Taurine-like Immunoreactivity Research Articles

Immunoreactivity for Taurine Characterizes Subsets of Glia, GABAergic and non-GABAergic Neurons in the Neo- and Archicortex of the Rat, Cat and Rhesus Monkey: Comparison with Immunoreactivity for Homocysteic Acid.

The cerebral cortex is an area rich in taurine (2-aminoethanesulphonic acid), but only limited information exists regarding its cellular distribution. We therefore examined taurine-like immunoreactivity in the cerebral cortex of the rat, cat and macaque monkey using antiserum directed against glutaraldehyde-conjugated taurine. Immunostaining was assessed at the light and electron microscopic level, and patterns obtained in light microscopic studies were compared to those produced with antiserum to gamma-aminobutyric acid (GABA) and homocysteic acid (HCA). In all three species, strong taurine-like immunoreactive perivascular endothelial cells, pericytes and oligodendrocytes were found. These cells were located throughout the neuropil, which itself showed a low level of immunoreactivity. In rats and cats, a small number of weakly taurine-enriched neurons were observed, particularly in superficial layers. In all cortical areas of the macaque, however, glial staining was matched by strong, selective staining of subpopulations of cortical neurons which were distributed in a bilaminar pattern involving layers II/III and VI. In addition, in primary visual cortex, area 17, immunopositive neurons were also present in sublayer IVCbeta, while in the hippocampus strongly taurine-positive neurons were most conspicuous in the granule cell layer of the dentate gyrus. In all regions, strongly taurine-positive neurons constituted only a subpopulation of the neurons occupying a given layer. Examination of adjacent sections for GABA immunoreactivity showed that the most strongly taurine-positive neurons in layers II/III were immunoreactive for GABA. The cells located in layers IVCbeta and VI, and the granule cells of the dentate gyrus, however, were GABA-negative. The morphological features of these latter groups suggested that the antiserum to taurine identifies subsets of spiny stellate, small pyramidal and dentate granule cells. None of these neurons showed immunoreactivity with antiserum to HCA in the primate; HCA-positive glia were found along the pial and white matter boundaries of the cortex, and showed no overlap with strongly taurine-positive glial elements. Although a transmitter role for taurine may be unlikely, particularly in view of its enrichment in subpopulations of both inhibitory and excitatory cells, the capacity of taurine to influence membrane-associated functions in excitable tissues, and its selective distribution demonstrated here, provides the potential for a contribution to communication between cortical cells.

Differential cellular distribution of two sulphur-containing amino acids in rat cerebellum. An immunocytochemical investigation using antisera to taurine and homocysteic acid.

An antiserum to homocysteic acid was raised in rabbits. Immunogens were prepared by coupling this amino acid to bovine serum albumin by means of glutaraldehyde and paraformaldehyde. When applied to semithin or ultrathin sections of rat cerebellum, the antiserum produced selective labelling of glial cells and processes, including the Bergmann fibers. No enrichment of immunoreactivity was detected in nerve terminals of the major excitatory fiber systems. The distribution of homocysteic acid-like immunoreactivity was very different from that of taurine (another sulphur-containing amino acid), as judged from consecutive semithin sections labelled with a postembedding immunoperoxidase procedure and from ultrathin sections labelled with a postembedding double immunogold procedure. Taurine-like immunoreactivity was concentrated in Purkinje cells and was low in glial elements. Our data suggest that the cerebellum contains a glial pool of homocysteic acid (and/or precursors that may undergo spontaneous oxidation to homocysteic acid) and that this amino acid is unlikely to act as a cerebellar transmitter.

Taurine-like immunoreactivity in photoreceptor cells and mushroom bodies: a comparison of the chemical architecture of insect nervous systems

Taurine is one of the most abundant amino acids found in the tissues of insect nervous systems. The distribution of taurine-like immunoreactivity was investigated in compound eyes, ocelli and mushroom bodies of Drosophila and Locusta. A comparison to the previously described taurine-like immunoreactivity in the brain of worker honeybees ( J. Comp. Neurol., 268 (1988) 60–70) showed that the photoreceptor cells of all three insect species are immunoreactive. Immunoreactive mushroom body intrinsic Kenyon cells were also found in all three species. The intensity of the immunoreactivity was however graded, depending on the species. While the majority of Kenyon cells were stained in Apis and Drosophila, the immunoreactivity of the locust mushroom body was mainly confined to the intrinsic neurons originating in the accessory calyx.

