Neuronal intermediate filament expression in rat dorsal root ganglia sensory neurons: an in vivo and in vitro study

Qualitative and quantitative examination was performed to evaluate the expression of peripherin and 200 kDa neurofilament in the sensory compartment of the peripheral nervous system of the rat both in vivo and in a new in vitro model. Under physiological conditions, these two neuronal intermediate filaments show different expression patterns in sensory neurons. To have a more complete comprehension of the role of these intermediate filaments and to fill in the blanks left in previously reported literature, we demonstrate in vivo using a morphological approach that peripherin and 200 kDa neurofilament define two distinct subpopulations within the dorsal root ganglia sensory neurons. Moreover, peripherin is specifically expressed in unmyelinated fibers while 200 kDa neurofilament is expressed in myelinated fibers. Additionally, in vitro analysis of RNA taken from dorsal root ganglia explants suggested that 200 kDa neurofilament is downregulated and peripherin is transiently expressed throughout sensory fiber regrowth. In particular, both neuronal intermediate filaments are downregulated immediately after sensory fiber axotomy thus suggesting that neither peripherin nor 200 kDa neurofilament has a role in the first steps of fiber regrowth. However, the upregulation of peripherin a few days after the beginning of fiber regrowth in vitro suggests that low levels of peripherin may be require to carry on the sequence of events involved in the correct regeneration and direction of sensory fibers.     

New insights into peripherin expression in cochlear neurons

Peripherin is an intermediate filament protein that is expressed in peripheral and enteric neurons. In the cochlear nervous system, peripherin expression has been extensively used as a differentiation marker by preferentially labeling the type II neuronal population at adulthood, but yet without knowing its function. Since the expression of peripherin has been associated in time with the process of axonal extension and during regeneration of nerve fibers in other systems, it was of interest to determine whether peripherin expression in cochlear neurons was a static phenotypic trait or rather prone to modifications following nerve injury. In the present study, we first compared the expression pattern of peripherin and beta III-tubulin from late embryonic stages to the adult in rat cochlea. The staining for both proteins was seen before birth within all cochlear neurons. By birth, and for 2 or 3 days, peripherin expression was gradually restricted to the type II neuronal population and their projections. In contrast, from postnatal day (P) 10 onwards, while the expression of beta III-tubulin was still found in projections of all cochlear neurons, only the type I population had beta III-tubulin immunoreactivity in their cell bodies. We next investigated the expression of peripherin in axotomized cochlear neurons using an organotypic explant model. Peripherin expression was surprisingly re-expressed in a vast majority of neurons after axotomy. In parallel, the expression and localization of beta III-tubulin and peripherin in dissociated cultures of cochlear neurons were studied. Both proteins were distributed along the entire neuronal length but exhibited complementary distribution, especially within the projections. Moreover, peripherin immunoreactivity was still abundant in the growth cone, whereas that of beta III-tubulin was decreasing at this compartment. Our findings are consistent with a model in which peripherin plays an important structural role in cochlear neurons and their projections during both development and regenerative processes and which is compatible with the assumption that frequently developmentally regulated factors are reactivated during neuronal regeneration.     

Microtubule-associated protein-2 and neurofilament immunoreactivity in neurons and small, intensely fluorescent cells of an amphibian cardiac ganglion.

The localization of two cytoskeletal proteins was analysed in the cell bodies and processes of ganglionic neurons and small, intensely fluorescent cells of the parasympathetic cardiac ganglion of Necturus maculosus (mudpuppy). Antibodies against microtubule-associated protein-2 and against the highly phosphorylated isoforms of high and middle molecular weight neurofilament subunits were used as somatodendritic and axonal markers, respectively. The ganglionic neurons, which usually have only one major process, and small, intensely fluorescent cells, which have several processes, showed distinctly different staining patterns with the two antibodies. In control and denervated ganglia, the ganglionic cell bodies and several hundred micrometers of the proximal processes were labeled with the antibody against microtubule-associated protein-2, whereas small, intensely fluorescent cells and processes showed a paucity of immunoreactivity. The neurofilament antibody labeled numerous axons in the ganglion but did not label the proximal part of the postganglionic process or small, intensely fluorescent cell processes. Denervation resulted in the presence of phosphorylated neurofilament subunit immunoreactivity in the soma and proximal process of the ganglionic neuron. These data suggest that (i) small, intensely fluorescent cells and ganglionic neurons in the mudpuppy cardiac ganglion contain distinctly different cytoskeletal proteins, (ii) the proximal part of postganglionic "axons" contains dendrite-like and not axon-like cytoskeletal proteins, and (iii) deafferentation promotes the localization of phosphorylated forms of neurofilament subunits in the soma and proximal process of parasympathetic ganglionic neurons.     

