Selective retrograde transport of D-aspartate in spinal interneurons and cortical neurons of rats

Retrograde labeling of neuronal elements in the brain and spinal cord has been investigated by autoradiographic techniques following injections of D-[3H]aspartate (asp), [3H] gamma-aminobutyric acid (GABA) or horseradish peroxidase (HRP) in the medulla and spinal cord of rats. Twenty-four hours after D-[3H]asp injections focused upon the cuneate nucleus, autoradiographic labeling is present over fibers in the pyramidal tract, internal capsule and over layer V pyramids in the forelimb representation of the sensorimotor cortex. After [3H]GABA injections in the same nucleus no labeling attributable to retrograde translocation can be detected in spinal segments, brain stem or cortex. Conversely, injections of 30% HRP in the cuneate nucleus label neurons in several brain stem nuclei, in spinal gray and in layer V of the sensorimotor cortex. These observations give further support to the proposed existence of a selective retrograde transport of D-[3H]asp and are consistent with the available evidence which indicates that the corticodorsal column nuclei path use glutamate and/or aspartate as neurotransmitter(s). D-[3H]Asp injections focused on the dorsal horn at cervical segments label a fraction of perikarya of the substantia gelatinosa and a sparser population of larger neurons in laminae IV to VI for a distance of 3-5 segments above and below the injection point. No brain stem neuronal perikarya appear labeled following spinal injections of D-[3H]asp although autoradiographic grains overlie pyramidal tract fibers on the side contralateral to the injection. This labeling however has not been observed rostral to lower pontine levels nor over cortical neurons at any of the survival times used in the present experiments (6-72 h). As in cases with cuneate injections this pattern of labeling contracts with that obtained after spinal injections of either [3H]GABA or HRP. Although labeling of neocortical neurons has not been observed after spinal injections of D [3H]asp, possibly as a result of the length of corticospinal axons, retrograde labeling of these elements for at least some distance may be taken as suggestive of a special affinity of their terminals for glutamate and/or aspartate.     

Combined immunocytochemistry and autoradiographic retrograde axonal tracing for identification of transmitters of projection neurons

A double labeling method is described which combines immunocytochemistry for identification of the neurotransmitter serotonin with autoradiographic retrograde axonal tracing using wheat germ agglutinin (WGA), N-[acetyl-³H]. The permanence, sensitivity, and distinctness of the two labels provide a valuable means for analyzing transmitter-identified projection neurons in the central nervous system. Combined immunohistochemical/autoradiographic preparations, following injections of WGA, N-[acetyl-³H] in the caudate-putamen of mice, revealed large numbers of serotonergic and fewer non-serotonergic raphe-striatal projection neurons.     

The intercollicular pathway in the golden hamster: An anatomical study

The intercollicular pathway of the hamster was studied by means of a combination of horseradish peroxidase (HRP), autoradiographic, and double-labeling (nuclear yellow-HRP) techniques. Small deposits of HRP marked significant numbers of cells in the contralateral colliculus only when the injection site included the laminae ventral to the stratum opticum. Anterior deposits labeled many more neurons than injections into the caudal part of the tectum. Of the cells labeled by our injections, 18.2% were located in the superficial collicular laminae (stratum griseum superficiale and stratum opticum), and the remainder (81.8%) were in the deep layers. A wide variety of morphological cell types contributed axons to the intercollicular projection, and in a given animal the loci of the labeled neurons were generally symmetrical with the injection site. Small deposits of [³H]-leucine resulted in contralateral labeling only when the injection included the deep collicular laminae. The transported label was most dense in the stratum griseum intermediale and stratum griseum profundum, and its location was generally homotopic with the injection site. Experiments in which collicular HRP deposits were combined with large cervical spinal or pontine reticular injections of Nuclear Yellow indicated that intertectal neurons did not, in most cases, contribute axon branches to the spinal or pontine reticular projections of the colliculus. Receptive field data obtained at the time of the HRP and/or [³H]-leucine deposits demonstrated that the collicular representations of the ipsilateral and at least 45° of the contralateral hemifields were encompassed by intercollicular connections. This was also true for the somatosensory representation of the entire head and a portion of the neck.     

