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J H Neale

Publications and source records attributed to J H Neale.

At least 19 recordsLinked to original sources
PubMed1992

Uptake, metabolism, and release of N-[3H]acetylaspartylglutamate by the avian retina.

N-Acetylaspartylglutamate (NAAG) is a nervous system-specific dipeptide that is released from retinal neurons on depolarization. In the present study, extracellular metabolism, uptake, and release of [3H]NAAG were examined in the chick retina. After in vitro incubation with NAAG radiolabeled in the glutamate moiety, [3H]glutamate and [3H]NAAG increased in retinal cells through time- and temperature-dependent processes, which were reduced in the absence of extracellular sodium. Coincubation of cells with [3H]NAAG and aspartylglutamate or phosphate resulted in the decreased extracellular appearance of [3H]glutamate, produced by hydrolysis of radiolabeled NAAG, and a consequent increased availability of [3H]NAAG for transport into the retinal cells. When this tissue was incubated with radiolabeled NAAG, glutamate, glutamine, or aspartate under similar conditions, only [3H]NAAG served as a significant source for the appearance of intracellular [3H]NAAG. These data support the conclusion that [3H]NAAG can be transported into retinal cells, whereas [3H]glutamate transport is the predominant process after release of this amino acid from NAAG by extracellular peptidase activities. After uptake, [3H]NAAG entered a cellular pool, from which the peptide was secreted under depolarizing conditions and in a calcium-dependent manner.

Animals↗
PubMed1991

N-acetylaspartylglutamate immunoreactivity in neurons of the monkey's visual pathway.

The acidic dipeptide N-acetylaspartylglutamate (NAAG) was identified immunohistochemically within neurons of the visual pathways of two adult macaque monkeys which had undergone midsagittal sectioning of the optic chiasm 6 or 9 years earlier. In both temporal and nasal retinae, amacrine cells, including some displaced amacrine cells, expressed NAAG immunoreactivity. In temporal but not nasal retina, retinal ganglion cells were stained, as were their dendrites in the inner plexiform layer, and their axons in the optic nerve fiber layer. In nasal retina, the ganglion cells had degenerated because they were axotomized by the optic chiasm section. In the target regions of the retinal ganglion cells, the superior colliculus and the lateral geniculate nucleus (LGN), both neuropil and cell bodies were stained. In LGN, staining was confined to layers 2, 3, and 5, that is, to the layers innervated by the intact ipsilateral pathway. Immunoreactivity was also seen in the cells of layers 2, 3A, 4B, 5, and 6 of area 17 and layers 3 and 5 of area 18. The neuropil was stained in all layers of area 17, but more heavily in layers 1, 2, 4B, the bottom of 4C beta, 5B, and 6B. Within 4C the staining was patchy; in tangential sections there were alternating bands of light and dark label which matched the ocular dominance bands demonstrated by cytochrome oxidase histochemistry in adjacent sections. This banding pattern is consistent with the presence of NAAG in geniculocortical terminals of the intact ipsilateral pathway and the absence of such terminals for the contralateral pathway, which had undergone transneuronal degeneration due to the optic chiasm sectioning. Overall, our results for monkey are very similar to those in cat and suggest that NAAG or a structurally related molecule may have a prominent role in the communication of visual signals at retinal, thalamic, and cortical levels.

Animals↗
PubMed1991

Effect of optic nerve transection on N-acetylaspartylglutamate immunoreactivity in the primary and accessory optic projection systems in the rat.

Evidence has been presented in recent years that support the hypothesis that N-acetylaspartylglutamate (NAAG) may be involved in synaptic transmission in the optic tract of mammals. Using a modified fixation protocol, we have determined the detailed distribution of NAAG immunoreactivity (NAAG-IR) in retinal ganglion cells and optic projections of the rat. Following optic nerve transection, dramatic losses of NAAG-IR were observed in the neuropil of all retinal target zones including the lateral geniculate nucleus, superior colliculus, nucleus of the optic tract, the dorsal and medial terminal nuclei and suprachiasmatic nucleus. Brain regions were microdissected and NAAG levels measured by a radioimmunoassay (RIA) (IC50: NAAG = 2.5 nM, NAA = 100 microM; smallest detectable amount = 1-2 pg/assay). Large decreases (50-60%) in NAAG levels were detected in the lateral geniculate, superior colliculus and suprachiasmatic nucleus. Moderate losses (25-45%) were noted in the pretectal nucleus and the nucleus of the optic tract. Smaller changes (15-20%) were detected in the paraventricular nucleus and the pretectal area. These results are consistent with a synaptic communication role for NAAG in the visual system.

