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.

Radioautography of the optic tectum of the goldfish after intraocular injection of ( 3 H)proline.

Radioautography of the optic tectum of the goldfish, performed after injection of [(3)H]proline into the contralateral eye, effectively resolves several distinct layers of retinal synapses. Silver grains are found unilaterally over nerve tracts containing efferent fibers from the tectum, a result that suggests intercellular migration of labeled molecules. The low background and high specific grain density obtained with [(3)H]proline radioautography indicate the usefulness of this technique for the elucidation of neuroanatomical connections in the visual system.

Camptothecin blocks memory of conditioned avoidance in the goldfish.

Intracranial injection of 10 to 75 micrograms of camptothecin, a plant alkaloid that blocks RNA synthesis in eucaryotic cells, blocks incorporation of tritiated uridine into RNA in the goldfish brain. Injection of 10 to 50 micrograms of the drug within 1.5 hours of training results in greatly diminished memory, tested 1 week later. Injection of the drug 5 or 24 hours after training produces no measurable effect on retention of the learned response.

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.

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.

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.

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.

Comparative distribution of N-acetylaspartylglutamate and GAD67 in the cerebellum and precerebellar nuclei of the rat utilizing enhanced carbodiimide fixation and immunohistochemistry.

The most prevalent peptide in the nervous system, N-acetylaspartylglutamate (NAAG), specifically activates N-methyl D-aspartate (NMDA) receptors and a subclass of metabotropic glutamate receptors. One action of this peptide may be to modulate the release of other neurotransmitters, including gamma-aminobutyric acid (GABA). The present study describes the cellular distribution of NAAG, relative to GABA, in the cerebellum and precerebellar nuclei as a foundation for further physiological investigations. Numerous cells of origin for mossy fibers, including many of the larger neurons of the pontine nuclei, lateral reticular nuclei, vestibular nuclei, reticulotegmental nuclei, and spinal grey, were moderately to strongly stained for NAAG. Many NAAG-labeled fibers were clearly visible in the cerebellar peduncles and central white matter. Mossy fibers and mossy endings were among the most prominent NAAG-immunoreactive elements in the cerebellar cortex. Most neurons in the inferior olive were not stained for NAAG, and only sparse, lightly immunoreactive, climbing fiber-like endings could be identified in restricted regions of the cortical molecular layer. Purkinje neurons ranged from nonreactive to moderately positive, with the great majority being unstained. Cerebellar granule cells did not exhibit any NAAG immunoreactivity. A population of neurons in the deep cerebellar nuclei was highly immunoreactive for NAAG. Additionally, many neurons of the red nucleus were intensely stained for NAAG. Comparisons with staining for the 67 kD form of glutamic acid decarboxylase in serial sections revealed complementary distributions, with NAAG in excitatory pathways and cell groups, and glutamic acid decarboxylase in inhibitory systems. These findings suggest a significant functional involvement of NAAG in the excitatory afferent and efferent projection systems and provide an anatomical basis for investigations into the interactions of NAAG and GABA in the cerebellum.

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.

Enhanced carbodiimide fixation for immunohistochemistry: application to the comparative distributions of N-acetylaspartylglutamate and N-acetylaspartate immunoreactivities in rat brain.

To improve carbodiimide-based immunohistochemistry, carbodiimide-mediated coupling of radiolabeled N-acetylaspartylglutamate (NAAG) to bovine serum albumin was assayed in vitro. Various perfusion protocols, based on assay results, were tested for their ability to improve the immunohistochemical localization of two nervous system-specific molecules, NAAG and N-acetylaspartate (NAA) in the spinal cord, medulla, hippocampus, and cerebral cortex of the rat. Coupling of [3H]-NAAG to BSA in vitro was optimal with 100 mM carbodiimide and 1 mM N-hydroxysuccinimide in water at 37 degrees C. Optimal fixation of tissue was defined as permitting the identification of the NAAG and NAA in neuronal somata, dendritic arborizations, fine axons, and synaptic terminals with minimal diffuse background immunoreactivity. These conditions were obtained at 37 degrees C with 6% carbodiimide, 1 mM N-hydroxysuccinimide, and 5% dimethylsulfoxide perfused transcardially. Strong NAAG and NAA immunoreactivities were co-distributed in the majority of neurons in the spinal cord. Large-diameter spinal sensory afferents were stained for NAAG in the dorsal horn. The dorsal column nuclei were immunoreactive for NAAG and NAA, but only NAA staining was observed in the nucleus of the solitary tract. In cerebral cortex and hippocampus, NAAG and NAA immunoreactivities appeared to be exclusive, with NAAG staining observed in interneurons throughout all cortical layers, and NAA immunoreactivity present in most pyramidal neurons.