Identification of GABA neurons in rat cortical cultures by GABA uptake autoradiography

Autoradiographic studies of rat cortical cultures were conducted with tritiated transmitters and related drugs. Autoradiographs prepared from cultures incubated in [³H]GABA showed selective labeling: dense accumulations of silver grains over the somas and all processes of approximately 30–50% of the neuronal population, few grains over the non-neuronal cells. This labeling was blocked by diaminobutyric acid (DABA) and sodium-free media but not by β-alanine and thus has the characteristics of GABA uptake in other neuronal systems. There were no obvious differences in the size, shape, number of processes or distribution in the culture between neurons which accumulated GABA and those which did not. Similar cultures incubated in either [³H]glycine or [³H]glutamate and processed for autoradiography resulted in a much different distribution of silver grains than that seen for [³H]GABA. Following incubation in [³H]glycine, silver grains were distributed uniformly over all cells in the culture, both neuronal and non-neuronal. This distribution suggests a metabolic and not a neurotransmitter role for glycine in the cultures, as would be expected of neuronal cells derived from cerebral cortex. Glutamate incubations resulted in the appearance of silver grains over only the non-neuronal cells with very few over the neuronal population. Autoradiograms were also prepared following incubation in the potent GABA receptor agonist [³H]muscimol. These autoradiograms were indistinguishable from those obtained following [³H]GABA incubation. Thus, a finite population of neurons was densely labeled, the labeling was blocked by the GABA uptake inhibitors DABA, nipecotic acid, guvacine and Na⁺-free media, while substances which interact with the GABA receptor, bicuculline methiodide, THIP, isoguvacine and the noncompetitive antagonist, picrotoxin, were without effect. These results demonstrate that the affinity of muscimol for the GABA uptake site far outweighs its affinity for the GABA receptor site in autoradiographic experiments where intact cells are employed, presumably because its binding to receptors is fleeting. Therefore, muscimol autoradiography may not be informative about GABA receptor localization.These autoradiographic studies suggest that nearly half the neurons in our culture system are GABA neurons but disclosed no morphological handle for GABA neurons.     

Possible excitatory amino acid afferents to nucleus raphe dorsalis of the rat investigated with retrograde wheat germ agglutinin and d-[H]aspartate tracing

Evidence for excitatory amino acid afferents to nucleus raphe dorsalis (NRD) has been found with retrograde tracing techniques. For neuroanatomical definition of afferent sources to NRD, rats received stereotaxic injections of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) or implantations of crystal WGA-HRP in glass micropipettes. Retrogradely transported WGA-HRP was visualized with the tetramethyl-benizidine method, and afferents to NRD were identified from 20 different brain regions. Large numbers of labeled cells appeared in te lateral hypothalamus, lateral habenular nucleus, ventral tegmental area, periaqueductal gray, parabrachial nuclei and nucleus raphe magnus. Important inputs were also noted from dorsomedial hypothalamus and the area surounding the perihypoglosal nucleus. Smaller numbers of WGA-HRP labeled cells appeared in bed nucleus of stria terminals, diagonal band of Broca, cuneiform nucleus, superior vestibular nucleus, pontine periventricular gray, and some hypothalamic and reticular areas. Another group of rats received microinjections of d-[³H]aspartate (d[³H]Asp) and autoradiography consistently revealed retrogra labeling of cell bodies in 4 of the regions indicated by the WGA-HRP experiments as afferents to NRD. The most prominent aggregation of d-[³H]Asp-labeled cells was found in the lateral habenular nucleus, indicating that this input operates with an exitatory amino acids as transmitter. Significant numbers of d-[³H]Asp-labeled cells were also found in substantia nigra, periaqueductal and pontine periventricular gray. After large d-[³H]Asp injections involving NRD as well as surrounding areas, labeled cells were observed in several additional areas. Some of these areas were considered as afferents to sorrounding periaqueductal gray or dorsal tegmental nuclei, while others may represent NRD afferents ith relatively lower affinity for d-[³H]Asp. Several afferents to NRD failed to label with d-[³H]Asp, including diagonal band of Broca, hypothalamic areas, ventral tegmental area, parabrachial nuclei, locus coeruleus and reticular areas.     