A pathogenic peripherin gene mutation in a patient with amyotrophic lateral sclerosis

Peripherin is a neuronal intermediate filament protein that is expressed chiefly in motor neurons and other nerve cells that project into the peripheral nervous system. Transgenic mice that over-express peripherin develop motor neuron degeneration, suggesting that mutations in peripherin could contribute to the development of motor neuron disease. In this paper, we report the identification of a homozygous mutation in the peripherin gene (PRPH) in a patient with amyotrophic lateral sclerosis (ALS). The mutation resulted in a substitution of aspartate with tyrosine at amino acid position 141, which is located within the first linker region of the rod domain. Immunocytochemical analysis of the spinal cord of the patient upon autopsy revealed distinctive large aggregates within the cell bodies of residual spinal motor neurons that contained peripherin and was also immunoreactive with antibodies to the neurofilament proteins. In order to study the effect of the mutation on peripherin assembly, we performed transient transfections. Unlike wild-type peripherin, which self-assembles to form a filamentous network, the mutant peripherin was prone to form aggregates in transfected cells, indicating that the mutation adversely affects peripherin assembly. Moreover, the neurofilament light (NF-L) protein was not able to rescue the mutant protein from forming aggregates. These data imply that mutation of PRPH is a contributing factor for ALS.     

Immunocytochemical localization of the intermediate filament protein peripherin in adult mouse adrenal chromaffin cells in culture

Peripherin is the main intermediate filament protein in sympathetic neurons. Immunoreactivity to peripherin was studied in mouse adrenal chromaffin cells after 6 days in culture, and compared to immunoreactivity to tyrosine hydroxylase used as a general marker of chromaffin cells in culture. Most of the cells immunoreactive to tyrosine hydroxylase were rounded, with a glandular phenotype and a few of them had processes. The cells reactive to peripherin only constituted a small proportion of the chromaffin cells (2%), and most of them sent out processes. However, not all the cells with processes were reactive for peripherin. These results did not change in the presence of nerve growth factor. The discussion focuses on the significance of the sub-population of cells reactive to peripherin. We suggest that these cells resemble the small granule chromaffin cells, regarded as an intermediate cell type between glandular cells and neurons. The cells that expressed peripherin here are compared to those selected to form the PC12 clone. The presence of peripherin in only a few of the cells sending out neurite-like processes is discussed in relation to the expression of other neurofilament proteins in developing cells and to the influence of non-chromaffin cells.     

Hypophosphorylated neurofilament subunits in the cytoskeletal and soluble fractions of cultured bovine adrenal chromaffin cells

The neurofilament proteins in cultured bovine adrenal chromaffin cells are in a hypophosphorylated state, as determined by the co-migration of the 160,000 and 210,000 molecular weight subunits with in vitro dephosphorylated bovine brain subunits on sodium dodecyl sulfate polyacrylamide gels. In addition, chromaffin cells were not stained by anti-heavy neurofilament subunit that binds only to phosphorylated epitopes. Pulse-labeling with 32Pi in the presence and absence of the protein synthesis inhibitor emetine indicated that some neurofilament protein phosphorylation occurred co-translationally and/or immediately after synthesis of the proteins. Pulse-chase experiments showed that the three neurofilament proteins rapidly attained their maximal phosphorylation levels, as multiple forms of either of the respective subunits were not seen after a one hour chase. We found that Triton X-100-soluble forms of high molecular weight neurofilament and middle molecular weight neurofilament subunits were present in chromaffin cells, and they also co-migrated with standard neurofilament proteins dephosphorylated in vitro. However, there were differences between the phosphopeptide maps of cytoskeleton-associated and soluble middle molecular weight neurofilament subunit, suggesting that the localization of phosphate moieties rather than extent of phosphorylation influences the association of the subunit with neurofilaments. Double immunofluorescence staining of cell cultures with antibody to the 70,000 molecular weight subunit and with anti-vimentin showed that chromaffin cells do not express vimentin.     

