Fred Walberg

Origin and distribution of glutamate decar☐ylase in substantia nigra of the cat

The topographical distribution of glutamate decar☐ylase (GAD) in substantia nigra in unoperated and operated cats was studied in samples microdissected from freeze-dried tissue sections. The concentration of GAD, the enzyme synthesizing γ-aminobutyric acid (GABA), was highest in the medial part of pars reticulata, and decreased in the mediolateral direction. In pars compacta, on the other hand, the highest enzyme activity was found in the lateral part which merges with pars reticulata, and it decreased gradually in the latero-medial direction. The activity of GAD was always lower in the medial part of pars compacta, which contains the highest concentration of cell bodies. GAD in substantia nigra decreased after lesions in putamen, nucleus caudatus, globus pallidus and nucleus entopeduncularis. The loss of enzyme activity was strictly localized and was related to the site of termination of the degenerating striato-nigral fibers. The reduction of GAD in substantia nigra following lesions of globus pallidus or nucleus entopeduncularis may be ascribed to the interruption of striato-nigral fibers passing through these regions. The results thus indicate that the fibers of the GAD-containing axon terminals in substantia nigra of the cat originate in putamen and nucleus caudatus. Subcellular fractionation showed that about 85% of GAD and about 25% of lactate dehydrogenase were present in particles (probably synaptosomes) from substantia nigra in unoperated animals. Electron microscopic examination revealed that 11.5% of the tissue volume of pars reticulata was occupied by boutons compared to 5.9% for pars compacta. The concentration of GABA in pars reticulata was found to be 9 mM. From these data the intraterminal concentration of GABA was estimated to be at least 60 mM, probably over 100 mM. DOPA decar☐ylase was mainly found in pars compacta. Acetylcholin-esterase showed a very high activity in substantia nigra, the highest concentration being found in the medial part of pars reticulata. In contrast, the concentration of choline acetyltransferase was very low. The ratio of acetylcholinesterase activity to choline acetyltransferase activity was 1000. DOPA decar☐ylase and the cholinergic enzymes were little affected by the above described lesions.

Metabolic compartmentation of glutamate and glutamine: Morphological evidence obtained by quantitative immunocytochemistry in rat cerebellum

An electron microscopic, double-labelling immunocytochemical procedure was used to assess the level of fixed glutamate and glutamine in different cell profiles in ultrathin sections of rat cerebellar cortex. The procedure was based on sequential immunolabelling with two rabbit antisera, using gold particles of different sizes as markers and formaldehyde vapour as a means to avoid interference between the two incubations. Model sections containing a series of known concentrations of the respective amino acids (aldehyde--fixed to rat brain protein) were incubated together with the tissue material. These revealed a close to linear relationship between gold particle density and antigen concentration throughout the range of biological relevance. The ratio between the density of the two categories of gold particles was calculated for the individual profile types. This ratio showed a 20-fold variation, with the highest glutamate/glutamine ratios obtained for putative excitatory terminals (terminals of parallel fibres in the outer part of the molecular layer, followed by mossy and climbing fibre boutons) and the lowest for glial cells (Bergmann glia, astrocytes in the granule cell layer, and oligodendrocytes). Granule cell bodies and dendrites, and cell bodies and processes of putative GABAergic cells (Purkinje, basket and Golgi cells) displayed intermediate ratios. The ratios corresponded to millimolar ratios (mM fixed glutamate/mM fixed glutamine) ranging from 4.5 to 0.2, tentatively assessed by adjusting for differences in labelling efficiency of the two antigens. Our results show that the compartmentation of glutamate and glutamine, an issue previously addressed mainly in the test tube, can be studied in morphologically intact preparations at a resolution that matches the complexity of CNS tissue. The data indicate that glutamate is effectively converted to glutamine in all categories of glial cells, and that glutamate synthesis prevails in each of the three types of excitatory terminals in the cerebellar cortex. Terminals of putative GABAergic cells form a distinct low glutamate/low glutamine compartment.

Cerebellar corticovestibular fibers in the cat

Abstract Lesions in various parts of the cerebellar vermal cortex were made in cats and the ensuing preterminal and terminal degeneration in the vestibular nuclei studied in silver-impregnated sections by the methods of Nauta and Glees. The results obtained by the two methods are concordant. The majority of the cerebellovestibular fibers are derived from the anterior lobe vermis; a considerably smaller number of fibers take origin from the posterior lobe. The corticovestibular projection is ipsilateral. The fibers from the anterior lobe pass rostral and lateral to, or through the rostrolateral part of the fastigial nucleus; the fibers from the posterior lobe dorsal and ventral to the nucleus. All fibers leave the cerebellum via the inferior cerebellar peduncle and terminate in approximately the dorsal halves of the lateral and inferior vestibular nuclei. The former nucleus receives by far the majority of the fibers. A somatotopical arrangement of the fibers is indicated inasmuch as the fibers from the forelimb region of the anterior lobe vermis end chiefly in the forelimb region of the ipsilateral lateral vestibular nucleus. Although isolated lesions of the hind-limb region of the anterior lobe have not been performed, it is probable that the same pattern exists for the fibers from this to the corresponding area of the lateral vestibular nucleus. The fibers terminate on all kinds of cells, but the majority make contact with large cells, especially their dendrites.

