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.

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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.

The pathway from the mamillary complex to the cerebellum via the pontine nuclei has been studied using several anterograde and retrograde tracing techniques in the cat. We have also compared the pontine terminal regions of fibres from the mamillary complex and from the cingulate gyrus. Implantations of crystalline horseradish peroxidase wheat germ agglutinin (HRP-WGA) in the mamillary complex and lesions of the cingulate gyrus were combined in the same animal with injections of HRP-WGA, rhodamine-B-isothiocyanate (RITC), and Fluoro-Gold in different parts of the cerebellar hemisphere. Fibres from both the mamillary complex and the cingulate gyrus terminate mainly within a transversely oriented, c-shaped band in the ipsilateral, rostral pontine nuclei. Within this band the terminal fields of fibres from the mamillary complex and the cingulate gyrus form a mosaic-like pattern of partly overlapping patches. Pontine regions receiving a mamillary input project mainly to the ventral paraflocculus, and to a lesser degree to the dorsal paraflocculus, but apparently not to the uvula or crus II. Judging from the literature it seems highly unlikely that other parts of the cerebellar hemispheres received projections from these pontine regions. Fibres from the ventral paraflocculus were shown to terminate in the parvicellular part of the lateral cerebellar nucleus only. The present findings would seem to imply that inputs from the mamillary complex and a related cortical region, the cingulate gyrus, are partly integrated, partly kept separate at the precerebellar level. This would ensure that small groups of cells in the rostral pontine nuclei receive a specific set of afferents. Conceivably, the information transmitted to the cerebellum by these groups of pontine cells might be related to functions of the mamillary complex, such as learning, motivation, and spatial memory.

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.

Summary: This study was undertaken to elucidate the roles of neurons and glial cells in the handling of gluta mate and glutamine, a glutamate precursor, during cere bral ischemia. Slices (400--6 00 fLm) 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 con trol slices that had not been subjected to "ischemic" con ditions, were then fixed and embedded. Ultrathin sec tions were processed according to a postembedding im munocytochemical method with polyclonal antibodies raised against glutamate or glutamine, followed by colloi dal 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 dep rivation caused a reduced glutamate immunolabeling in neuronal somata, while that of glial processes increased. Following 1 h of recovery, the glutamate labeling of neu ronal somata declined further to very low values, com pared to control slices. The glutamate labeling of glial cells returned to normal levels following recovery. InThe pathogenesis of ischemic brain damage is not fully understood, and a number of neurotoxic sub stances has been implicated (Krause et aI. , 1988; Siesjo, 1988;Ginsberg, 1990; Siesjo et aI. , 1990 503axon terminals, no consistent change in the level of glu tamate 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 dep rivation. This treatment was also associated with a re duced glutamine immunolabeling in glial cells. Reversed glutamate uptake due to perturbations of the transmem brane ion concentrations and membrane potential proba bly contributes to the loss of neuronal glutamate under "ischemic" conditions. The increased glutamate labeling of glial cells under the same conditions can best be ex plained by assuming that glial cells resist a reversal of glutamate uptake, and that their ability to convert gluta mate into glutamine is compromised due to the energy failure. The persistence of a nerve terminal pool of glu tamate is compatible with recent biochemical data indi cating that the exocytotic glutamate release is contingent on an adequate energy supply and therefore impeded dur ing ischemia.

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.

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