Distribution of five peptides,three general neuroendocrine markers,and two synaptic-vesicle-associated proteins in the spinal cord and dorsal root ganglia of the adult and newborn dog: An immunocytochemical study

This study describes the immunocytochemical distribution of five neuropeptides (calcitonin gene-related peptide [CGRP], enkephalin, galanin, somatostatin, and substance P), three neuronal markers (neurofilament triplet proteins, neuron-specific enolase [NSE], and protein gene product 9.5), and two synaptic-vesicle-associated proteins (synapsin I and synaptophysin) in the spinal cord and dorsal root ganglia of adult and newborn dogs. CGRP and substance P were the only peptides detectable at birth in the spinal cord; they were present within a small number of immunoreactive fibers concentrated in laminae I–II. CGRP immunoreactivity was also observed in motoneurons and in dorsal root ganglion cells. In adult animals, all peptides under study were localized to varicose fibers forming rich plexuses within laminae I–III and, to a lesser extent, lamina X and the intermediolateral cell columns. Some dorsal root ganglion neurons were CGRP- and/or substance P-immunoreactive. The other antigens were present in the spinal cord and dorsal root ganglia of both adult and newborn animals, with the exception of NSE, which, at birth, was not detectable in spinal cord neurons. Moreover, synapsin I/synaptophysin immunoreactivity, at birth, was restricted to laminae I–II, while in adult dogs, immunostaining was observed in terminal-like elements throughout the spinal neuropil. These results suggest that in the dog spinal cord and dorsal root ganglia, peptide-containing pathways complete their development during postnatal life, together with the full expression of NSE and synapsin I/synaptophysin immunoreactivities. In adulthood, peptide distribution is similar to that described in other mammals, although a relative absence of immunoreactive cell bodies was observed in the spinal cord.     

Phosphorylated neurofilament epitopes in neuronal perikarya in the septum, mesencephalon and dorsal root ganglia of mammals and birds

Summary We and other researchers have previously described the presence of axon-specific phosphorylated neurofilament epitopes in the cell bodies of three neuronal types in the rat: bipolar septofimbrial neurons and the large light A-type cells in the dorsal root ganglia and the mesencephalic nucleus of the Vth nerve. This spontaneous presence of phosphorylated neurofilaments at the level of the perikaryon contrasts with the induced appearance of these epitopes in axotomized neurons. We have undertaken a study of this phenomenon in rat, mouse, gerbil, rabbit, pig and chicken to analyse its species distribution. Phosphorylated neurofilament positive perikarya could be detected in the dorsal root ganglia and mesencephalic nucleus of the Vth nerve in all analysed species. Although this labelling has been shown to be specific for A-type cells in rat, in pig small cells were preferentially labelled, whereas the largest cells were mostly completely devoid of label. In the septofimbrial nucleus, phosphorylated neurofilament positive perikarya were seen in rat, mouse, gerbil and rabbit. In the pig, only a phosphatase-insensitive neurofilament antibody labelled these neurons. In the chicken, the labelling was completely absent. These observations establish the widespread species distribution of perikaryal phosphorylated neurofilament epitopes in the dorsal root ganglia and mesencephalic nucleus of the Vth nerve. In the septofimbrial nucleus however, this phenomenon seems to be restricted to rodents and lagomorphs. We discuss possible explanations for these cytoskeletal singularities in dorsal root ganglia, the mesencephalic nucleus of the Vth nerve and septofimbrial neurons.     

