Dendritic Staining Research Articles

Degeneration of purkinje cells in parasagittal zones of the cerebellar vermis after treatment with ibogaine or harmaline

The indole alkaloids ibogaine and harmaline are β-carboline derivatives that cause both hallucinations and tremor. Reports that ibogaine may have potent anti-addictive properties have led to initiatives that it be tested for the treatment of opiate and cocaine addiction. In this study, ibogaine-treated rats were analysed for evidence of neurotoxic effects because human clinical trials of ibogaine have been proposed. We recently found that ibogaine induces a marked glial reaction in the cerebellum with activated astrocytes and microglia aligned in parasagittal stripes within the vermis. 28 Based on those findings, the present study was conducted to investigate whether ibogaine may cause neuronal injury or degeneration. The results demonstrate that, after treatment with ibogaine or harmaline, a subset of Purkinje cells in the vermis degenerates. We observed a loss of the neuronal proteins microtubule-associated protein 2 and calbindin co-extensive with loss of Nissl-stained Purkinje cell bodies. Argyrophilic staining of Purkinje cell bodies, dendrites and axons was obtained with the Gallyas reduced silver method for degenerating neurons. Degenerating neurons were confined to narrow parasagittal stripes within the vermis. We conclude that both ibogaine and harmaline have selective neurotoxic effects which lead to degeneration of Purkinje cells in the cerebellar vermis. The longitudinal stripes of neuronal damage may be related to the parasagittal organization of the olivocerebellar climbing fiber projection. Since these drugs produce sustained activation of inferior olivary neurons, we hypothesize that release of an excitatory amino add from climbing fiber synaptic terminals may lead to excitotoxic degeneration of Purkinje cells.

The involvement of eosinophils in the patch test reaction to aeroallergens in atopic dermatitis: its relevance for the pathogenesis of atopic dermatitis.

Atopic dermatitis (AD) is considered a T-cell mediated disease. Activated T-cells, mainly of the CD4-subtype, are abundantly present in lesional AD skin. Although not many intact eosinophils are present, deposits of eosinophil derived major-basic-protein (MBP) and eosinophil-cationic-protein (ECP) suggest eosinophil involvement. After patch testing AD patients with aeroallergens, an eczematous reaction develops after 24-48 hr at the site of application. This patch test reaction shows macroscopic resemblance to lesional AD skin and does not take place in normal individuals, asthma and allergic rhinitis patients. Lymphocytes together with eosinophils infiltrate into the dermis 2-6 hr after allergen application. Twenty-four to forty-eight hours after patch testing, eosinophils are in an activated state since they release ECP (being EG2-positive). At this point in time eosinophils have also infiltrated the epidermis. Here they are EG2-negative. Forty-eight to seventy-two hours after patch testing the eczematous reaction decreases. This coincides with disappearance of eosinophils from both the dermis and the epidermis; then, a dendritic staining pattern can be observed in the epidermis with anti-eosinophil peroxidase. Thus, eosinophils infiltrate the dermis and epidermis after patch testing AD patients with aeroallergens and release part of their granular constituents. Recent in vitro investigations revealed that eosinophils from the circulation of AD patients react more powerfully in in vitro test systems such as chemiluminescence, chemotaxis and endothelial adherence and transmigration. It is very likely that this activated (= primed) state is caused by the influence of lymphocyte-derived cytokines like IL-3, IL-5 and GM-CSF, since activated lymphocytes in the circulation (and tissue) may release these cytokines. The primed state of the eosinophils may facilitate tissue infiltration. The subsequent activation of eosinophils within the tissue leading to mediator release and the function of these mediators need to be further elucidated. The close similarity between the cellular events after a patch test reaction to aeroallergens in AD patients and those present in lesional AD skin suggests that the patch test reaction may be a helpful in vivo model to study the pathogenesis of AD. The prominent involvement of lymphocytes and eosinophils in this reaction also suggests some similarity with late phase reactions (LPR) observed in the skin after intracutaneous allergen challenge.

