Redistribution of glutamate and glutamine in slices of human neocortex exposed to combined hypoxia and glucose deprivation in vitro.

This study was undertaken to elucidate the roles of neurons and glial cells in the handling of glutamate and glutamine, a glutamate precursor, during cerebral ischemia. Slices (400-600 microns) from human neocortex obtained during surgery for epilepsy or brain tumors were incubated in artificial cerebrospinal fluid and subjected to 30 min of combined hypoxia and glucose deprivation (an in vitro model of brain ischemia). These slices, and control slices that had not been subjected to "ischemic" conditions, were then fixed and embedded. Ultrathin sections were processed according to a postembedding immunocytochemical method with polyclonal antibodies raised against glutamate or glutamine, followed by colloidal gold-labeled secondary antibodies. The gold particle densities over various tissue profiles were calculated from electron micrographs using a specially designed computer program. Combined hypoxia and glucose deprivation caused a reduced glutamate immunolabeling in neuronal somata, while that of glial processes increased. Following 1 h of recovery, the glutamate labeling of neuronal somata declined further to very low values, compared to control slices. The glutamate labeling of glial cells returned to normal levels following recovery. In axon terminals, no consistent change in the level of glutamate immunolabeling was observed. Immunolabeling of glutamine was low in both nerve terminals and neuronal somata in normal slices and was reduced to nondetectable levels in nerve terminals upon hypoxia and glucose deprivation. This treatment was also associated with a reduced glutamine immunolabeling in glial cells. Reversed glutamate uptake due to perturbations of the transmembrane ion concentrations and membrane potential probably contributes to the loss of neuronal glutamate under "ischemic" conditions. The increased glutamate labeling of glial cells under the same conditions can best be explained by assuming that glial cells resist a reversal of glutamate uptake, and that their ability to convert glutamate into glutamine is compromised due to the energy failure. The persistence of a nerve terminal pool of glutamate is compatible with recent biochemical data indicating that the exocytotic glutamate release is contingent on an adequate energy supply and therefore impeded during ischemia.

Subcortical projections to the pontine nuclei in the cat.

In a systematic attempt to trace all projections from the brainstem and diencephalon to the pontine nuclei of the cat, implantations and injections of horseradish peroxidase-wheat germ agglutinin (HRP-WGA) or Fluoro-Gold were placed in the pontine nuclei of 21 cats. In most of the cases there was no evidence of spread of tracer outside the pontine nuclei. Retrogradely labeled cells in the brainstem and diencephalon were carefully mapped and counted. The number labeled cells in the brainstem and diencephalon ranged from 24 in cases with very small implantations to 3,490 in cases with large injections in the pontine nuclei (counts from every fifth section). The labeled cells are located bilaterally with an ipsilateral preponderance. After large injections, 25-38% of the labeled cells were located in the brainstem reticular formation, 10-16% in the pretectal nuclei, 10-15% in the hypothalamus, 7-9% in the zona incerta, 3-9% in the fields of Forel, 4-5% in the nucleus locus coeruleus, 3-5% in the ventral lateral geniculate body, 2-4% in the superior colliculus, 3% in the periaqueductal gray, and 14-15% in other parts of the brainstem. Judging from cases with small tracer deposits entirely confined to the pontine nuclei, there appear to be two types of subcortical inputs: Projections from the reticular formation, the nucleus locus coeruleus, the periaqueductal gray, and the raphe nuclei are widespread, presumably reaching all parts of the pontine nuclei, while projections from a diversity of other sources are localized, reaching limited parts of the pontine nuclei only or predominantly.

Demonstration of topographically organized projections from the hypothalamus to the pontine nuclei: an experimental anatomical study in the cat.