Nipecotic acid: preferential accumulation in the cochlea by GABA uptake systems and selective retrograde transport to brainstem

[³H]Nipecotic acid was shown to be preferentially accumulated by the same cochlear structures which selectively accumulate [³H]γ- aminobutyric acid ([³H]-GABA), including the terminals of a subset of olivocochlear neurons. With both amino acids, olivocochlear fibers selectively transported label in a retrograde direction, from cochlea to brainstem. However, only [³H]nipecotic acid produced dense labeling, and labeling of cell bodies in the superior olive, presumably because it is metabolized very slowly. Nipecotic acid appears to provide a selective retrograde tracer, specific to neurons whose terminals exhibit preferential GABA uptake.     

Cysteine sulfinic acid-induced release ofd-[H]aspartate and [C]GABA in hippocampus slices: the role of sodium channels and cAMP

Cysteine sulfinic acid, a putative transmitter in the brain induces release ofd-[³H]aspartate and [¹⁴C]GABA without the help of any general depolarizing agent. Tetrodotoxin partially blocks the release ofd-[³H]aspartate and completely blocks the induced release of [¹⁴C]GABA. Withdrawal of Ca²⁺ from the medium does not affect thed-[³H]aspartate release, but increases the extent of inhibition by tetrodotoxin. In contrast, removal of Ca²⁺ increases the cysteine sulfinic acid-induced [¹⁴C]GABA release, which remains totally blocked by the toxin.Anemonia sulcata toxin type II, which slows down Na⁺ channel inactivation, acts in synergism with cysteine sulfinic acid to increase the rate of release of both of the labeled amino acids. Comparison of glutamate with cysteine sulfinic acid in the same experiments indicates a different action pattern of the two acidic amino acids. Forskolin plus isobutyl methyl xanthine, which are known to raise intracellular cyclic adenosine monophosphate (cyclic AMP) levels, caused little release of the labeled amino acids on their own, but strongly enhanced the cysteine sulfinic acid-induced release. The experiments conducted by double labeling withd-[³H]aspartate and [¹⁴C]GABA, revealed several characteristic differences between the glutamatergic and the GABAergic neurons. It is tentatively concluded that cysteine sulfinic acid brings about excitation of the glutamatergic as well as the GABAergic neurons, leading to opening of Na⁺ channels which play a role in the release in both systems. Cyclic AMP, presumably by initiating phosphorylation of a specific component, has a remarkable potentiating effect on the release.     

Selective retrograde labeling of neurons of the cat vestibular ganglion with [H]d-aspartate

d-[2,3-³H]Aspartate ([³H]d-Asp) was injected in the cat vestibular nuclei. Labeling patterns resulting from retrograde axonal transport by the vestibular nerve fibers were observed in the vestibular ganglion neurons and also in the nerve fibers. The selectivity of such labeling, related to the neurotransmitter's specificity, is strongly indicated.     

Colocalization of excitatory and inhibitory neurotransmitter markers in striatal projection neurons in the rat