The expression of the protein p68/70 within the goldfish visual system suggests a role in both regeneration and neurogenesis

Previous attempts to characterize the molecular events that support successful regeneration of axotomized goldfish retinal ganglion cells (RGCs) have led to the discovery of an acidic protein doublet in retina that displays an increased incorporation of 35S-methionine following axotomy, and is known to be axonally transported within the optic nerve. This protein is termed p68/70, reflecting its relative migration in 10% SDS-PAGE. In the present study, an affinity-purified polyclonal antibody to p68/70 (anti-p68/70) was developed and used to assess the species, tissue, and cellular distribution of p68/70. The antibody cross-reacted with homogenates of brain and other tissues from goldfish and closely related fish species. While each goldfish tissue tested expressed p68/70, the levels varied over a 30-fold range, with the highest amounts in brain, egg, and ovary. Immunolabeling of goldfish retina revealed prominent staining of RGC somata, dendrites, and axons. During regeneration, the immunoreactivity of the RGC somata and axons increased dramatically. Intense immunolabeling was also observed in the germinal neuroepithelial cells and rod precursors and in all retinal layers near the peripheral margin, in the region of recently differentiated neurons. In the tectum, the germinal zone was also highly labeled. The elevated expression of p68/70 in each of these areas known to mediate neuronal growth within the goldfish visual system suggests that p68/70 plays a role in axonal growth, regrowth, and possibly in neural development as well.

Immunohistochemical changes of neuronal calcium-binding proteins parvalbumin and calbindin-D-28k following unilateral deafferentation in the rat visual system

The neuron-specific calcium-binding proteins, parvalbumin and calbindin-D-28k, were studied in the subcortical visual system of normal and unilaterally deafferented albino rats. Immunohistochemistry with monoclonal antibodies was used on vibratome sections through optic tract (OT), dorsal lateral geniculate nucleus (dLGN), olivary pretectal nucleus (OPN), and superior colliculus (SC). In controls, OT stained strongly for parvalbumin and weakly for calbindin-D-28k. The dLGN contained a plexus of parvalbumin-positive fibers. In dLGN, calbindin-D-28k-antibodies showed strong labeling of some neurons with long dendrites and weak staining of the cytoplasm in other neurons. In OPN, parvalbumin stained a ring of neurons and terminals in the shell region, whereas calbindin-D-28k was contained in medial cell populations. In SC, parvalbumin was contained in fibers, terminals, and neurons throughout the visual layer. Calbindin-D-28k showed a laminar distribution of neurons with a predominance in deep portions of superficial grey matter and in ventral portions of stratum opticum. Following unilateral deafferentation induced by optic nerve section, retinal axons showed immunohistochemical changes related to Wallerian degeneration and target neurons reacted by changes of calcium-binding proteins. Parvalbumin and calbindin-D-28k immunostaining decreased during Wallerian degeneration of OT. In the deafferented dLGN, immunohistochemical labeling for calbindin-D-28k declined in strongly stained neurons from 4 to 21 days after lesion. Measurement of dendritic length per number of cells or per area of dLGN showed a significant decline for the contralateral side at 4, 8 and 21 days (ANOVA, P < 0.05). In deafferented OPN, terminal-like staining for parvalbumin decreased and neuronal labeling was enhanced. In deafferented SC, the neuronal and dendritic staining for parvalbumin increased beginning from Day 1 on and persisting at Day 21, whereas fibers and terminal-like elements decreased in staining. Measurement of parvalbumin-positive neurons per area of SC showed a significant increase of labeling in the contralateral side from Day 1 to Day 21 (ANOVA, P < 0.05). These studies show that cellular responses to deafferentation of visual neurons involve a regulation of calcium-binding proteins. The decline in staining for calbindin-D-28k in dLGN may relate to reduced retinal afferent activity. The progressive cellular changes in parvalbumin staining may be related to unmasking of intrinsic neurons after removal of parvalbumin-containing, afferent fibers and terminals. Additionally, the changes of parvalbumin labeling in SC neurons may reflect a plastic reorganization of local circuits known to occur in rat SC in response to deafferentation.

Dynamic post-translational modification of tubulin in rat cerebral cortical neurons extending neurites in culture: Effects of taxol