Distribution of taurine-like immunoreactivity in cerebellum of kittens from taurine-supplemented and taurine-deficient mothers

Using an antibody prepared against taurine conjugated to bovine serum albumin with glutaraldehyde, the distribution of taurine in cerebellum of newborn and 8-week-old kittens from mothers fed 0, 0.02, 0.05, or 1% dietary taurine has been determined. In general, taurine-like immunoreactivity was greater in kittens from mothers fed the greatest amounts of taurine, as was the total cerebellar taurine concentration. The most notable feature in newborn kitten cerebellum was a dense band of staining in the inner molecular layer adjacent to the Purkinje cell layer, which corresponds to the short Purkinje cell dendrites. In cerebellum of 8-week-old kittens, taurine-like immunoreactivity was present in Purkinje cells and their dendrites, most granule cells, and a few interneurons in the molecular layer of the 0.02, 0.05, and 1% groups. The cerebellum of the 0% group was distinctive in that virtually no neurons were reactive, appearing as ‘ghosts’ against the background, and both white matter and the granule cell layer contained large numbers of reactive astrocytes. The presence of such large numbers of reactive astrocytes and the immunoglobulin within the brain suggests an impairment of the blood-brain barrier in such taurine-deficient kittens.

Ultrastructural description of glutamate‐, aspartate‐, taurine‐, and glycine‐like immunoreactive terminals from five rat brain regions

The ultrastructural localization of putative excitatory (glutamate, aspartate) and inhibitory (taurine, glycine) amino acid neurotransmitters is described in several selected rat brain regions. In general, axon terminal profiles immunoreactive for excitatory amino acids formed asymmetric synapses with non-immunoreactive small diameter dendritic profiles or dendritic spines. In the cerebellum, both mossy fiber terminals and parallel fiber terminals were immunoreactive for glutamate and aspartate. In the hippocampus, mossy fiber terminals within the stratum lucidum of the CA3 region were immunoreactive for glutamate. Localization of glutamate and aspartate to cerebellar parallel and mossy fibers, as well as the identification of glutamate in hippocampal mossy fibers, is consistent with the excitatory nature of these fibers as described in previous physiological studies. Glutamate-like immunoreactive terminals were also identified in subnucleus caudalis of the spinal trigeminal nucleus and in the dorsal horn of the spinal cord. Immunoreactive axon terminals for two putative inhibitory neurotransmitters, glycine and taurine, displayed a greater number of morphological variations in synaptic structure. In the cerebellum, taurine-like immunoreactivity was present in both basket cell axon terminals which formed symmetric synapses with Purkinje cell neurons, and in a few mossy fiber terminals which formed asymmetric synapses with dendritic spines. In the area dentata of the hippocampus, taurine-like immunoreactive profiles formed asymmetric synapses with dendritic elements. Glycine-like immunoreactive terminals formed symmetric synapses with cell perikarya in both the ventral horn of the spinal cord and in the cochlear nuclei, and on axon terminals in the spinal trigeminal and cochlear nuclei. In contrast, some glycine-like immunoreactive terminals formed asymmetric synapses with distal dendritic profiles in the spinal cord and spinal trigeminal nucleus. The localization of taurine to cerebellar basket cell axons and glycine to axon terminals that synapse on ventral horn motor neuron perikarya is consistent with the hypothesis that these amino acids are functioning as inhibitory neurotransmitters at these synapses. Taurine localization to cerebellar mossy fibers and to fibers in the molecular layer of the dentate gyrus may be more consistent with a proposed neuromodulator role of taurine.