Animals↗
PubMed1991

Immunohistochemical localization of N-acetylaspartate in rat brain.

N-acetylaspartate (NAA) is one of the most prevalent compounds in the mammalian nervous system. As such, NAA largely contributes to the major peak on water-suppressed proton magnetic resonance spectra. Highly specific antibodies to NAA demonstrate that this compound is discretely localized in a substantial number of neurons throughout the extent of the rat CNS. N-acetylaspartylglutamate (NAAG) is a structurally related neuronal dipeptide which is less widely distributed than NAA. NAAG and NAA immunoreactivities were extensively colocalized in many brainstem areas, where NAAG containing neurons were more numerous than in forebrain structures.

Animals↗
PubMed1989

Two forms of the GABAA receptor distinguished by anion-exchange chromatography.

The GABAA receptor complex was solubilized from rat brain membranes in Triton X-100, enriched by 1012-S affinity chromatography, and subjected to DEAE anion-exchange chromatography. Two forms were distinguished by their differential elution during this HPLC with a KCl gradient. They displayed similar [3H]muscimol- and [3H]flunitrazepam-binding characteristics, as well as [3H]flunitrazepam-binding inhibition by CL 218872. Rechromatography of these distinct ionic forms indicated that they were not in dynamic equilibrium during chromatography. Resolution of these two pharmacologically similar populations of GABAA receptor by anion-exchange HPLC suggests that they differ in charge densities, a condition which may reflect differing glycosylation or phosphorylation states of the complex.

Animals↗
PubMed1989

Type I and type II gamma-aminobutyric acid/benzodiazepine receptors: purification and analysis of novel receptor complex from neonatal cortex.

The gamma-aminobutyric acid (GABA) type A receptor was purified several thousandfold by affinity chromatography from rat cerebellum, adult cortex, and neonatal cortex. Competition for the benzodiazepine binding site by CL 218872 indicated that cerebellar receptors were predominantly type I, adult cortical receptors were a mixture of subtypes, and neonatal cortex was enriched in type II receptor. The receptor purified from neonatal cortex contained predominantly a 54-kilodalton (kDa), beta-subunit-like protein, whereas receptors from cerebellum and adult cortex contained nearly equal amounts of a 50-kDa, alpha-subunit-like protein and a 54-kDa polypeptide. Peptide maps of trypsin-digested 54-kDa subunits from cerebellum, adult cortex, and neonatal cortex exhibited very similar profiles, a result indicating considerable homology between these proteins in the receptor subtypes. A 59-kDa subunit protein was detected in the receptor complex purified from neonatal cortex. Like the 50-kDa, alpha-subunit of the type I receptor, this protein was photolabeled with [3H]flunitrazepam. The photolabeled peptide fragments, produced by trypsin digestion of these alpha 50- and alpha 59-subunits, exhibited the same retention times on reverse-phase HPLC. A less highly purified GABAA receptor preparation from adult rat spinal cord possessed characteristics that were very similar to those of the receptors purified from neonatal cortex.

Animals↗
PubMed1989

Immunological identification of multiple alpha-like subunits of the gamma-aminobutyric acidA receptor complex purified from neonatal rat cortex.