Dissociated embryonic (E18-E20) rat cortical neurons were grown in culture and double-labelled by immunofluorescence with antibodies directed against tyrosinated (YL 1/2), detyrosinated (SUP GLU), and acetylated (6-11B-1) alpha-tubulin. Within 90 min of plating, neurons extended growth cones that were YL 1/2+ but SUP GLU- and 6-11B-1-. The neurite that forms behind the advancing growth cone is also, initially, YL 1/2+ and SUP GLU-/6-11B-1-. However, when it has attained a length of about half the cell body diameter, it becomes SUP GLU+ and 6-11B-1+. The effects of the microtubule polymerizing agent taxol (15 microM) on growth cone and neurite alpha-tubulins was investigated. Taxol, as reported previously, caused the formation of microtubule loops in the central domain of the growth cone, a loss of filopodia, and the collapse of the growth cone onto the loops. The taxol effects peaked at 60 min, when over 85% of neurites showed microtubule loops, and declined thereafter, so that at 420 min in taxol, only about 23% of neurites had microtubule loops. Over this period there was an inverse correlation between the presence of microtubule loops and growth cones. Taxol had striking effects on the intensity of SUP GLU and 6-11B-1 staining in neurons. In 48 h cultures, a 30 min exposure to taxol enhanced the SUP GLU and 6-11B-1 staining of dendrites and axons and produced a loss of YL 1/2 staining in axons. Immunoblotting experiments confirmed that there was an overall reduction in YL 1/2 immunoreactivity and an increase in SUP GLU immunoreactivity. These observations support previous suggestions that the neurite microtubules are assembled in the growth cone and post-translationally modified in the neurite and, in addition, imply that growth cones can overcome the effects of taxol in the continued presence of the compound.

Purkinje cell antibodies in a patient with cerebellar disorder: detection of responsible antigenic proteins

A 34-year-old male patient developed a neurological disorder and signs of cerebellar degeneration, with antibodies against Purkinje cells in the serum, a syndrome previously described as "paraneoplastic cerebellar atrophy". Antibody reaction of the patient's serum was demonstrated by immunohistochemistry on sections through the rat and human cerebellum. Purkinje cells demonstrated granular staining of cytoplasmic proteins and proximal dendrites with nuclear sparing. In an immunoblot, the antibodies from the patient's serum reacted with proteins from an extract of rat cerebellum. Only a few distinct proteins from the complex mixture of cerebellar proteins were found to bind with the serum antibodies when using a combination of affinity chromatography and sodium dodecyl sulphate (SDS) gel electrophoresis. The molecular masses of the proteins differed significantly from those identified in patients reported in the literature. Protein denaturation by SDS and 2-mercaptoethanol resulted in a decrease of antibody binding capacity. After immunosuppressive therapy and plasmapheresis, the reaction of the patient's serum with Purkinje cells was greatly diminished; however, only slight clinical improvement was observed. No sign of neoplasm could be found with repeated examinations. The immunological aspects of this case suggest that cerebellar degeneration may be linked to a previously unreported autoimmune response.

Nerve growth factor receptor expression on dendritic reticulum cells in follicular lymphoid proliferations

Using an antibody to the nerve growth factor receptor (NGFR), we examined dendritic reticulum cells (DRCs) immunohistochemically in 62 formalin-fixed, paraffin-embedded lymph nodes from patients with reactive follicular hyperplasia or with various types of lymphoma. A dendritic staining pattern within germinal centers was present in 25 of 26 routinely processed lymph nodes with reactive follicular hyperplasia. In contrast, dendritic staining with anti-NGFR was present within neoplastic follicles in only three of 28 follicular lymphomas. Staining of benign, residual centers with anti-NGFR was present in mantle zone lymphoma and Hodgkin's disease. These findings suggest a possible role for the NGFR in the maturation and/or activation of normal DRCs. The loss of NGFR expression in most follicular lymphomas indicates that DRCs are altered as part of the neoplastic process. The possibility that DRCs may play a role in the pathogenesis of follicular lymphoma is suggested.

Spatial distributions of cytoskeletal proteins and the nerve growth factor receptor in septal transplants in oculo: Protection from abnormal immunoreactivity by hippocampal co-grafts