GABA, glycine, aspartate, glutamate and taurine in the vestibular nuclei: an immunocytochemical investigation in the cat.

SummaryThe distributions of five amino acids with well-established neuroexcitatory or neuroinhibitory properties were investigated in the feline vestibular complex. Consecutive semithin sections of plastic-embedded tissue were incubated with antisera raised against protein-glutaraldehyde conjugates of GABA, glycine, aspartate, glutamate and taurine. This approach allowed us to study the relative densities of the different immunoreactivities at the level of individual cell profiles. The results indicate that in the vestibular nuclei, neuronal colocalization of two or more neuroactive amino acids is the rule rather than an exception. Colocalization was found of immunoreactivities for GABA and glycine; glycine, aspartate and glutamate; glycine and aspartate, and glutamate and aspartate. GABA immunoreactive neurons were generally small and were found scattered throughout the vestibular complex. Glycine immunoreactive neurons were similarly distributed, except in the superior nucleus where the latter type of neuron could not be detected. Neuronal profiles colocalizing immunoreactivities for GABA and glycine occurred in all nuclei, but were most numerous in the lateral nucleus. The vast majority of the neurons showed noteworthy staining for glutamate and aspartate, although the level of immunoreactivities varied (e.g., the large neurons in the lateral and descending nuclei were more intensely aspartate immunoreactive than the smaller ones). Taurine-like immunoreactivity did not occur in neuronal cell bodies but appeared in Purkinje cell axons and in glial cell profiles. The functional significance of the complex pattern of amino acid colocalization remains to be clarified. In particular it needs to be distinguished between metabolic and transmitter pools of the different amino acids. The present results call for caution when attempts are made to conclude about transmitter identity on the basis of amino acid contents alone.

Cerebellar afferent projections from the vestibular nuclei in the cat: an experimental study with the method of retrograde axonal transport of horseradish peroxidase.

SummaryDetails of cerebellar afferent projections from the vestibular nuclei were investigated by the method of retrograde axonal transport of horseradish peroxidase (HRP) in the cat. The distribution of labeled cells in the vestibular nuclei following HRP injections in various parts of the cerebellum indicates that vestibular neurons in the medial and descending nuclei and cell groups f and x project bilaterally to the entire cerebellar vermis, the flocculus, the fastigial nucleus and the anterior and posterior interpositus nuclei. In addition, labeled cells (giant, medium and small) were consistently found bilaterally in the superior and lateral vestibular nuclei following HRP injections in the nodulus, flocculus, fastigial nucleus, and following large injections in the vermis. No labeled cells were observed in cases of HRP injections in crus I and II, the paramedian lobule, paraflocculus and lateral cerebellar nuclei. The present findings indicate that secondary vestibulocerebellar fibers project to larger areas in the cerebellum and originate from more subdivisions and cell groups of the vestibular nuclear complex than previously known.

Aspartate-like and glutamate-like immunoreactivities in the inferior olive and climbing fibre system: A light microscopic and semiquantitative electron microscopic study in rat and baboon (Papio anubis)

A post-embedding immunogold procedure was used to analyse, in a semiquantitative manner, the distributions of aspartate-like and glutamate-like immunoreactivities in the inferior olive and climbing fibre system in rats and baboons. The neurons in the inferior olive were uniformly labelled for aspartate as well as glutamate, indicating a 100% co-localization of these two amino acids in the cell bodies. The level of glutamate-like immunoreactivity in the climbing fibre terminals was similar to that in the parent cell bodies, as judged by a computer-assisted calculation of gold particle densities. In contrast, the level of aspartate-like immunoreactivity in the climbing fibre terminals was only one-seventh of that of the olivary neurons. No differences were found between the hemispheres and vermis. Nerve terminals in the inferior olive were generally moderately labelled with the aspartate antiserum, as were cell bodies of astrocytes. With a few exceptions, the results obtained in baboons were similar to those in rats. Notably, no evidence was found of an enrichment of aspartate-like immunoreactivity in climbing fibres. The present results do not support previous data suggesting that aspartate is the transmitter of the climbing fibres but indicate that glutamate or another excitatory compound should be considered as candidate for this role. Our findings show that the presence of aspartate-like immunoreactivity in cell bodies is an unreliable indicator of transmitter identity.