Immunoreactivity for phosphorylated 200-kDa neurofilament subunit is heterogeneously expressed in human sympathetic and primary sensory neurons

This study was undertaken to investigate whether human sensory and sympathetic neurons contain phosphorylated neurofilament proteins, and whether they may be classified on the basis of this property, as in other mammalian species. The distribution of the phosphorylated 200-kDa neurofilament protein subunit (p200-NFP) was investigated in lumbar sympathetic and dorsal root ganglia by means of the RT97 monoclonal antibody (against p200-NFP). The intensity of immunostaining, and the size of neuronal body profiles were measured in order to define different neuron subclasses. In dorsal root ganglia, most of the neuronal profiles (96%) were p200-NFP immunoreactive, and the intensity of immunostaining was not related to neuronal perikarya size. In the lumbar paravertebral sympathetic ganglia, virtually all neurons displayed p200-NFP immunoreactivity, and the intensity of immunolabelling was also independent of the size of the neuronal somata. These results demonstrate heterogeneity in the expression of p200-NFP immunoreactivity in human sympathetic and sensory neurons. In contrast to other mammalian species, RT97 immunolabelling cannot be used as a discriminative marker for the two main types of human primary sensory neurons. On the other hand, our findings provide evidence for the occurrence of phosphorylated neurofilaments within peripheral neuron cell bodies.     

The appearance of trigeminal ectopic ganglia within the surface ectoderm in the chick embryo

Aberrant ganglion cell morphogenesis associated with the ophthalmic placode was observed within the epidermis of the chick embryo at stages 20 through 26. This process was studied using immunohistochemistry and scanning and transmission electron microscopy. Clusters of ganglion cells were covered by epidermis and seen as epidermal nodules distributed diffusely in the supraorbital region. The ganglion cell clusters found within these nodules were not delineated by basement membrane from the epidermis, and were provisionally termed ectopic ganglia. The cell bodies within the ganglia possessed neurites containing microtubules and neurofilament protein. The cell bodies were also immunoreactive to monoclonal antibodies, E/C8 and HNK-1, indicating neuronal phenotypes. The neurites were often associated with the ophthalmic nerve or its branches as well as being distributed in the ectoderm. The ectopic ganglia seemed to represent the delayed gangliogenesis of a part of the ophthalmic placode which failed to detach from the ectoderm during ophthalmic ganglion formation.     

Slow transport of freely movable cytoskeletal components shown by beading partition of nerve fibers in the cat

To account for the transport in nerve fibers of tubulin and neurofilament proteins in slow component a, the Structural Hypothesis holds that these proteins are assembled into microtubules and neurofilaments in the cell bodies and the cytoskeletal organelles then moved down in the fibers as part of an interconnected matrix at a uniform rate of about 1 mm/day. The Unitary Hypothesis, on the other hand, considers these proteins to be carried down within the fibers as soluble components or as freely movable small polymers or subunits turning over locally in the stationary cytoskeleton. To differentiate between the two hypotheses, cat L7 dorsal roots were taken at times from 7 to 25 days after their L7 dorsal root ganglia were injected with [3H]leucine to assess the labeling of the cytoskeleton by the use of beading and autoradiography. Beading was induced by a mild stretch and after fast-freezing and freeze-substitution of the roots for histological preparation, the beads were seen in the fibers as a series of expanded regions alternating with constrictions. In the constrictions the cytoskeleton was compacted into an area as small as 5% that of the normal axon, with the axoplasmic fluid and displaceable (freely movable) components squeezed from the constrictions into the adjoining expansions. Roots taken after 7 and 14 days, times consistent with slow component a downflow, were assessed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and their content of tubulin and neurofilament proteins shown to constitute 40-50% of all the labeled proteins present. In autoradiographs of dorsal roots taken at those times, numerous grains due to radioactivity were located over the non-constricted regions of the fibers. Few or no grains were present over the constrictions after 7 days. The findings are in accord with the labeled tubulins and neurofilament proteins being present in soluble form in the fibers and expressed from the constrictions into the expansions of the beaded fibers. In contrast, a number of fibers in roots taken at 14-20 days after injection showed somewhat higher grain densities over the constrictions, and more so after 25 days, indicating uptake of labeled subunits into the cytoskeletal organelles at later times. The results are consistent with the downflow of tubulin and neurofilament proteins as soluble components which drop off in the axon to turn over locally in their respective cytoskeletal organelles.     

Aspartate-like immunoreactivity in primary afferent neurons

There is now good evidence that amino acids act as neurotransmitters in primary afferent neurons of dorsal root ganglia. Glutamate is the primary candidate for such a role, and there are reasons to believe that release of glutamate may be accompanied by the release of other neuroactive substances. Using immunocytochemical techniques, we have tested the hypothesis that some dorsal root ganglion neurons contain elevated levels of aspartate as well as glutamate. Antisera raised against conjugates of aspartate or glutamate were used for this purpose. Blocking experiments confirmed that these antibodies were specific to their antigens in cryostat sections of dorsal root ganglia.