Intraocular grafts of embryonic rat septum and co-grafts of septum plus hippocampus were studied with immunohistochemical markers after one and six months (short term) and 12 months (long term) of survival. Neurons in all the septal tissues expressed the epitope for the rat β -nerve growth factor receptor in sections reacted with the monoclonal antibody 192-IgG. Stained fibers traversed the interface of the short and long term co-grafts and 192-IgG-positive processes were most prominent in the septum when combined with the hippocampal formation. In contrast, labeled processes were sparse and the perikarya of positive neurons appeared shrunken in the long term single septal transplants. Axon and dendrite profiles in the grafts were examined with antibodies that recognize the phosphorylated heavy neurofilament unit (RT97) and the high molecular weight microtubule-associated protein termed MAP 2, respectively. In the short term single and double grafts, characteristic arrays of RT97-positive processes defined the tissues and axonal tracts connecting the septum with the hippocampus. Typical immunostaining of the neuronal somas and the dendrite arbors were outlined with the MAP 2 antibody. After one year in oculo, extensive changes in the patterns of axonal and dendritic immunoreactivity were noted in the isolated septal grafts. Abnormalities identified with the RT97 antibody included hypertrophied axons, short fragments of kinked axons and neurofilaments in the neuronal perikarya. The formation of circular “abnormal fiber aggregates” composed of densely packed abnormal and normal axonal processes were also distinctive in only the long term single septal transplants. In addition, a reduction in the density of dendrites and the presence of truncated arbors stained with the MAP 2 antibody suggested that regression of the dendrites had occurred. These spatial modifications in axonal and dendritic staining were not present in the septal portion of the combined preparations. In astrocytes, an increase in the antigenicity to glial fibrillary acidic protein paralleled the age of the transplant and was most extensive in the septal grafts. The results illustrate that intraocular co-grafts of hippocampus protect septal neurons and glial cells from abnormal changes in immunoreactivity to antibodies directed against cytoskeletal proteins and exemplify the long term supportive effects of the hippocampus on the morphology of septal neurons, including neurons that express the receptor for nerve growth factor.

Pac 1: An epitope associated with two novel glycoprotein components of isolated postsynaptic densities and a novel cytoskeleton-associated polypeptide

A monoclonal antibody has been raised which recognizes an epitope, PAC 1 (postsynaptic density and cytoskeleton enriched), which is specifically associated with two novel glycoprotein components of forebrain postsynaptic density preparations and a novel neuronal cytoskeletal-associated polypeptide. The monoclonal antibody has been used to study the cellular and subcellular localization of these molecules and for the partial characterization of all three PAC 1 antigens in the rat. The PAC 1 epitope is present on two concanavalin A binding glycoproteins of apparent molecular weights 130,000 (pgp130) and 117,000 (pgp l 17). Both species are enriched in preparations of rat forebrain postsynaptic densities and to a lesser extent in synaptic membranes. The epitope is also expressed by a polypeptide of 155,000 mol. wt, cp155. This molecule is highly enriched in cytoskeleton rather than membrane preparations. Enzymic removal of N-linked carbohydrate lowers the molecular weights of the PAC 1 glycoproteins pgp130 and pgp117 by 11,000 and 14,000 respectively, and suggests that cp155 is not glycosylated. Detergent, alkaline and salt extractions of postsynaptic densities and synaptic membranes indicate that pgpl30 and pgp117 are integral membrane glycoproteins and are tightly bound components of postsynaptic density preparations. Immunocytochemical studies of adult rat forebrain show prominent staining of pyramidal cell dendrites and perikarya. There is no evidence of glial staining. Electron microscope studies show staining of microtubules together with punctate deposits of plasma membrane-associated reaction product. Several criteria have been used to show that pgp130 and pgp117 do not correspond to other known neuronal glycoproteins of similar molecular weight. We conclude that the PAC 1 epitope is expressed by two novel synaptic glycoproteins which are very probably integral components of the postsynaptic density and by a novel neuronal cytoskeleton-associated protein.

Immunohistochemical study of microtubule-associated protein 2 and ubiquitin in chronically aluminum-intoxicated rabbit brain

Experimental neurofibrillary change was produced in rabbit brain by daily subcutaneous aluminum tartrate injection for 40 days. The production of experimental neurofibrillary changes was confirmed by immunostaining with antibodies against neurofilament triplet proteins and the brain tissue was studied immunohistochemically with antibodies against microtubule-associated protein (MAP) 2 and ubiquitin. The hippocampal neurons of the chronically aluminum-intoxicated rabbit brain showed diminished staining of dendrites by anti-MAP2 antibody. The length of anti-MAP2-positive dendrites in hippocampus was significantly shorter than that of the control brain. In the cortex somata of a subset of pyramidal neurons were intensively stained by anti-MAP2 antibody, while the MAP2 immunoreactivity of distal dendrites was diminished. The immunostaining by anti-ubiquitin antibody revealed the positive staining of the neurons bearing experimental neurofibrillary changes in the lower brain stem nuclei. It is speculated that MAP2 dislocation and ubiquitination are accompanying phenomena of the production of experimental neurofibrillary changes in chronically aluminum-intoxicated rabbit brains.