An estimation of the concentration of γ-aminobutyric acid and glutamate decar☐ylase in the inhibitory Purkinje axon terminals in the cat

Abstract In the present study an estimate was made of the concentration of GABA within the Purkinje axon terminals. The values were based on data obtained from amino acid and enzyme measurements, subcellular fractionation, and quantitative morphological analyses in normal and operated animals. Boutons constituted 4–5% of the tissue volume of the two regions, and their density was estimated to be 6–9 × 1010/cu.cm. The proportion of Purkinje axon terminals within the cerebellar nuclei and the dorsal part of the lateral vestibular nucleus was 12–33% of the total bouton number. The concentration of GABA was between 50 and 100 m M; and the activity of GAD permitted, at a maximum, 350–1000 m M GABA to be synthesized/h.

Is lectin-coupled horseradish peroxidase taken up and transported by undamaged as well as by damaged fibers in the central nervous system?

Uptake and transport of horseradish peroxidase-wheat germ agglutinin conjugate (HRP-WGA) in intact and damaged passing fibers were studied by injections of the medulla and pons in 11 cats. Injections with evidence of damage to olivocerebellar fibers and cranial nerve fibers invariably lead to retrograde labeling of neurons in the inferior olive and cranial motor nuclei. With staining around--but apparently no damage of--cranial nerve root fibers, no labeling was found in their motor nuclei. Injections limited to the medullary pyramid with slight fiber damage and limited staining lead to faint retrograde labeling of a small number of cells in the ipsilateral sensorimotor cortex. More extensive staining and fiber damage of the pyramid gave a higher number of labeled cells in the ipsilateral sensorimotor cortex. From these experiments we conclude that HRP-WGA is taken up and transported retrogradely with subsequent significant cell labeling in damaged but not in intact fibers. Anterograde transport of HRP-WGA in fibers passing through the injected area was found to take place only for a very short distance, as judged from cases with injections of either the pons or the medullary pyramid interrupting many corticospinal fibers.

The lateral reticular nucleus in the cat—I. An experimental anatomical study of its spinal and supraspinal afferent connections

The localization of the neurons from which the main ascending and descending projections to the lateral reticular nucleus originate has been studied in nine cats, using the retrograde axonal transport of horseradish peroxidase injected within that nucleus. The ascending spinoreticular neurons are widely distributed from the cervical to the sacral segments of the spinal cord. These neurons, which are of different sizes, are mainly located within Rexeds laminae VI, VII and VIII, but they spread both dorsally to laminae II–VI as well as ventrally to lamina IX. Labeled neurons of a size similar to motoneurons are particularly found within lamina IX, intermingled with the motoneurons. The spinal projection to the lateral reticular nucleus is crossed and uncrossed, with the ipsilateral spinoreticular neurons being located more dorsally within the grey matter of the spinal cord than the contralateral spinoreticular neurons. Moreover, while neurons with a crossed ascending projection are almost equally distributed along the whole rostro-caudal extension of the spinal cord, those with an uncrossed projection are predominantly located within the cervical segments of the spinal cord. Additional evidence indicates that the spinoreticular projection to the lateral reticular nucleus is somatotopically organized. In addition to the spinoreticular projection, the lateral reticular nucleus receives a crossed rubroreticular projection and a crossed fastigioreticular projection originating from the rostro-ventralmost part of the nucleus. A few neurons in the interposite nuclei also project to the lateral reticular nucleus.

Mode of termination of primary vestibular fibres in the lateral vestibular nucleus an experimental electron microscopical study in the cat

SummaryFollowing transection of the vestibular nerve in cats, the electron microscopical changes occurring in the lateral vestibular nucleus were studied after survival periods of 2–11 days. Material for study was taken from the rostroventral part of the nucleus of Deiters since this is known to receive the primary vestibular fibres.Degeneration of terminal boutons is evident two days after the lesion. Degenerating boutons show an increased electron optic density, mitochondrial changes and a loss of synaptic vesicles. They are often surrounded by a pericellular space filled with flocculent (probably protein) material. At three days and later this space is occupied by processes of astrocytes or of a type of phagocytic cells which surround or engulf the degenerating boutons. Nine to eleven days after the lesion almost all degenerating boutons have disappeared. There is evidence of phagocytosis of axons and myelin sheaths by astrocytes but mainly by phagocytes of yet undetermined origin. The “filamentous type” of bouton degeneration has not been observed.Degenerating boutons are found on neuronal perikarya and on proximal as well as on thin distal dendrites and on spines. They are common on small and medium-sized cells, but have also been seen on some giant cells. The degenerating boutons do not form series of synaptic complexes. Degenerating fibres and boutons have so far been found only ipsilateral to the lesion.The findings confirm and extend those made in corresponding experiments with silver impregnation procedures, but emphasize the limitations of the latter methods as regards conclusions concerning synaptic contacts.