Aspartate immunoreactivity was found in approximately 30% of neurons in cervical dorsal root ganglia. The relation between cell size and staining intensity for aspartate was examined using quantitative video microscopy: the great majority of cells immunopositive for aspartate were small (15–30 μm in diameter); about 85% of these cells stained for aspartate, although staining intensities varied over a wide range. By reacting consecutive sections with anti-aspartate and anti-glutamate it was shown that elevated levels of aspartate were found in the same cells which contained elevated levels of glutamate. By measuring the staining intensity of individual cells for both aspartate and glutamate, it was also shown that there was a positive correlation between staining intensity and, presumably, concentration of the two amino acids. The presence of high levels of aspartate in terminals located in the superficial laminae of the dorsal horn was verified by pre- and post-embedding immunocytochemistry with the electron microscope. Aspartate was demonstrated in scalloped terminals, including dark scalloped terminals believed to be associated with unmyelinated fibers of nociceptors.

This evidence supports the hypothesis that aspartate as well as glutamate is present in the cell bodies and terminals of nociceptive primary afferents, and may be released by the terminals of these afferents to activate neurons in the superficial laminae of the dorsal horn.     

Influence of peripheral and central targets on subpopulations of sensory neurons expressing calbindin immunoreactivity in the dorsal root ganglion of the chick embryo

The influence of central and peripheral target tissues on the expression of calbindin D-28k by sensory neurons of the chick dorsal root ganglia was tested under various experimental conditions. Firstly, dorsal root ganglia of chick embryos were transplanted at two stages of development onto the chorioallantoic membrane of a host embryo for a period of 4 or 8 days. In dorsal root ganglia grafted at E12, 20% of the ganglion cell bodies were immunoreactive to calbindin 4 and 8 days later; the percentage of calbindin-immunostained neurons in grafted dorsal root ganglia was similar to that observed in control dorsal root ganglia of the same embryonic age (E16 or 20). In contrast, when grafted dorsal root ganglia were taken from a donor embryo at E8, no calbindin-immunoreactive neuron was found 4 or 8 days later. However, when dorsal root ganglia at E8 were cotransplanted with musculature cells, 14% of the grafted ganglion cell bodies were again immunoreactive to calbindin 4 or 8 days later. Secondly, peripheral targets of sensory neurons were suppressed by excision of one hindlimb. After excision at E6, virtually all the ipsilateral dorsal root ganglia cells were free of calbindin immunoreaction after 6 days of reincubation. In contrast, when the excision was performed at E11, calbindin was expressed in about 9% of the nerve cell bodies. Thirdly, central connections were destroyed by cauterization of the lumbosacral spinal cord at E6 or E11. Six days after deprivation of central connections, the percentage of calbindin-immunoreactive ganglion cells was the same as in control dorsal root ganglia of the same age.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distribution and origin of nerve fibers in the rat temporomandibular joint capsule

 The distribution and origin of nerve fibers containing neuropeptides and NOS projecting to the temporomandibular joint capsule (TMJ) of the rat were studied by retrograde tracing in combination with immunocytochemistry. Numerous nerve fibers were seen in the TMJ as revealed by the neuronal marker protein gene product 9.5. Nerve fibers containing neuropeptide Y (NPY), vasoactive intestinal peptide (VIP), pituitary adenylate cyclase activating peptide (PACAP), substance P (SP), calcitonin gene-related peptide (CGRP), and nitric oxide synthase (NOS) were seen in the synovial membrane, the joint capsule and entering the articular disc. Injection of the retrograde tracer True Blue (TB) into the TMJ resulted in the appearance of numerous labeled nerve cell bodies in the trigeminal and superior cervical ganglia, and moderate numbers in the nodose, the otic, the sphenopalatine, the stellate and the dorsal root ganglia at levels C2–C5. Most of the TB-labeled cell bodies in the superior cervical and stellate ganglia contained NPY. In the trigeminal ganglion, numerous TB labeled cell bodies contained CGRP and a minor population stored SP, a few cell bodies were seen to store NOS or PACAP. In the sphenopalatine and otic ganglia, TB labeled cell bodies contained NOS or VIP. In the nodose ganglion, labeled cell bodies contained CGRP; other labeled cell bodies harbored NOS. In the cervical dorsal root ganglia, the majority of the labeled cell bodies stored CGRP and smaller populations stored SP and PACAP. Thus, the innervation of the TMJ is complex and many different ganglia are involved. Accepted: 13 October 1997     