Trauma‐induced golgi‐like staining of neurons: A new approach to neuronal organization and response to injury

A new esterification-silver approach to the directed staining of the dendritic trees of traumatized neurons is described. Stained neurons compare favorably to those labeled with silver chromate Golgi impregnations in the visualization of dendritic arbors. Cells in all parts of the brain, including the hypothalamus, hippocampus, cerebral cortex, cerebellum, striatum, spinal cord, thalamus, and olfactory bulb, can be detected after focal trauma to that region. Selection of neurons to be stained is made by increasing their affinity for silver with many different types of directed neuronal trauma, including micropipette wounds, pressure, surgical incision, chemical cytotoxicity, and impact trauma. Trauma to one area of the brain results in dendritic arbors staining only in the injured area; other areas of the brain are free of dendritic staining. Injury can be produced either in vivo or in vitro. In vitro injury to neurons allows a high degree of localization and facilitates the analysis of neuronal response to trauma in the absence of complicating factors such as blood flow and secondary injury. The selective affinity for silver staining in this approach is increased very rapidly, allowing detection of traumatized cells fixed minutes after injury. Brains of all mammals used appear to stain similarly, including the rat, mouse, pig, and human. Axons, although labeled less frequently than dendritic arbors, are induced to stain just as rapidly as dendrites. The ability to visualize a large part of the dendritic tree after trauma allows the segregation of neuronal subtypes on the basis of their differential response to injury. Subpopulations of cells in the same area of the brain appear to respond differently to trauma. Differential response of neurons to trauma can easily be detected. For instance, slight variations in trauma can be used to label selectively the major subpopulations of neurons in the hippocampus, including pyramidal cells, interneurons near the pyramidal cell layer, or granule cells. Similarly, neurons of the hypothalamic paraventricular and arcuate nuclei respond to compression trauma much more dramatically than other cell types in the same region of the hypothalamus. Hypothalamic neurons were studied extensively, particularly in regions that are difficult to routinely stain with Golgi impregnations, including the arcuate, paraventricular, supraoptic, and suprachiasmatic nuclei. Trauma to these areas was made in vitro after removal of the brain from the skull, allowing easy access to the ventral surface of the brain.(ABSTRACT TRUNCATED AT 400 WORDS)

Distribution of calcium‐activated protease calpain in the rat brain

Calpain is a calcium-activated neutral protease that degrades a number of cytoskeletal proteins. It may participate in the maintenance of the cytoskeleton and in the rapid turnover of structural proteins associated with synaptic plasticity. Calpain may also be involved in the neurodegeneration that accompanies aging and age-related diseases. To aid in the interpretation of disease-related alterations in staining patterns, the present study examined calpain's normal distribution in the mammalian brain and spinal cord. A monoclonal antibody was employed with the avidin-biotin-peroxidase immunocytochemical technique on samples of rat tissue. Glia (astrocytes, microglia) and virtually all neurons were immunopositive, although neuronal processes exhibited varying staining patterns. The axonal staining pattern depended upon either the origin or destination of the process: those axons remaining within the brain (e.g., corpus callosum) were only lightly immunoreactive, whereas spinal cord and peripheral axons (trigeminal nerve) were more darkly labeled. The architecture of the dendritic tree determined the dendritic staining pattern: neurons with prominent apical and basal dendritic trees (e.g., pyramidal cells) were immunolabeled along their entire extent; labeling of multipolar cells (e.g., hilar cells of dentate gyrus) was limited to the proximal dendrites. The ubiquitous distribution of calpain argues against a primary role for the enzyme in the regional pattern of neuronal death seen in Alzheimer's disease. An alteration in the concentration, localization, or inhibition of the enzyme could, however, lead to the abnormal accumulations of cytoskeletal elements seen with the disease.

Glutamate-positive neurons and axon terminals in cat sensory cortex: a correlative light and electron microscopic study.

Immunocytochemical methods were used to perform a correlative light and electron microscopic study of neurons and axon terminals immunoreactive to the antiglutamate (Glu) serum of Hepler et al. ('88) in the visual and somatic sensory areas of cats. At the light microscopic level, numerous Glu-positive neurons were found in all layers except layer I of both cortical areas. On the basis of the dendritic staining of Glu-positive cells, two major morphological categories were found: pyramidal cells, which were the most frequent type of immunostained neuron, and multipolar neurons, which were more numerous in layer IV of area 17 than in any other layer. A large number of Glu-positive neurons, however, did not display dendritic labelling and were considered unidentified neurons. Counts of labelled neurons were performed in the striate cortex; approximately 40% were Glu-positive. Numerous lightly stained punctate structures were observed in all cortical layers: the majority of these Glu-positive puncta were in the neuropil. After resectioning the plastic sections for electron microscopy it was observed that: 1) the majority of neurons unidentifiable at light microscopic level were indeed pyramidal neurons except in layer IV of area 17, where many stained cells were probably spiny stellate neurons. Some Glu-positive neurons, however, exhibited clear ultrastructural features of nonspiny nonpyramidal cells; 2) all synaptic contacts made by Glu-positive axon terminals were of the asymmetric type, but not all asymmetric synaptic contacts were labelled. The vast majority of postsynaptic targets of Glu-positive axons were unlabelled dendritic spines and shafts. The present results provide further evidence that Glu (or a closely related compound) is probably the neurotransmitter of numerous excitatory neurons in the neocortex.