GABA, glycine, aspartate, glutamate and taurine in the vestibular nuclei: an immunocytochemical investigation in the cat

The distributions of five amino acids with well-established neuroexcitatory or neuroinhibitory properties were investigated in the feline vestibular complex. Consecutive semithin sections of plastic-embedded tissue were incubated with antisera raised against protein-glutaraldehyde conjugates of GABA, glycine, aspartate, glutamate and taurine. This approach allowed us to study the relative densities of the different immunoreactivities at the level of individual cell profiles. The results indicate that in the vestibular nuclei, neuronal colocalization of two or more neuroactive amino acids is the rule rather than an exception. Colocalization was found of immunoreactivities for GABA and glycine; glycine, aspartate and glutamate; glycine and aspartate, and glutamate and aspartate. GABA immunoreactive neurons were generally small and were found scattered throughout the vestibular complex. Glycine immunoreactive neurons were similarly distributed, except in the superior nucleus where the latter type of neuron could not be detected. Neuronal profiles colocalizing immunoreactivities for GABA and glycine occurred in all nuclei, but were most numerous in the lateral nucleus. The vast majority of the neurons showed noteworthy staining for glutamate and aspartate, although the level of immunoreactivities varied (e.g., the large neurons in the lateral and descending nuclei were more intensely aspartate immunoreactive than the smaller ones). Taurine-like immunoreactivity did not occur in neuronal cell bodies but appeared in Purkinje cell axons and in glial cell profiles. The functional significance of the complex pattern of amino acid colocalization remains to be clarified. In particular it needs to be distinguished between metabolic and transmitter pools of the different amino acids. The present results call for caution when attempts are made to conclude about transmitter identity on the basis of amino acid contents alone.

Taurine-like immunoreactivity in the bullfrog sympathetic ganglia and sciatic nerves: Comparison to autoradiographic study

Taurine-like immunoreactivity in the bullfrog sympathetic ganglia and sciatic nerves: Comparison to autoradiographic study

Taurine-like immunoreactivity in the bullfrog sympathetic ganglia and sciatic nerves: Comparison to autoradiographic study

Taurine-like immunoreactivity in the bullfrog sympathetic ganglia and sciatic nerves: Comparison to autoradiographic study

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.

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.

Immunocytochemical evidence suggests that taurine is colocalized with GABA in the Purkinje cell terminals, but that the stellate cell terminals predominantly contain GABA: a light- and electronmicroscopic study of the rat cerebellum

The distributions of taurine-like and GABA-like immunoreactivities in the rat cerebellum were compared by analysis of consecutive semithin and ultrathin sections, postembedding labeled with the peroxidase-antiperoxidase technique or with an indirect immunogold procedure, respectively. Taurine-like immunoreactivity was selectively enriched in Purkinje cell bodies, dendrites and spines, and boutons in the cerebellar nuclei exhibiting ultrastructural features typical of Purkinje cell terminals. The stellate and basket cell bodies and terminals were very weakly labeled. A computer assisted quantitative assessment of the net immunogold labeling revealed that the mean gold particle density in the Purkinje cell terminals was about 70% higher than that in the Purkinje cell dendrites, and about 14 times higher than that in the stellate/basket cell terminals in the molecular layer. Stellate, basket and Purkinje cell terminals emerged as intensely immunoreactive in adjacent sections processed with an antiserum against conjugated GABA. These findings indicate, contrary to recent electrophysiological data, that GABA is a more likely transmitter candidate than taurine in the stellate cells. The apparent colocalization of GABA and taurine in the terminals of Purkinje cells raises the possibility that these terminals are capable of releasing two different inhibitory amino acids.