The principle neuronal output of the neostriatum comes from medium spiny neurons that project from the caudate/putamen to the globus pallidus and substantia nigra. Although current evidence generally indicates that γ-aminobutyric acid (GABA) is the principal neurotransmitter in this pathway, this cannot account for the excitatory synaptic activity present among cultures of striatal neurons or the short latency excitatory postsynaptic potentials which often proceed or obscure inhibitory activity evoked by striatal stimulation. In this study, retrograde transport of [³H]D -aspartate has been used to demonstrate striatopallidal and striato-nigral neurons that possess a high-affinity uptake system for glutamate and aspartate and are therefore putatively glutamatergic. Injections of [³H]D -aspartate into the globus pallidus or substantia nigra, pars reticularis of the rat retrogradely labeled mediumsized neurons throughout the rostral-caudal extent of the neostriatum. To characterize this population further, adjacent sections were immunoreacted with antibodies to either GABA, glutamic acid decarboxylase (GAD), calbindin, or parvalbumin prior to autoradiographic processing. Under these conditions, autoradiographically labeled neurons displayed positive immunoreactivity for GABA, GAD, or calbindin. Autoradiographic label did not colocalize with parvalbumin immunoreactivity. The colocalization of anatomical markers of GABAergic and glutamatergic neurotransmission raises the possibility that both neurotransmitters are functionally expressed within single striatal projection neurons. © 1994 Wiley-Liss, Inc.     

Glutamatergic connections of the auditory midbrain: Selective uptake and axonal transport of D-[3H]aspartate

D-[³H]aspartate was used to identify potential glutamatergic connections of the chinchilla inferior colliculus (IC). High-affinity uptake of D-[³H]aspartate is considered a selective marker for glutamatergic synapses, and neurons retrogradely labeled from such injections are believed to use glutamate, or a closely related compound, as a transmitter. Injections of D-[³H]aspartate suggest that glutamatergic endings in the IC arise primarily from intrinsic connections, the opposite IC, layer 5 of temporal cortex, nucleus sagulum, and lateral lemniscal nuclei. Neurons giving rise to the principal sensory (lemniscal) projections to the IC, i.e., those from the cochlear nuclei, superior olive, and the majority of projections from the lateral lemniscal nuclei, did not label in these experiments, indicating that their synapses do not recognize D-[³H]aspartate as a suitable substrate and may use inhibitory or other excitatory transmitters. After IC injections, fiber and diffuse labeling was found ipsilaterally in the medial geniculate body, superior colliculus, and dorsolateral pontine nuclei, contralaterally in the IC, and bilaterally in the superior olive and cochlear nuclei. Such labeling was attributed to anterograde transport of D-[³H]aspartate within the efferent collaterals of labeled IC neurons, suggesting that many of the IC's efferent projections may also be glutamatergic. This interpretation was confirmed in separate experiments in which D-[³H]aspartate, injected in the medial geniculate body, retrogradely labeled neurons in the IC as well as in layer 6 of temporal cortex. Finally, the mesencephalic trigeminal nucleus and tract labeled in some cases and may have local glutamatergic connections. © 1996 Wiley-Liss, Inc.     

Selective retrograde labeling with D-[3H]-aspartate in afferents to the mammalian superior colliculus

The selectivity previously reported for retrograde labeling patterns obtained following D-[3H]-aspartate injections and proposed to be related to the transmitter specificity of the labeled pathways was tested in afferents to the superior colliculus (SC) of rats and rabbits. In rats selectivity was assessed by comparing retrograde perikaryal labeling patterns observed in D-[3H]-aspartate experiments with those found after administration of a nonselective tracer, horseradish-peroxidase-labeled wheat germ agglutinin (HRP-WGA), and of the tritiated neurotrasmitter gamma-aminobutyric acid (GABA). Following D-[3H]-aspartate injections into the SC labeling was intense in a large number of cortical and hypothalamic neurons. Other afferents to the SC, however, such as those originating from the ventrolateral geniculate nucleus, the pars reticulata of the substantia nigra, the locus coeruleus, the pontine nuclei, or the retinal ganglion cells, were not labeled. Similar results were obtained in rabbits. In cats, the analysis was focused on the cerebral cortex, since in an earlier investigation no retrograde labeling had been detected in the visual cortex following D-[3H]-aspartate injections in the SC. In the present work, however, retrogradely labeled neurons were observed in various cortical areas including a few in visual cortex. This report shows selective retrograde labeling for a part of the afferents to the SC. This selectivity does not display major differences among the mammalian species studied. Moreover, according to the information available about the distribution of neurotransmitters in the brain, the findings reported here favour the idea that D-[3H]-aspartate is a retrograde tracer selective for glutamatergic and/or aspartatergic pathways.     