Subpopulations of neurons in the guinea-pig inferior mesenteric ganglia distinguished by the differential distribution of neurofilament triplet epitopes

The distribution of the neurofilament protein triplet was examined in neurochemically identified subpopulations of neurons in the guinea-pig inferior mesenteric ganglion. A majority of the catecholamine-containing nerve cell bodies also contained the neurofilament protein triplet. However, a major proportion of the noradrenergic, neuropeptide Y-immunoreactive neurons did not contain neurofilament protein triplet immunoreactivity. Furthermore, a specific subpopulation of neurons that lacked catecholamines and were associated with the hypogastric nerve could be distinguished by the unusual feature of cell body content of post-translationally modified neurofilament protein triplet epitopes. These studies indicate that neurons in the inferior mesenteric ganglia can be distinguished by the presence of specific neurofilament protein triplet epitopes, and thus this class of intermediate filament proteins may confer specific properties to the neurons in which it is contained.     

Substance P, neurofilament, peripherin and SSEA4 immunocytochemistry of human dorsal root ganglion neurons obtained from post-mortem tissue: a quantitative morphometric analysis

Summary Immunocytochemical studies on lumbar dorsal root ganglia obtained at routine postmortem 24–36 h after death were carried out, and neuronal cross-sectional areas measured. The subjects were elderly (76–81 years), of both sexes, had died from heart attack or haemorrhage, and had no clinical evidence of clinical neuropathy or of disease known to be associated with neuropathy. The data were consistent between ganglia from the three subjects. There were striking similarities with data from other species. Two populations of cell profiles with overlapping size distributions were distinguished with an anti-neurofilament antibody, neurofilament-rich (45% of cell profiles) with a large mean area and neurofilament-poor with a smaller mean area. Anti-substance P and anti-peripherin antibodies both labelled a population with a small mean area, with extensive co-localization between them. There were also some differences between these human dorsal root ganglia and dorsal root ganglia from some other species. More neuronal profiles were labelled for substance P in humans (44%) than in rat (20%). More neuronal profiles were labelled for SSEA4 (stage specific embryonic antigen 4) in human (40.5%) than in rat dorsal root ganglia (10%), and the SSEA4-positive profiles were relatively smaller in human than in rat. No selective accumulation of lipofusin in profiles of large cells was apparent. This study also shows that quantitative morphometric analysis of immunocytochemically labelled dorsal root ganglion neuronal profiles can be carried out successfully on human sensory ganglia obtained at post-mortem. This is the first demonstration of the two main subgroups of dorsal root ganglia neurones with neurofilament-rich and poor somata in human tissue. The size distributions of neurons with neurofilament, substance P and peripherin are consistent with these neuronal populations having similar functional properties to those described in other species. From the known sensory and fibre loss with aging, it is speculated that the loss of some large diameter neurones with myelinated fibres and low mechanical thresholds, might account for the high percentage of neurones expressing substance P.     

Expression of neurofilament proteins by horizontal cells in the rabbit retina varies with retinal location

Summary Classical neurofibrillar staining methods and immunocytochemistry with antibodies to the light, medium and heavy chain subunits of the neurofilament triplet have been used forin situ andin vitro investigation of the organization of neurofilaments in A- and B-type horizontal cells of the adult rabbit retina. Surprisingly, their expression and organization within a cell is dependent on its location along the dorso-ventral axis of the retina. A-type horizontal cells in superior retina consistently stained with a wide variety of neurofibrillar methods to reveal neurofibrillar bundles, which immunocytochemistry showed to contain all three neurofilament subunits. A-type horizontal cells in inferior retina were uniformly refractory to neurofibrillar staining, although they expressed all three subunits. However, there was less of the light and medium subunits; the organization of the filaments into bundles (neurofibrils) is minimal. B-type horizontal cells could not be stained with any neurofibrillar method and were not recognizable byin situ immunocytochemistry. However, B-type cells could be seen to express all three subunitsin vitro, but the expression of the light and medium subunits was weak. There was only a slight difference between B-type cells taken from superior and inferior retina. Combined with the results of recent transfection studies, these findings suggest that the amount of the light neurofilament subunit present in a horizontal cell determines its content of neurofibrillar bundles, and that rabbit horizontal cells may contain more neurofilament protein, particularly of the heavy subunit, than is used for neurofilament formation. The results also show that determination of the neurofilament protein content of a population of nerve cells can be critically dependent on examination of single isolated cells. Isolated rabbit horizontal cells provide a promising system for studies of the mechanical, molecular and biochemical properties of neurofilaments.     