Quantitative assessment of taurine-like immunoreactivity in different cell types and processes in rat cerebellum: an electronmicroscopic study based on a postembedding immunogold labelling procedure

Ultrathin sections of plastic-embedded rat cerebella were incubated with an antiserum against conjugated taurine and subsequently treated with a secondary antibody coupled to colloidal gold. The density of gold particles in various cellular profiles was calculated with the assistance of a computer. In the cerebellar cortex the highest density was found in the somata, dendrites, and dendritic spines of the Purkinje cells, supporting parallel light-microscopical observations in postembedding stained semithin sections from the same tissue blocks. The remaining profiles could be divided into three groups according to their immunolabelling intensity, in descending order: 1) somata and processes of granule and Golgi cells; 2) somata and processes of stellate, basket, and glial cells, and 3) mossy fiber terminals. In a representative experiment, the structures in the first and second groups showed gold particle densities in the range of 19-25%, and 4-11%, respectively, of that in the Purkinje cell somata (values corrected for background) whereas the particle density in the mossy fiber terminals was not significantly above background level. In the cerebellar nuclei, taurine-like immunoreactivity was concentrated in terminals that typically established symmetric or intermediate type contacts with weakly labelled dendrites and cell bodies. These terminals, which shared the ultrastructural features of Purkinje cell terminals, showed an average gold particle density that was about 60% higher than that of the Purkinje somata. For specificity control, ultrathin sections containing a series of different amino acid conjugates were incubated in the same drops of sera as the tissue sections. The highly selective labelling of the taurine conjugate indicated that the distribution of gold particles in the tissue was not confounded by crossreactivity with GABA, glutamate or other common amino acids but adequately reflected the distribution of fixed taurine. For additional control of specificity, the taurine antiserum was applied to the soluble fraction of a rat brain extract separated by thin layer chromatography. In this system the taurine antiserum stained a single spot that comigrated with free taurine. The present results suggest that all cell types and processes in the rat cerebellum (with the exception of the mossy fiber terminals) contain taurine. However, the concentration of taurine appears to vary considerably among the different cell types and may also differ between different parts of the same neuron.

Taurine-like immunoreactivity in the brain of the honeybee.

Taurine (2-aminoethanesulfonic acid) is one of the most abundant free amino acids in the insect central nervous system. We have investigated the distribution of taurine-like immunoreactivity in the brain of the honeybee with an antiserum recognizing fixed taurine. Taurine-like immunoreactivity appeared within neuronal perikarya, neurites, and terminals, whereas glial cells were unlabelled. All photoreceptor cells of the compound eyes and the ocelli were stained. So were the fibers of the anterior superior optic tract, which connects the optic lobes to the mushroom bodies in the median protocerebrum. In the mushroom bodies the majority of intrinsic Kenyon cells showed high levels of taurine-like immunoreactivity. The lateral antennoglomerular tract, which interconnects the mushroom bodies with the antennal lobes, was also intensely stained. In the antennal lobes, strong labelling was observed within a few fibers that invade a set of posterior glomeruli from the posterior margin. Sensory projections from the antennal nerve into the antennal lobes showed only intermediate levels of staining. Sensory projections into the dorsal lobe were devoid of taurine-like immunoreactivity. Labral, mandibular, maxillary, and labial nerves, which innervate the various parts of the feeding apparatus, contain a set of five to eight heavily stained fibers. A comparison of taurine-like immunoreactivity with glutamate- and GABA-like immunoreactivities in the brain of the honeybee indicates that the three amino acids are enriched in distinct neuronal populations.

Immunohistochemical and biochemical studies of substances with taurine-like immunoreactivity in the brain.

Taurine-like immunoreactivity (TLI) in the brain was studied by immunohistochemical and biochemical approaches. The immunohistochemical examination, using different fixatives or taurine antisera preabsorbed with crossreactive substances, demonstrated that TLI staining in the striatum differed from that in the cerebellum. The results suggested that TLI staining should be attributed to unknown substances as well as the amino acid taurine. Immunohistochemical results using a new antiserum raised against γ-glutamyl-taurine supported this assumption. Furthermore, we attempted to extract TLI-positive materials from bovine brain by ion-exchange chromatography and high performance liquid chromatography. A novel immunoreactive substance was isolated and it was distinct immunochemically from either taurine, γ-glutamyl-taurine or glycyl-taurine.