Direct projections from the centre median-parafascicular complex to the subthalamic nucleus in the cat and rat

Single injections of a mixture of [³H] leucine and L-[³H] made into the centre median-parafascicular complex (CM-Pf) of the cat. The ipsilateral subthalamic nucleus (STN) was most heavily labeled at its ros-tral pole and moderately in the ventral and ventromedial portions of its rostral third, At middle subthalamic levels, label was more sparse and disappeared over the caudal third of the nucleus. Labeled fibers appeared to outline the borders of the STN throughout its rostral half. Control in-jections of isotope into several other thalamic andmesodiencephalic regions produced no terminal labeling in the cat STN. After horseradish peroxidase (HRP) injections into the cat STN, a small number of the CM-Pf neurons were labeled retrogradely. The labeled neurons were scattered diffusely in the CM-Pf and were not obviously distinguishable from other unlabeled neurons in the nucleus. Single injections of L-[³H] leucine were made into the CM-Pf of the rat. Anterograde labeling was seen in the rostral half of the ipsilateral STN. Heavy labeling was present throughout the most rostral STN, became less prominent more caudally, and was absent from the caudal half of the STN. HRP injections in the rat CM-Pf produced a distribution of anterograde labeling in the STN similar to the isotope injections. It was concluded that a small number of scattered CM-Pf neurons project primarily to rostral region of the STN. Thus, the CM-Pf is in a unique position to regulate the basal ganglia by way of a newly established thalamosubthal-amic pathway as well as a widespread, conspicuous thalamostriate pathway in those two mammalian species.     

Some noradrenergic neurons of locus ceruleus-olfactory pathways contain neuropeptide-Y

We report a specific tracing technique for studying projections of noradrenergic neurons that contain other transmitters. Autoradiography after retrograde axonal transport of tritiatednoradrenaline ([³H]NA) was combined with immunocytochemical detection of endogeneous NA or neuropeptide Y (NPY). The specificity of [³H]NA retrograde labeling was dependent on the concentration of [³H]NA injected at the terminal region. At 2 x 10⁻³ M, the specificity of [³H]NA retrograde labeling was confirmed by immunodetection of endogenous NA in radiolabeled cell bodies of the locus ceruleus. Combination of autoradiography of [³H]NA retrograde labeling with immunodetection of NPY demonstrated that a some scattered locus ceruleus noradrenergic neurons (about 26%) projecting to the olfactory bulb do contain NPY.     

Diazepam enhances the action but not the binding of the GABA analog, THIP

GABA (4-aminobutyric acid) and its bicyclic analog THIP (4,5,6,7-tetrahydroisoxazolo-[4,5-c]-pyridin-3-ol) produced membrane hyperpolarization and increased chloride ion conductance of mouse spinal cord neurons in cell culture. Above 1 nM diazepam enhanced the actions of both GABA and THIP with similar potency and efficacy. Diazepam has been shown to enhance the binding of [³H]GABA to rat brain membranes over similar concentration ranges, with the EC₅₀ values for enhancement of [³H]GABA binding and increase in membrane conductance being similar. In contrast, binding of [³H]THIP has been shown to be unaltered by diazepam under a variety of conditions. The possible reasons for such a discrepancy between these electrophysiological and neurochemical results with THIP are discussed.     