Neurotransmitters, receptors, and neuropeptides in the accessory optic system: an immunohistochemical survey in the pigeon (Columba livia).

Immunohistochemical techniques were used to survey the distribution of several conventional transmitters, receptors, and neuropeptides in the pigeon nucleus of the basal optic root (nBOR), a component of the accessory optic system. Amongst the conventional neurotransmitters/modulators, the most intense labeling of fibers/terminals within the nBOR was obtained with antisera directed against glutamic acid decarboxylase (GAD) and serotonin (5-HT). Moderately dense fiber plexuses were seen to label with antibodies directed against tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT). GAD-like immunoreactivity (GAD-LI) was found in many small and medium-sized perikarya within the nBOR. Some of the medium-sized cells were occasionally positive for ChAT-LI. Cell body and dendritic staining was also commonly seen with the two tested antisera against receptors-anti-GABA-A receptor and anti-nicotinic acetylcholine receptor. The antisera directed against various neuropeptides produced only fiber labeling within the nBOR. The densest fiber plexus staining was observed with antiserum against neuropeptide Y (NPY-LI), while intermediate fiber densities were seen for substance P (SP-LI) and cholecystokinin (CCK-LI). A few varicose fibers were labeled with antisera against neurotensin (NT), leucine-enkephalin (L-ENK), and the vasoactive intestinal polypeptide (VIP). Unilateral enucleation produced an almost complete elimination of TH-LI in the contralateral nBOR. SP-LI and CCK-LI were also decreased after enucleation. No apparent changes were seen for all other substances. These results indicate that a wide variety of chemically-specific systems arborize within the nBOR. Three of the immunohistochemically defined fiber systems (TH-LI, SP-LI, and CCK-LI fibers) were reduced after removal of the retina, which may indicate the presence of these substances in retinal ganglion cells. In contrast, the fibers exhibiting ChAT-LI, GAD-LI, 5-HT-LI, NPY-LI, NT-LI, L-ENK-LI, and VIP-LI appear to be of nonretinal origin. Two different populations of nBOR neurons exhibited GAD-LI and ChAT-LI. However, these two populations together constituted only about 20% of the nBOR neurons.

Colocalization of taurine- and cysteine sulfinic acid decarboxylase-like immunoreactivity in the hippocampus of the rat.

It is proposed that taurine is an inhibitory neurotransmitter/neuromodulator in the CNS. The present study localized taurine-containing neurons within the rat hippocampus with the use of a monoclonal antibody against conjugated taurine (Tau2) in conjunction with an antiserum against cysteine sulfinic acid decarboxylase (CSADC), a synthesizing enzyme for taurine. Taurine-like immunoreactivity Tau-LI) and CSADC-LI were colocalized in neurons of the dentate gyrus, CA1(/CA2), CA3, and CA4. Of all the cells examined, pyramidal basket cells within the granule cell layer of the dentate gyrus were most intensely stained with both Tau2 and CSADC. Granule cells were also double-labeled with Tau-LI and CSADC-LI. Cell nuclei and dendrites in the CA1 region stained more intensely with Tau2 than somata. CSADC-LI was colocalized with Tau-LI within these neurons. Light staining with both Tau2 and the CSADC antiserum was inconsistently present in CA3 and CA4 neurons and was found to be highly dependent on the type of fixation and delay to fixation. Tau-LI was more consistently present in increased numbers of neurons in CA3 when glutaraldehyde was added to the paraformaldehyde fixative solution. Hippocampi which were immersion-fixed in paraformaldehyde following a 0-, 6-, or 24-hour postmortem delay exhibited a lack of Tau2 staining in the CA3 region in the majority of animals studied, similar to some paraformaldehyde perfusion-fixed rats. These studies suggest that taurine was present in the majority of neurons within the major cell layers of the rat hippocampus, but Tau-LI was more easily lost from neurons in the CA3 region following delay to fixation. The localization of Tau-LI in excitatory neurons such as granule cells and pyramidal cells is not consistent with its proposed inhibitory transmitter role. However, the prominent Tau2 staining in dendrites of the CA1 region provides anatomical support for the hypothesis that taurine may be released from dendrites in the CA1 region and may function as a neuromodulator of calcium flux in these pyramidal neurons.