Antibodies were prepared against a synthetic peptide corresponding to amino acid sequences 174-203 of the bovine gamma-aminobutyric acidA (GABAA) receptor alpha 1-subunit. The antibodies recognized this synthetic alpha 1-peptide, but failed to react with the homologous peptide sequence, 170-199, of the bovine beta 1-subunit. On Western blots, anti-alpha 1-subunit antibody recognized a 50-kilodalton (kDa) protein in affinity-purified receptor preparations from adult rat cortex and cerebellum. In receptor purified from neonatal cortex, the anti-alpha 1-antibody reacted with 50-kDa, 53-54-kDa, and 59-kDa proteins. After digestion with endoglycosidase F, these three protein bands retained differing electrophoretic mobilities. The 50-kDa and 59-kDa subunits of affinity-purified neonatal receptor, which were photoaffinity-labeled with [3H]flunitrazepam, were immunoprecipitated to different extents by alpha-subunit antibody. These data suggest the existence in GABAA receptor from neonatal cortex of three proteins (50 kDa, 53 kDa, and 59 kDa) which have immunological homology to alpha 1-subunit of bovine GABAA receptor. The presence of an alpha- and a beta-like subunit with similar mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis may account for the relatively high concentration of protein in the 53-54-kDa band which has been observed in receptor purified from neonatal cortex. The presence of multiple alpha-like subunits may be related to the presence of a relatively high concentration of type II GABA receptor in this tissue.

Amino Acid Sequence↗
PubMed1988

Calcium-dependent release of N-acetylaspartylglutamate from retinal neurons upon depolarization.

N-Acetylaspartylglutamate (NAAG) is present in high concentrations specifically in the nervous system. Its neuronal distribution, presence in synaptic vesicles and its excitatory actions support the hypothesis that this dipeptide participates in communication between neurons. Following the incorporation of [3H]glutamate by frog retinal cells in vivo, the release of radiolabeled glutamate, GABA and NAAG was studied during acute incubation of the retina in vitro. Release of the radiolabeled amino acids and dipeptide was stimulated by elevated extracellular potassium. The release required the presence of extracellular calcium. These data are the first which demonstrate the release of NAAG following biosynthesis from a radiolabeled precursor and are consistent with synaptic release of this dipeptide.

Animals↗
PubMed1987

Cellular localization of N-acetylaspartylglutamate in amphibian retina and spinal sensory ganglia.

Antisera were produced against N-acetylaspartylglutamate (NAAG) and were used to localize the molecule within the retina and spinal sensory ganglia of Rana pipiens. NAAG immunoreactivity (IR) in the retina was confined to a subpopulation of amacrine and bipolar cells. The dipeptide was present in the perikarya of these cells and their neurites which terminated in two discrete bands of the inner plexiform layer. Some NAAG-IR was also present in the outer plexiform layer and the inner segment layer. In spinal ganglia, a subpopulation of relatively large sensory neuron cell bodies expressed NAAG-IR. These data are consistent with the hypothesis that this dipeptide has a function which is specific to discrete subclasses of neurons. In the amphibian retina, the NAAG distribution can be related to the reported involvement of the N-methyl-D-aspartate receptor in neurotransmission at the level of amacrine and ganglion cells.

Animals↗
PubMed1987

Immunohistochemistry and biosynthesis of N-acetylaspartylglutamate in spinal sensory ganglia.

N-Acetylaspartylglutamate (NAAG) is a nervous system-specific dipeptide which has been implicated in chemical neurotransmission. Antisera were prepared against NAAG in order to study its cellular distribution. When these antisera were applied to tissue sections of rat spinal sensory ganglia, NAAG-like immunoreactivity was detected within a subpopulation of relatively large neuronal cell bodies in cervical, lumbar, and thoracic ganglia. In order to confirm the presence of NAAG within these neurons, the dipeptide was extracted and purified from spinal ganglia using high-performance liquid chromatography and its composition confirmed by amino acid analysis. Further, the biosynthesis of NAAG was studied in vitro by following the incorporation of either [3H]glutamine or [3H]glutamate into the glutamate residue of the purified dipeptide. [3H]Aspartate was not incorporated efficiently into NAAG under these conditions, suggesting a precursor role for the large N-acetylaspartate pool. The incorporation of radiolabeled amino acids into newly synthesized NAAG by spinal sensory ganglia was not inhibited by incubation of the cells with anisomycin or cycloheximide at concentrations which significantly inhibited protein synthesis. These data suggest that NAAG is present in a subpopulation of primary afferent spinal neurons and that its biosynthesis is mediated by a dipeptide synthetase.