Ribozyme-mediated reduction of the GABA(A) receptor alpha1 subunit.

As an approach to understanding the role of the alpha1 subunit of the GABA(A) receptor, ribozymes were designed to reduce expression of this subunit protein by hydrolysis of alpha1 subunit message and antisense inactivation. The ribozyme cleavage sites were selected through homology comparison of all known murine GABA(A) receptor subunits at the amino acid and nucleotide sequence level. Two ribozymes were designed and synthesized: one against the extracellular domain and the other against the cytoplasmic domain. These ribozymes were cloned in a mammalian expression plasmid, pZeoSV2 (+). Cleavage of both extracellular and cytoplasmic domain transcripts by the respective ribozymes was observed when each ribozyme was tested against in vitro transcribed mRNA. The stable cell line, 122, expressing recombinant human GABA(A) alpha1, beta2 and gamma2S subunits of receptor was stably transfected with the cytoplasmic domain ribozyme (cy) alone and with both the cytoplasmic (cy) and extracellular domain (ex) ribozyme expression plasmids. Northern analysis showed a 55-60% reduction of alpha1 mRNA in clones of cells transfected with either the single ribozyme (Cy) or with both ribozymes (EC). The alpha1 protein level was reduced 75% in a stable Cy clone and more than 90% in a stable EC clone when compared with alpha1 expression in 122 cells and the vector transfected (Zeo) cells. Electrophysiological analysis revealed that the GABA(A) receptor properties were very similar in 122 cells and in stable clones in which the subunit protein expression had been greatly reduced. No significant difference was detected in the potentiation of the receptor response by either bretazenil or zolpidem. These data demonstrate the efficacy of the ribozyme approach in dramatically reducing GABA(A) subunit protein levels in transfected cells and identify those elements that will be important to the application of similar ribozymes to knock-down transmitter receptor subunit proteins under inducible promoters in transgenic mice.

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.

N-acetylaspartylglutamate catabolism is achieved by an enzyme on the cell surface of neurons and glia.

N-Acetylaspartylglutamate (NAAG) is a nervous system-specific, acidic dipeptide which is released from neurons in a manner consistent with a function in synaptic neurotransmission. The hydrolysis of NAAG to produce glutamate and N-acetylaspartate was analyzed in cell cultures prepared from murine brain cells. Peptidase activity against NAAG was found in cultures which contained both neurons and glia, as well as in cultures of glia alone. Several lines of evidence were obtained in support of the hypothesis that this peptidase activity is predominantly bound to the extracellular face of the plasma membranes of these cells. Glutamate released from NAAG accumulated in the extracellular medium. Extracellular application of peptidase inhibitors effectively reduced NAAG hydrolysis. Peptidase activity was not secreted into the cell culture medium by intact cells, and lysed cells did not release detectable peptidase activity beyond that obtained with intact cells. Replacement of extracellular sodium with choline inhibited peptide uptake while stimulating apparent extracellular NAAG hydrolysis by intact cells in culture. Finally, the steady rise in extracellular glutamate as a consequence of NAAG hydrolysis by these brain cells, including glia, supports the conclusion that glutamate uptake is not tightly coupled to peptidase activity and thus that NAAG serves as a significant source of glutamate in the synaptic space following depolarization-induced peptide release.

Benzodiazepine receptor binding by membranes from brain cell cultures following chronic treatment with diazepam.