Immunohistochemical and biochemical studies of substances with taurine-like immunoreactivity in the brain

Immunohistochemical and biochemical studies of substances with taurine-like immunoreactivity in the brain

Antisera against taurine: quantitative characterization of the antibody specificity and its application to immunohistochemical study in the rat brain.

An antiserum against taurine was generated in rabbits by immunization with taurine that was conjugated to carrier protein via glutaraldehyde. When the antiserum was applied to immunohistochemistry, structures with taurine-like immunoreactivity were observed in both neuronal and glial components of rat brain. The specificity of the serum was quantitatively examined using an enzyme-linked immunoassay method, which has been newly developed for detecting such small molecules as amino acids. The best serum obtained had a high titer against taurine. It showed low cross-reactivity with taurine metabolites and with other amino acids (less than 0.5%), while considerably higher reactivities were noted in the case of taurine-containing peptides such as gamma-glutamyl-taurine and glycyl-taurine. The results indicate that much care should be taken with taurine peptides in the evaluation of immunohistochemical results using taurine antiserum.

Demonstration of taurine-like immunoreactive structures in the rat brain

An antiserum was raised in a rabbit by immunization with taurine bound to bovine serum albumin. The antibody was purified by passage over an immunoabsorbent column (formyl-cellulofine-taurine) and it did not cross-react significantly with glutamate, aspartate, glycine, GABA (0.4%), glutamine, proline, cysteine, β-alanine, cysteic acid, carnosine or homocarnosine in enzyme-linked immunosorbent assays and nitrocellulose paper immunoblots. Immunocytochemical studies employing the peroxidase-antiperoxidase immunohistochemical technique revealed that many cerebellar Purkinje cells showed taurine-like immunoreactivity. Labelled axons could be followed within the white matter up to the deep cerebellar nuclei, where numerous puncta were observed. Immunoelectron microscopic examination revealed that labelled puncta were presynaptic terminals, and axo-dendritic or axo-somatic symmetrical synapses were observed on deep cerebellar nuclear neurons.

Potassium-evoked release of taurine from synaptosomal fractions of rat cerebral cortex

Rat cerebellar Purkinje cells show a high level of taurine-like immunoreactivity. Light-microscopic immunocytochemisty indicated that the level of taurine in these cells was substantially decreased in animals that had survived for 4 h after an intraperitoneal injection of distilled water. This treatment resulted in a 15–20% reduction in plasma osmolality. The changes in the Purkinje cells were accompanied by an increased immunolabeling of neighboring glial cells (Golgi epithelial cells). The changes in both cell types were reversed in animals whose plasma osmolality had been normalized by injections of hypertonic saline 4 h after the water loading. Adjacent sections incubated with a GABA antiserum did not exhibit any overt changes in response to the hypo-osmotic stress.Quantitative electron-microscopic analysis of ultrathin sections subjected to postembedding immunogold cytochemistry indicated that the Purkinje cells had lost 50–60% of their taurine contents after water loading and that the loss affected all intracellular compartments, including mitochondria and cytoplasmic matrix. The loss of taurine immunoreactivity from Purkinje cells was accompanied by an estimated 70–80% increase in the contents of immunoreactive taurine in adjacent glial cells.Biochemical recordings of tissue amino acids in a parallel series of animals revealed a 12% reduction in cerebellar taurine contents 4 h after water loading (value corrected for changes in specific gravity). This reduction had progressed to 32% after 8 h and was only partly prevented by normalization of plasma osmolality. The tissue levels of GABA and several other amino acids showed a decrease similar to that of taurine, while glutamine displayed a considerable increase after water loading.Our findings indicate that acute reductions in plasma osmolality cause a flux of taurine from Purkinje cells to glia, and that this flux is reversed upon normalization of plasma osmolality. These changes are superimposed on a decrease in the biochemically recorded tissue level of taurine. Unlike the cellular redistribution, this decrease was not reversible within the time frame of the present study, and it was not specific for taurine. Cellular redistribution of taurine may represent a rapid adjustment to osmotic perturbations in vivo. In addition, it may reflect a higher priority for neuronal compared with glial volume regulation.