In 22 cats implantations and injections of horseradish peroxidase-wheat germ agglutinin (HRP-WGA) or Fluoro-Gold were placed in the pontine nuclei or the hypothalamus. The occurrence and distribution of labeled cells in the hypothalamus and of labeled terminal fibers in the pontine nuclei were mapped. Following implantations of HRP-WGA ventromedially in rostral parts of the pontine nuclei, 22-44% of all labeled cells in the brainstem and diencephalon are found in the medial mamillary nucleus ipsilateral to the implantation. Some labeled cells are also found in the supramamillary, premamillary, anterior mamillary, and tuberomamillary nuclei. Thus, labeled cells in the hypothalamus make up 33-54% of all labeled cells in the brainstem and diencephalon in such cases. In contrast, implantations and injections in mediocaudal parts of the pontine nuclei result in labeling of cells mainly in the posterior, dorsal, and lateral hypothalamic areas (terminology of Bleier: The Hypothalamus of the Cat. Baltimore: Johns Hopkins Press, '61). In these cases the labeled cells in the hypothalamus make up 16-25% of all labeled cells in the brainstem and diencephalon. Implantations in more lateral parts of the pontine nuclei label only a few cells in the hypothalamus. Following implantations of HRP-WGA in restricted parts of the hypothalamus, fibers from the medial mamillary nucleus were found to terminate ventromedially at all rostrocaudal levels of the pontine nuclei, ipsilateral to the implantation. In the rostralmost part of the pontine nuclei, the terminal labeling forms a dense, transversely oriented, c-shaped band. Fibers from the posterior and dorsal hypothalamic areas terminate medially and dorsomedially in the caudal third of the pontine nuclei. Sparse terminal labeling is also seen in lateral parts of the pontine nuclei and medially at more rostral levels. In two cases with small implantations of HRP-WGA ventromedially in rostral parts of the pontine nuclei, labeled cells are found both in the medial mamillary nucleus and the cingulate gyrus. Thus, it seems possible that fibers from the medial mamillary nucleus and the cingulate gyrus converge upon a restricted area ventromedially in rostral parts of the pontine nuclei.

Projections to the pontine nuclei from choline acetyltransferase-like immunoreactive neurons in the brainstem of the cat.

By use of retrograde transport of horseradish peroxidase-wheat germ agglutinin (HRP-WGA) in combination with monoclonal antibodies against choline acetyltransferase (ChAT), we show that putative cholinergic inputs to the feline pontine nuclei originate from cells in the dorsolateral pontine tegmentum. These cells form a loosely arranged continuum that nevertheless may be subdivided into two groups on the basis of differences in cell morphology. One group consists of double-labeled cells in the periventricular gray substance medial to, and partly merging with, the nucleus locus coeruleus. The other group consists of double-labeled cells surrounding the brachium conjunctivum. In two cats with tracer injections in the pontine nuclei, 81% and 84%, respectively, of the retrogradely labeled cells in the dorsolateral pontine tegmentum are ChAT-like immunoreactive (ChAT-LI). In the same experiments, many ChAT-LI cells, but no retrogradely labeled cells, are seen in the basal telencephalon. The pontine nuclei contain a plexus of thin ChAT-LI fibers with varicosities resembling en passant as well as terminal boutons. These ChAT-LI fibers appear to branch extensively and cover all parts of the pontine nuclei. Following injections of rhodamine-B-isothiocyanate (RITC) in the thalamus and Fluoro-Gold in the pontine nuclei and surrounding regions in the same animal, all retrogradely labeled cells in the dorsolateral pontine tegmentum are labeled with both tracers, whereas most cells in the paramedian pontine reticular formation are labeled either with RITC or Fluoro-Gold. Thus it appears that all cells in the dorsolateral pontine tegmentum that project to the pontine nuclei also project to the thalamus. In analogy with findings by others in the dorsal lateral geniculate nucleus, we suggest that the putative cholinergic projections to the pontine nuclei may serve to modulate transmission of cerebellar afferent information in accordance with the behavioral state of the animal.

An electron microscopic, immunogold analysis of glutamate and glutamine in terminals of rat spinocerebellar fibers.