Amino Acids↗
PubMed1986

Localization of dynorphin gene product-immunoreactivity in neurons from spinal cord and dorsal root ganglia.

Using spinal cord and dorsal root ganglion cell cultures, we have studied the immuno-histochemical distribution of several peptide products of the dynorphin gene. With antibody directed toward the midregion of dynorphin A, peptide-immunoreactivity was found exclusively in the cell bodies of spinal cord neurons. Antibody directed toward the amino- or carboxy-terminus of dynorphin A revealed peptide-immunoreactivity in the neurites, as well as perikarya. Spinal cord neurons also expressed dynorphin B- and alpha-neo-endorphin-immunoreactivities in both cell bodies and neurites. Dorsal root ganglion neurons cultured from embryonic tissue expressed dynorphin A-(1-13)-, dynorphin A-(9-17)- and dynorphin B-immunoreactivities in their perikarya. Sensory neurons obtained from dissociated adult ganglia similarly expressed dynorphin-immunoreactivity immediately upon inoculation into culture. Embryonic and adult murine sensory ganglia from the sacral region more frequently expressed dynorphin than did cells obtained from other spinal levels. Expression of dynorphin-immunoreactivity by sensory neurons was not influenced by elevated levels of Nerve Growth Factor or spinal cord conditioned medium. These data indicate that intrinsic spinal cord neurons may modulate sensory and spinal function in rather subtle ways via the expression of several different opioid peptide products of the dynorphin gene, in addition to the opioid peptides produced by the proenkephalin A gene. Beyond this, the observation of dynorphin-related peptides in dorsal root ganglion neurons suggests that these opioid peptides may have a specialized role in primary afferent neurotransmission.

Animals↗
PubMed1978

Enkephalin-containing neurons visualized in spinal cord cell cultures.

Neuronal cells, axons, and terminals containing immunoreactive enkephalin have been visualized in cultures of dissociated fetal spinal cord. These cultures may provide a valuable system in which to explore the effects of chronic drug treatment on the physiology of enkephalin-containing cells and their interactions with other cells.

Animals↗
PubMed1978

Opiate peptide modulation of amino acid responses suggests novel form of neuronal communication.

Mouse spinal neurons grown in tissue culture were used to study the electrophysiological pharmacology of the opiate peptide leucine-enkephalin. Enkephalin depressed glutamate-evoked responses in a noncompetitive manner independent of any other effects on membrane properties. The results demonstrate a neuromodulatory action of opiate peptide functionally distinct from the conventional neurotransmitter class of operation.

Cells, Cultured↗
PubMed1977

Choline acetyltransferase activity of spinal cord cell cultures increased by co-culture with muscle and by muscle-conditioned medium.

Activity of the enzyme choline acetyltransferase (CAT), which mediates the synthesis of the neurotransmitter, acetylcholine, was increased up to 20- fold in spinal cord (SC) cells grown in culture with muscle cells for 2 wk. This increase was directly related to the duration of co-culture as well as to the cell density of both the SC and muscle involved and was not affected by the presence of the acetylcholine receptor blocking agent, alpha-bungarotoxin. Glutamic acid decarboxylase (GAD) activity was often markedly decreased in SC-muscle cultures while the activities of acetylcholinesterase and several other enzymes were little changed. Increased CAT activity was also observed when SC cultures were maintained in medium which had been conditioned by muscle cells or by undifferentiated cells from embryonic muscle. Muscle-conditioned medium (CM) did not affect the activities of SC cell GAD or acetylcholinesterase. Dilution or concentration of the CM directly affected its ability to increase SC CAT activity , as did the duration and timing of exposure of the SC cells to the CM. The medium could be conditioned by muscle cells in the presence or absence of serum, and remained effective after dialysis or heating to 58 degrees C. Membrane filtration data were consistent with the conclusion that the active material(s) in CM had a molecular weight in excess of 50,000 daltons. We conclude that large molecular weight material that is released by muscle cells is capable of producing a specific increase in CAT activity of SC cells.

Acetyltransferases↗