Intracellular lucifer yellow injection in fixed brain slices combined with retrograde tracing, light and electron microscopy

The present paper contains a full methodological description of iontophoretic Lucifer Yellow injections in fixed brain slices in mammals. In brief, cortical tissue was either perfused or immersion-fixed in paraformaldehyde. After Vibratome sectioning, tissue slices were transferred to epifluorescence microscopes equipped with long distance objectives. Under visual guidance, neurons were selectively impaled with Lucifer Yellow-filled electrodes and intracellularly injected until all dendrites appeared brightly fluorescent. Excellent dendritic staining was obtained in both perfusion-fixed cat visual cortex and immersion-fixed human brain biopsies. Dendritic spines, varicosities and growth cones could be readily discerned. Filling of axonal collaterals was, however, incomplete. Callosally projecting neurons in cat visual cortex were retrogradely traced with a mixture of the fluorescent tracers Fast Blue and DiI. Subsequently the morphology of labelled cells was determined by intracellular Lucifer Yellow injection. Although the Fast Blue fluorescence had become undetectable in filled neurons the granular red appearance of DiI was still discernible. Hence the neuronal composition of even relatively sparse projections can be demonstrated. To obtain permanent preparations, dye-filled neurons were immersed in a diaminobenzidine solution and irradiated with epifluorescent illumination until all visible fluorescence had faded. Photo-oxidation resulted in the intracellular formation of a homogeneously distributed brown reaction product visible with the light microscope. Brief osmication enhanced the staining contrast, thus providing a Golgi-like image. Subsequent electron microscopy of photo-converted cells showed the fine granular nature of the electron opaque reaction product, thus revealing numerous cytological features. The precipitate was homogeneously distributed throughout the entire cytoplasm and nucleus, extending into dendrites and axon. Any apparent leakage of the label into the extracellular space was not observed. Intracellular staining in fixed tissue yields a high number of neurons with extensive filling of dendritic arbors. Photo-oxidation provides stable, non-fading preparations with the option of subsequent electron microscopy. In addition, the technique can be combined with immunocytochemistry and a variety of fluorescent tracer substances. These features, combined with its high selectivity and relative methodological simplicity, render the method to be a promising alternative to classical neuroanatomical approaches.

Ultrastructural description of taurine-like immunoreactive cells and processes in the rat hippocampus.

A monoclonal antibody against taurine conjugated to KLH was used to identify and describe taurine-like immunoreactive processes in the rat hippocampus. Tissue from perfused rats was processed for immunohistochemical visualization of taurine and embedded for electron microscopy. Representative tissue samples from three regions, the dentate gyrus, CA3, and CA1, were sectioned, examined, and photographed. In the dentate gyrus, both granule cells and pyramidal basket cells were taurine-like immunoreactive. Some axon terminals in the dentate gyrus molecular layer as well as some mossy fiber boutons in the hilus were also taurine-like immunoreactive. In the CA3 region both pyramidal neurons and glial cells were taurine-like immunoreactive A few small-diameter axon terminals in stratum radiatum and some mossy fiber boutons in stratum lucidum were taurine-like immunoreactive. In CA1, pyramidal neurons and some glia were intensely taurine-like immunoreactive. A few immunoreactive axon terminals were seen in stratum radiatum and stratum oriens. In all regions, dendritic staining predominated. Our results support the hypothesis that while taurine may act as a neurotransmitter in a small portion of hippocampal terminals, its main function is probably as a neuromodulator or ionic regulator.