Murine brain cells differentiated for 19 days in culture before treatment for 5 days with 10 microM diazepam. Radioligand binding to the benzodiazepine receptors on membranes obtained from these cultures was determined by the filtration assay method. Decreased binding was observed in the membranes from treated cultures relative to untreated cells. However, this decrease appears to be due, at least in part, to competition from residual drug in the assay system despite extensive cell and membrane rinses. These data emphasize the difficulty in ascribing the mechanism of benzodiazepine tolerance observed clinically with chronic treatment to receptor down-regulation as determined by binding assays.

Ultrastructural localization of N-acetylaspartylglutamate in synaptic vesicles of retinal neurons.

Localization of N-acetylaspartylglutamate (NAAG) in a variety of central and peripheral neurons, as well as its receptor-mediated activation of membrane conductance have led to speculation that this peptide has a role in chemical neurotransmission. We previously identified NAAG in retinal neurons of several species, including the grass frog, and now have determined its ultrastructural distribution within the plexiform layers of this amphibian retina. NAAG immunoreactivity was localized within vesicles in synaptic endings of presumptive amacrine and bipolar neurons in the inner plexiform layer. Additionally, the peptide was present in vesicles within ribbon synapses in the outer plexiform layer, a result suggestive of release from photoreceptor cells. These data support the hypothesis that NAAG is secreted at points of synaptic contact between neurons, including retinal amacrine, bipolar and photoreceptor cells.

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.

Rapidly transported proteins in sensory, motor and sympathetic nerves of the isolated frog nervous system.

The synthesis and rapid axonal transport of [35S]methionine-labelled proteins has been studied using the isolated frog spinal cord and peripheral nervous system. Polyaerylamide gel electrophoresis in sodium dodecylsulfate of synthesized and transported proteins revealed similar labelling patterns of proteins transported in the sensory, motor and sympathetic systems. The relative labelling pattern of transported proteins which accumulated at ligatures in peripheral nerves was different from those obtained from ganglia or nerves and roots when they were incubated in labelled methionine. When compared with methionine-labelled protein profiles of rapid axonal transport in other species and systems, a common set of rapidly transported proteins emerges. The approximate molecular weights of these common proteins include (in 1000 daltons): 18,24-29, 34-36, 57, 65-68, 100 and 130. These proteins may represent fundamental macromolecules involved in the general maintenance of the function of nerve processes.

Immunohistochemical localization of the pro-opiomelanocortin-gene product, glycylglutamine, in the intermediate pituitary.

Glycylglutamine, the carboxyterminal sequence of beta-endorphin1-31, is produced as a free dipeptide during the posttranslational synthesis of beta-endorphin1-27. Antisera which recognize glycylglutamine were raised in rabbits and used for immunohistochemistry. With these antisera, glycylglutamine immunoreactivity was demonstrated in cells of the rat intermediate pituitary. In contrast, anterior pituitary cells, which exhibited beta-endorphin immunoreactivity, did not react with the anti-glycylglutamine sera. The conclusion that the antisera distinguished glycylglutamine immunoreactivity from beta-endorphin1-31 immunoreactivity is based upon cellular specificity, fixation requirements and blocking studies. The antisera demonstrated the differential expression of this dipeptide product of the proopiomelanocortin prohormone. The efficacy of carbodiimide as an immunohistochemical fixative for small molecules is also shown.

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.

N-acetylaspartylglutamate immunoreactivity in neurons of the cat's visual system.

The acidic dipeptide, N-acetylaspartylglutamate (NAAG) was identified immunohistochemically within neurons of the cat's visual system. In the retina, NAAG-like immunoreactivity was observed in some horizontal and amacrine cells at the inner and outer margins of the bipolar cell layer. NAAG-like immunoreactivity was also observed in many retinal ganglion cell bodies, their neurites, and the neuropil of their target areas, the lateral geniculate nucleus (LGN) and the superior colliculus. Additionally, peptide immunoreactivity was also seen in the projection neurons of the LGN, in cells of the pulvinar nucleus, and in the pyramidal cells of layers III and V in areas 17, 18 and 19 of the cerebral cortex. These data 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.