a permeable collagen diaphragm 6 microns thick prevents Schwann cell proliferation; (b) treatment of intact neurites with trypsin before preparation of neurite membranes abolishes the ability of these membranes to be mitogenic for Schwann cells; and (c) the mitogenic activity of neurite homogenates is exclusively localized in the particulate rather than the soluble fraction of the homogenate. The mitogenic component on the neurite surface is heat labile, and is inactivated by aldehyde fixation. Preliminary data suggest that the mitogenic effect of neurite on Schwann cells is not mediated by 3',5'-cyclic AMP.

N-Acetylaspartylglutamate like immunoreactivity (NAAG-L) was identified in retinal ganglion cell bodies and their axons. The presence of the dipeptide in ganglion cell projection areas, the lateral geniculate nucleus (LGN) and superior colliculus (SC), was confirmed following NAAG purification from these tissues by a high-performance liquid chromatographic method. NAAG-L was identified in the optic tract as well as within fibers and puncta in the LGN and SC. The hypothesis that NAAG is present within ganglion cell axons in the brain was tested by unilateral enucleation which resulted in loss of NAAG and NAAG-L within the contralateral LGN and SC.