Uptake [H]GABA by oligodendrocytes in dissociated brain cell culture: A combined autoradiographic and immunocytochemical study

Uptake of [³H]GABA by dissociated mixed cell cultures of fetal mouse brain was studied using light microscopic autoradiography. Major cell types in the cultures were identified and quantified by immunocytochemical localization of reliable cell type-specific antigenic markers. In 12 days in vitro (DIV) cultures [³H]GABA uptake was predominantly into neurons and oligodendrocytes, whilst at 28 DIV the only surface cells labeled were oligodendrocytes. This was confirmed by complement-dependent antibody-mediated cytotoxicity against galatocerebroside-positive oligodendrocytes. There was a moderate labeling of almost all flat cells, the majority of which were glial fibrillary acidic protein (GFAP)-positive astrocytes. Heavily labeled astrocytes were only occasionally observed. Oligodendrocytes accumulated [³H]GABA more rapidly than astrocytes but slower than neurons. Oligodendroglial labeling was predominantly over the cell body, whereas neuronal labeling was more uniformly distributed over cell body and processes. The uptake was inhibited by diaminobutyric acid (DABA) and nipecotic acid, but not by β-alanine, and thus had similar characteristics to neuronal rather than astroglial uptake. Oligodendrocytes did not accumulate [³H]β-alanine, which labeled only astrocytes. Oligodendroglial [³H]GABA uptake was Na⁺-dependent and sensitive to ouabain, but was only slightly enhanced by aminooxyacetic acid (AOAA), whereas astroglial uptake was not sensitive to ouabain but was markedly enhanced by AOAA. The results indicate that oligodendrocytes, in addition to astrocytes, may also be involved in the modification of neuronal function by the uptake and inactivation of neuroactive substances.     

GABA binding and bicuculline in spinal cord and cortical membranes from adult rat and from mouse neurons in cell culture

Sodium-independent [³H]GABA and [³H]muscimol binding was determined in adult rat cerebral cortical and spinal cord membranes and in membranes from fetal mouse cortical and spinal cord neurons in primary dissociated cell culture. In adult rat cerebral cortical membranes, [³H]GABA bound to two sites (K_d=8nM,B_max=0.62pmol/mg protein; K_d=390nM,B_max=3.9pmol/mg protein) whereas the GABA agonist, [³H]muscimol, bound only to a high affinity site (K_d=5.6nM,B_max=1.9pmol/mg protein). In adult rat spinal cord, only a low affinity site was seen with [³H]GABA (K_d=340nM,B_max=9.8pmol/mg protein) and only a high affinity site was seen with [³H]muscimol (K_d=5.6nM,B_max=0.25pmol/mg protein). The inability to measure a high affinity [³H]GABA binding site in spinal cord probably reflects the high ratio of low to high affinity sites in spinal cord (39:1). In membranes from mouse neurons in cel; culture, [³H]GABA bound to two sites on cortical neurons (K_d=9nM,B_max=0.24pmol/mg protein; K_d=510nM,B_max=1.3pmol/mg protein) and spinal cord neurons (K_d=13nM,B_max=0.12pmol/mg protein; K_d=640nM,B_max=3.2pmol/mg protein). Again, the ratio of low to high affinity sites in cultured mouse spinal cord neurons was high (27:1).The effects of the potent GABA antagonist, (+)bicuculline, on both low and high affinity [³H]GABA binding was determined. Bicuculline appeared to inhibit binding to both sites competitively but theK_i for inhibiting the high affinity site was 5 μM and for inhibiting the low affinity site was 115 μM. Bicuculline inhibited [³H]muscimol binding in both brain and spinal cord competitively withK_is of 4μM and 10 μM respectively. Bicuculline inhibition of [³H]muscimol binding in cultured neuronal membranes was similar to that in adult rat membranes.The binding of the potent GABA agonist, muscimol, only to the high affinity site in both adult rat and cultured mouse neuronal membranes suggests that the high affinity site is the physiologically relevant postsynaptic GABA receptor. The fact that bicuculline inhibits the high affinity site (but not the low affinity site) in concentrations similar to those needed to block GABA-responses in physiological experiments²⁸ supports this hypothesis.     