A semiquantitative, electron microscopic immunocytochemical procedure based on the use of colloidal gold particles as markers was employed to analyze the subcellular distribution of glutamate and glutamine, a major glutamate precursor, in a subpopulation of spinocerebellar mossy fiber terminals. These terminals were identified by anterograde transport of a horseradish peroxidase-wheat germ agglutinin conjugate, injected in the thoracic spinal cord. Gold particles signalling glutamate-like immunoreactivity were enriched over clusters of synaptic vesicles relative to organelle-free cytoplasmic matrix, and there was a strong positive correlation between gold particle and synaptic vesicle densities (correlation coefficient 0.94). Gold particles indicating glutamine-like immunoreactivity showed a much weaker correlation with vesicle density (correlation coefficient 0.36) and were about equally concentrated over cytoplasmic matrix as over clusters of synaptic vesicles. Compared with the mossy fibers, the putative GABAergic Golgi cell terminals exhibited a lower level of glutamate-like immunoreactivity, which was very weakly correlated with the vesicle density (correlation coefficient 0.27). The level of glutamine-like immunoreactivity in the Golgi cell terminals was similar to that in mossy fibers, but much lower than that in glial cells. The anterogradely labelled mossy fiber terminals were not enriched in immunoreactivities for aspartate or GABA. These results suggest that the level and subcellular distribution of glutamate in presumed glutamatergic terminals differs from that in terminals in which glutamate only serves metabolic or precursor roles, and that these differences can be exploited in immunocytochemical studies aimed at identifying glutamate-using neurons. In contrast, glutamine immunocytochemistry does not seem to be generally useful in this regard.

GABA and glycine as putative transmitters in subcortical pathways to the pontine nuclei. A combined immunocytochemical and retrograde tracing study in the cat with some observations in the rat.

Using the retrograde tracers horseradish peroxidase-wheatgerm agglutinin and gold particles conjugated to wheatgerm agglutinin apo-horseradish peroxidase in combination with an antiserum against glutaraldehyde-fixed GABA, it was examined whether the pontine nuclei of the cat receive projections from GABA-like immunoreactive neurons in the brainstem, diencephalon, or deep cerebellar nuclei, contributing to the GABA-like immunoreactive fibre plexus previously demonstrated in the pontine nuclei [Brodal et al. (1988) Neuroscience 25, 27-45]. Following tracer injections that covered both the pontine nuclei and the reticular tegmental nucleus in two cats, it was found that 125 out of 1166 (10.7%) and 29 out of 294 (9.9%) retrogradely labelled neurons in the cerebellar nuclei were GABA-like immunoreactive. In the same two experiments only six out of 2029 (0.3%) and 10 out of 1398 (0.7%) retrogradely labelled neurons in the brainstem and diencephalon were GABA-like immunoreactive. Among the regions in the brainstem and diencephalon known to project to the pontine nuclei, double-labelled cells were seen in the reticular formation, the periaqueductal gray, and the nucleus praepositus hypoglossi, but not in the zona incerta or the anterior pretectal nucleus, regions that have been shown to contain glutamate decarboxylase-like immunoreactive neurons projecting to the pontine nuclei in the rat [Border et al. (1986) Brain Res. Bull. 17, 169-179]. In order to test whether this is due to species differences, the same experimental approach was used in the rat, and it was found that 54 out of 3249 (1.7%) retrogradely labelled neurons in the brainstem and diencephalon were double-labelled. Notably, in the zona incerta 2% of the retrogradely labelled cells were also GABA-like immunoreactive, and in the reticular formation there was a higher proportion of double-labelled cells than was found in the cat. Additional sources were identified, that may contribute to the GABA-like immunoreactive fibre plexus in the pontine nuclei of the rat. This, in conjunction with the previous finding that the pontine nuclei of the rat contain only very few putative GABAergic neurons [Border and Mihailoff (1985) Expl Brain Res. 59, 600-614; Brodal et al. (1988) Neuroscience 25, 27-45], lead to the suggestion that the GABA-like immunoreactive fibre plexus in the pontine nuclei of the rat is predominantly of extrinsic origin, possibly representing a mosaic of the terminal fields of several subcorticopontine projections.(ABSTRACT TRUNCATED AT 400 WORDS)

Organization of cingulo-ponto-cerebellar connections in the cat.