Glycine immunoreactivity localized in the cochlear nucleus and superior olivary complex

Polyclonal antibodies were made in rabbits against glycine conjugated to bovine serum albumin with glutaraldehyde and were used for immunocytochemical studies in the cochlear nucleus and superior olivary nucleus of the guinea-pig. Antibodies selective for glycine were prepared by affinity chromatography. By dot-blot analysis this preparation showed a strong recognition of glycine conjugates and relatively little recognition of conjugates of most other amino acids tested. However, there was a significant reaction with conjugates of alanine and beta-alanine, and this cross-reaction could not be removed by affinity chromatography without eliminating the preparation's recognition of glycine. The affinity-purified preparation showed only a weak recognition of conjugates of gamma-aminobutyrate (GABA) which was detectable at high concentrations of primary antibody. Immunocytochemical studies showed several intensely staining cell bodies in the cochlear nucleus and superior olivary complex. Most immunoreactive cell bodies in the cochlear nucleus were in the dorsal cochlear nucleus, being present in both the superficial and deep layers. Scattered immunoreactive cells were present in the ventral cochlear nucleus. Intense staining of cell bodies was seen in the medial nucleus of the trapezoid body, and these cells appear to correspond to the principal cells of that nucleus. Punctate labelling, suggestive of immunoreactive presynaptic terminals, was also apparent, particularly in the ventral cochlear nucleus and lateral superior olive. In the ventral cochlear nucleus, immunoreactive puncta were found around unlabeled cell bodies, at times nearly covering the perimeter of the cell. A population of glycine-immunoreactive cell bodies in the superficial dorsal cochlear nucleus also labeled with anti-GABA antibodies as determined through double-labeling studies. However, glycine-positive cells in the deep dorsal cochlear nucleus were not labeled with anti-GABA antibodies, and some populations of GABA-positive cells in the superficial layers were not labeled with anti-glycine antibodies. In the hippocampus intense staining of cell bodies and puncta was seen with anti-GABA antibodies while essentially no staining was seen with anti-glycine antibodies. These results suggest that anti-glycine antibodies can be useful for immunocytochemical identification of glycinergic neurons. From this study several populations of putative glycinergic neurons are identified in the auditory nuclei of the brain stem using these antibodies. Some populations of GABA-containing neurons also contain high levels of glycine or a related molecule.     

The organization of 10 nm filaments and microtubules in embryonic neurons from spinal ganglia

Summary The distribution of tubulin and 10 nm filament protein: vimentin and the 70 K neurofilament component, was investigated in cultures of dorsal root ganglion cells by indirect immunofluorescence using antisera specific for these proteins. The neuronal cell bodies and the neurites in these cultures were brightly stained. Fibres stained with both tubulin and 10 nm filament antibodies were visible at branch points, whereas only microtubules extended into growth cones, including those that were in the process of bifurcation. Differences in the arrangement of microtubules and 10 nm filaments were also detected during colcemid-induced neurite retraction. After complete retraction, perinuclear coils of 10 nm filaments were found in the cell bodies whereas only very weak diffuse tubulin staining was visible. When regrowth commenced on removal of colcemid, microtubule organizing centres were visible in the cell bodies and were then rapidly obscured by bright staining which later extended into the neurite stump. Similarly, the 10 nm coils were replaced by fibrous staining which also projected into the growing neurite. These observations suggest that the extension of microtubules and 10 nm filaments is both co-ordinated and sequential, with microtubules having a dynamic role in growth and 10 nm filaments stabilizing the pattern of growth thereafter.