Effects of chronic treatment with a cyclic AMP-selective phosphodiesterase inhibitor, rolipram, on excitatory amino acid neurotransmission systems in young and aged rat brains

Summary Rolipram selectively inhibits cyclic AMP-specific phosphodiesterase, and leads to an increase in cyclic AMP levels in the brain. In this study, we investigated the effects of chronic rolipram treatment on excitatory and inhibitory amino acid neurotransmission systems in young and aged Wistar rat brains. We used in vitro autoradiography with [³H]MK-801, [³H]glycine, D-[³H]aspartate, and [³H]muscimol to label N-methyl-D-aspartate (NMDA) receptors, glycine modulatory sites, glutamate transport sites, and -aminobutyric acid-A (GABA) receptors, respectively. Rolipram (0.01 or 0.1 mg/kg, per os) or its vehicle (distilled water) was administered once a day for 4 weeks. The highest binding of [³H]MK-801, [³H]glycine, and d-[³H]aspartate was seen in the hippocampus in vehicle-treated rats. No significant differences in these binding activities were seen between young and aged rat brains. [³H]Muscimol binding was the highest in the cerebellum, and decreased in many brain regions in aged rats. The chronic rolipram treatment resulted in (1) an increase in [³H]MK-801 binding in the dentate gyrus in both young and aged rats, (2) remarkable reductions in D-[³H]aspartate binding in many regions of both young and aged rats, and (3) no or minimal changes in [³H]glycine and [³H]muscimol binding. These results suggest that the chronic rolipram treatment modifies the excitatory amino acid neurotransmission system.     

Selective labeling of spiral ganglion and granule cells with D-aspartate in the auditory system of cat and guinea pig

The present study sought to locate putative glutamatergic or aspartatergic pathways in the auditory system of cats and guinea pigs. We injected 0.06 to 3 mM D-[3H] aspartate (D-Asp) in the cochlear nucleus before preparation for light microscopic autoradiography. At short survival times (15 and 40 min) there was heavy labeling of astrocytic somata. Labeling patterns typical of cochlear nerve endings decorated neurons in the cochlear nucleus, e.g., cell bodies and dendritic trunks of octopus cells. Labeling patterns consistent with retrograde axonal transport by the parallel fibers of granule cells appeared in the molecular layer of the dorsal cochlear nucleus and in the external granular layer. Retrograde labeling of the cochlear nerve root fibers also occurred. Consistent with these results are companion biochemical findings on the rapidly dissected cochlear nuclei of guinea pigs. The dorsal, anteroventral, and posteroventral cochlear nuclei, each, evinced uptake of D-Asp. Subsequently, electrical stimulation of each nucleus released a portion of the accumulated amino acid. Most of this release probably came from synaptic endings. Another group of experiments compared autoradiographic localization of 0.06 to 3 mM D-Asp to that of horseradish peroxidase (HRP) 6 hr to 2 d after injections in the cochlear nucleus. Astroglial cell bodies were no longer labeled by D-Asp, but spiral ganglion cell bodies in the cochlea and granule cell bodies in the cochlear nucleus were. Perikarya of the periolivary and ventral cochlear nuclei projecting to the dorsal cochlear nucleus were labeled by HRP and not by D-Asp. Thus, comparisons with the HRP findings indicate that D-Asp labeling resulted from a selective retrograde transport. There was no evidence for a selective anterograde axonal transport. The present observations support the hypothesis that cochlear nerve fibers and granule cells may use L-glutamate and/or L-aspartate as a transmitter in the cochlear nucleus.     

Reduced d-[H]aspartate binding in Down's syndrome brains

The binding of d-[³H]aspartate to glutamate uptake sites was measured in post-mortem brains from subjects with Down's syndrome (DS) and age-matched controls. DS brains had substantially reduced d-[³H]aspartate binding in the frontal and temporal cortex, hippocampus and caudate nucleus. There was no correlation between the numbers of Alzheimer-like plaques and tangles or clinically-assessed dementia and d-[³H]aspartate binding in DS brains. The binding of [³H]N-(1-[2-thienyl]cyclohexyl)piperidine([³H]TCP) to postsynaptic N-methyl-d-aspartate sites was normal in DS brains. This study suggests that the reduction in glutamate uptake sites in DS is more substantial and widespread than in Alzheimer's disease.