This study deals with three different aspects of the organization of connections from the cingulate gyrus to the cerebellum. (1) With the use of wheat germ agglutinin-horseradish peroxidase as a retrograde tracer, the distribution of cingulate neurons projecting to the pontine nuclei was studied. Retrogradely labeled cells were found in layer 5 in all parts of the cingulate gyrus. Average densities of cingulo-pontine cells were similar in the different cytoarchitectonic subdivisions, although some density gradients were observed. The projection was found to be remarkably strong. Average densities of corticopontine cells in the cingulate gyrus ranged from 500-700 cells per mm2 cortical surface, and the total number of neurons was in the range of 75,000-105,000 (n = 4). (2) A topographical organization of terminal fields of fibers originating in different parts of the cingulate gyrus was demonstrated with the combined use of anterograde degeneration and anterograde transport of wheat germ agglutinin-horseradish peroxidase. Terminal fibers originating in different zones of the cingulate gyrus were distributed in a patchy mosaic within a narrow band along the ventromedial aspect of the pontine nuclei. (3) We confirm, with the combined use of lesions in the cingulate gyrus and injections of wheat germ agglutinin-horseradish peroxidase in the ventral paraflocculus, that there is considerable overlap between terminal fibers originating in the cingulate gyrus, and cells retrogradely labeled from the ventral paraflocculus. The role of the ventral paraflocculus as a receiver of "limbic" input is discussed.

Profiles enriched in GABA-like immunoreactivity participate in serial synapses in the pontine nuclei of baboon (Papio anubis).

Using a postembedding immunogold procedure with an antiserum against glutaraldehyde-fixed GABA, we demonstrate GABA-like immunoreactivity in two classes of synaptic profiles in the pontine nuclei of baboon. One is an axon terminal in symmetrical synaptic contact with small or medium-sized GABA-immunonegative dendrites, the other is a pale, vesicle-containing profile resembling a dendrite or dendritic process which participates in serial synaptic arrangements. These synaptic arrangements, or triads, consist of a GABA-like immunoreactive, pale vesicle-containing profile being postsynaptic to a GABA-immunonegative axon terminal, and presynaptic to a small or medium-sized GABA-immunonegative dendrite. In at least some of these triads, the GABA-immunonegative axon terminals also contact the GABA-immunonegative dendrite directly.

Immunocytochemical evidence for in vitro release of glutamate and GABA from separate nerve terminal populations in the rat pontine nuclei.

A quantitative electron microscopic immunocytochemical method was used to study the synaptic handling of glutamate and GABA in slice preparations from the rat pontine nuclei. Slices were subjected to a depolarizing stimulus (55 mM K+, 20 min) in the presence of a physiological or low Ca(2+)-concentration. Depolarization at physiological [Ca2+] evoked a depletion of glutamate-like immunoreactivity from nerve terminals that contain round vesicles and establish asymmetric synaptic contacts. When depolarization was induced in the presence of only 0.1 mM Ca2+ (10 mM Mg2+ added), the loss of glutamate was significantly reduced or abolished, indicative of a Ca(2+)-dependent component of glutamate release. By means of a double-labeling immunocytochemical method we could identify a population of nerve terminals that displayed strong GABA-like immunoreactivity, and a level of glutamate-like immunoreactivity that was low but yet clearly above background level. This type of terminal contains elongated or pleomorphic vesicles and establishes symmetric synaptic contacts. In these terminals, depolarization evoked a Ca(2+)-dependent depletion of GABA-like immunoreactivity, but failed to change the level of glutamate-like immunoreactivity. The present study demonstrates that two different types of nerve terminal in the rat pontine nuclei contain releasable pools of glutamate and GABA, respectively, and that the GABA-releasing terminals also contain a non-releasable pool of glutamate. The glutamate of the latter pool could act as precursor of GABA.