Colocalization of taurine- and cysteine sulfinic acid decarboxylase- like immunoreactivity in the cerebellum of the rat with monoclonal antibodies against taurine

Two monoclonal antibodies against fixative-modified taurine, Tau1 and Tau2, were produced, characterized, and used in the present study to analyze the distribution of taurine in the cerebellum of the rat. In addition, immunohistochemical colocalization experiments were performed to determine whether cerebellar neurons contain both taurine and its synthesizing enzyme, cysteine sulfinic acid decarboxylase (CSADC). In ELISAs, both Tau1 and Tau2 displayed high affinities for taurine conjugated to various carrier proteins and possessed some cross-reactivity for other amino acids which are present in lower concentrations in the brain than taurine. Tau2 was found to recognize only taurine and hypotaurine when paraformaldehyde was used to fix the amino acids to carrier proteins. With the use of glutaraldehyde fixation, Tau1 cross-reacted with conjugates of beta-alanine and hypotaurine and Tau2 cross-reacted strongly with conjugates of cysteic acid and hypotaurine and weakly with cysteine sulfinic acid. Despite different cross-reactivities, Tau1 and Tau2 exhibited almost identical patterns of neuronal staining in bands of Purkinje cells in the cerebellum. Staining of Purkinje cell dendrites was more prominent than staining of the soma. Light immunoreactivity was present in Golgi, stellate, and basket cells. A scattered population of granule cells displayed taurine-like immunoreactivity at the electron microscopic level. Immunostaining was identified in some terminals in the Purkinje cell layer and in a limited number of mossy fibers. Tau2-like immunoreactivity was colocalized with CSADC-like immunoreactivity in the cerebellar neurons described above. These immunoreactive cells may represent a subpopulation of neurons that contain a higher concentration of taurine than neighboring cells due to their ability to synthesize taurine. The intense immunoreactive staining of Purkinje cell dendrites provides support for the hypothesis that calcium-dependent release of taurine in the cerebellum may originate primarily from dendritic rather than synaptic processes and suggests a neuromodulator role for taurine in the cerebellum.

Chronic seizures and collateral sprouting of dentate mossy fibers after kainic acid treatment in rats

Kainic acid (KA) injections destroy hippocampal pyramidal cells, induce recurrent collateral sprouting of the hilar mossy fibers (MFs), and lead to chronic seizures. In the present study, rats were injected systemically with KA (14 mgkg) to determine whether the subsequent occurrence of seizures was correlated with MF sprouting, as indicated by Timm's staining of proximal dendrites. At 4 weeks, 56% of the KA-treated rats had MF sprouting in the temporal (ventral) hippocamopus, and 52% had shown seizures between 10 and 28 days. Both seizures and sprouting were seen in 32% of the treated animals. While Timm's staining of MFs in the inner molecular layer was not directly correlated with seizure scores, animals which exhibited chronic seizures had significantly more sprouting than animals which did not have seizures.

An immunocytochemical and biochemical study of the microtubule-associated protein MAP-2 during post-lesion dendritic remodeling in the central nervous system of adult rats

A monoclonal antibody against the microtubule-associated protein MAP-2 was used to examine the fate of this molecule during post-lesion dendritic remodeling in the hippocampus and septum of adult rats. Qualitative and quantitative immunocytochemical analyses were carried out in the dentate gyrus after unilateral destruction of the entorhinal cortex (EC). An increase in MAP-2 immunoreactivity was detected in dendritic processes located in the outer 2 3 of the ipsilateral molecular layer (ML) 2 days after the lesion, whereas dendritic staining decreased considerably in the inner 1 3 of the same ML. The increase of staining was also detected 4, 6 and 8 days after the lesion; it was accompanied by an increase in the immunoreactivity in the inner 1 3 of the ML. After that period, a progressive decrease in anti-MAP-2 staining toward control levels was detected along the whole extent of the ipsilateral ML. This was concurrent with alterations in dendritic orientation, and a decrease in stained dendrites in the inner 1 3 of the ML. By 30 days post-lesion anti-MAP-2 staining was almost identical to that of the contralateral ML, although the alterations in dendritic morphology were still present in the ipsilateral ML. Changes in MAP-2 levels were also evaluated by densitometry of Western blots or dot immunobinding of hippocampal extracts obtained at different post-lesion intervals. The results obtained revealed a pattern of change in MAP-2 levels identical to that observed with the immunohistochemical stain. A similar, immunocytochemical and biochemical, analysis conducted in the lateral septal nucleus after unilateral transection of the fimbria showed no changes in the distribution and/or content of MAP-2 at any post-lesion interval analyzed (2, 10 and 20 days post-lesion). The present observations show that post-lesion dendritic remodeling is concurrent with modifications in the levels and distribution of MAP-2. These modifications suggest that the dendritic cytoskeleton is dynamically changing in response to perturbation of the synaptic environment. In addition, our results indicate that these changes may only occur in those neurons which have the capability to remodel their post-synaptic surface in response to deafferentation.