Charles Gorenstein

Neuronal localization of pseudocholinesterase in the rat cerebellum: sagittal bands of Purkinje cells in the nodulus and uvula

The histochemical distribution of pseudocholinesterase was studied in the rat cerebellum using Koelles copper-thiocholine method. Throughout the cerebellum, pseudocholinesterase is uniformly localized in the endothelial cells of blood vessels and in the cell bodies and processes of the Bergmann glia. In addition, we demonstrate that exclusively in the ventral uvula and in the nodulus (lobules IXc and X of Larsell) pseudocholinesterase is localized in a small subpopulation of Purkinje cells. The cell bodies and dendrites of these labeled Purkinje cells form at least 4 distinct parallel bands extending along the sagittal plane of each of the lobules. Two broad bands on either side of the midline, approximately 800-900 microns wide and composed of 15-20 Purkinje cells have been designated as A bands. Two narrower bands, approximately 160-300 microns wide and composed of 5-7 Purkinje cells, on the lateral aspects of the lobules have been designated as B bands. The unique distribution of pseudocholinesterase in a small and anatomically restricted population of neurons suggests that in the cerebellum this enzyme may play a role in the metabolism of neuroactive substances.

The localization of GABAA receptors in mice with mutations affecting the structure and connectivity of the cerebellum.

The distribution of cerebellar [3H]muscimol binding sites was studied autoradiographically in normal C57BL/6J mice and in the weaver, reeler, Purkinje cell degeneration and staggerer mutant mice. In the normal 79-day-old mouse cerebellum, the highest concentration of [3H]muscimol binding sites was observed in the granule cell layer. A much lower grain density was present over the Purkinje cell and molecular layers and negligible numbers of binding sites were seen over the deep cerebellar nuclei and white matter. A significant decrease in [3H]muscimol labeling was observed over the cerebellar cortex of the 81-86-day-old weaver mutant; this was most pronounced in the vermis where granule cell loss was the greatest. Over the hemispheres, where fewer granule cells degenerate, a higher density of binding sites remained. In the 27-29-old reeler cerebellum, where Purkinje cells are malpositioned, no labeling was seen over the deep Purkinje cell masses. In the quasi-normal superficial cortex, labeling density over the surviving granule cell layer was only slightly decreased. In the 54-57-day-old Purkinje cell degeneration mutant, where essentially all Purkinje cells have disappeared by day 45, a 29% decrease in grain density over the granule cell layer was observed, while labeling was still present in the molecular layer. Virtually no [3H]muscimol labeling was detected over any part of the cerebellar cortex of the 25-27-day-old staggerer mutant (which lacks parallel fiber-Purkinje cell synapses), although clusters of surviving granule cells were present in significant numbers in the lateral aspects of the cortex. Our autoradiographic data indicate that GABAA receptors are associated with granule cells in both the molecular and granule cell layers. Furthermore, our results raise the possibility that the maintenance of receptor levels may be dependent upon synaptic contacts between the granule cell and its main postsynaptic target, the Purkinje cell.

Molecular forms of acetylcholinesterase in cerebral cortex and dorsal thalamus of developing rats

Histochemical studies show that primary sensory regions of rat cerebral cortex and dorsal thalamus display transient patterns of intense acetylcholinesterase (AChE) activity during early postnatal development. Sucrose gradient fractionation techniques were used to determine the molecular forms of AChE in developing rat brain at the time of onset (postnatal day 5), during peak expression (days 10-11), and after decline (day 18) of the transient AChE expression. Tissue from auditory and visual regions of cortex and from dorsal thalamus at each age examined contained 10S and 4S forms of AChE, similar to the pattern observed in mature brain. The 10S form was almost totally membrane bound; the 4S form was largely soluble. Hemithalamic lesions reduce both forms of AChE in cortex. These data indicate that transiently expressed AChE does not represent a unique or unusual form of the enzyme.

Examination of the transient distribution of lysosomes in neurons of developing rat brains.

We have previously observed that lysosomes redistribute from their normal location in neuronal cell bodies to the dendrites following an intracerebroventricular injection of an antimitotic such as colchicine, vinblastine or vincristine. In the present study, we have followed the developmental distribution of lysosomes in the brains of untreated rats, using a lysosomal marker enzyme, dipeptidylaminopeptidase II. A relatively high concentration of neuronal lysosomes was found in the dendrites of olfactory bulb mitral cell neurons and in hippocampal granule and pyramidal cell neurons from postnatal day 1 (P1) to P8. As the animals matured, the pattern of lysosomal enzyme distribution was reversed. Lysosomes became progressively less concentrated in the dendrites and more concentrated in neuronal cell bodies. In cerebellar Purkinje cells, lysosomes were only found in the cell bodies during the first week after birth. Between P9 and P19, lysosomes appeared in the dendrites of these neurons and, with maturity, progressively disappeared from the dendrites and were concentrated mainly in cell bodies. The presence of lysosomes in the dendrites of developing animals suggests that the transport of lysosomes to the dendrites, induced by microtubule poisons, mimics a physiological process which is normally present during development.

Redistribution of neuronal lysosomes induced by colchicine: an electron microscopic quantitative study.

We have previously demonstrated that a single injection of the microtubule poison colchicine, into the lateral cerebral ventricle of the rat, induced a rapid redistribution of the lysosomal marker enzymes, dipeptidylaminopeptidase II (Dpp II) and acid phosphatase, from their normal location in neuronal cell bodies out into the dendrites. In the present study, we have quantitatively analyzed this phenomenon at the electron microscopic level by identifying and counting the number of lysosomes and mitochondria in neuronal cell bodies and dendrites of control and colchicine-treated rats. Areas examined included the anterior dorsal (AD) thalamus, pontine nucleus, and facial nucleus. The results show that the cytoplasm of these neurons contains significantly fewer large lysosomes after treatment with colchicine while the dendrites become abnormally enriched with large and small lysosomes after treatment. Lysosomes were rarely seen in the axons of either control or colchicine-treated animals. A significant increase in the density and the shape of mitochondria was also observed in the dendrites following colchicine treatment. The data presented support the hypothesis that neurons contain a transport system which selectively translocates lysosomes, and possibly other organelles, into dendrites. The size, shape, and number of these organelles may change as a result of this transport.

Redistribution of lipofuscin in aged neurons induced by colchicine

The effect of a single, 40 micrograms, intracerebroventricular injection of colchicine on the distribution of neuronal lysosomes and lipofuscin granules in aged mice was studied. At the light microscope level we observed that colchicine induced a redistribution of dipeptidyl aminopeptidase II (Dpp II), a lysosomal and lipofuscin granule marker enzyme, from the cell bodies of neurons to the dendrites; cell bodies became depleted of Dpp II while dendrites became enriched with this enzyme. Quantitation of this phenomenon at the electron microscope level demonstrated that colchicine induced a rapid and significant decrease in the density of lysosomes and lipofuscin granules from the somata of neurons whereas in dendrites we observed a significant increase in the density of these organelles.

Fate of lysosomes transported to the dendrites by a colchicine-induced mechanism.

Lysosomes in hypoglossal motoneurons were retrogradely labeled with fluorescent latex microspheres and their distribution as well as that of acid phosphatase was examined after a single 50 microgram intracerebroventricular (i.c.v.) injection of colchicine. In saline injected controls the fluorescent label was distributed mainly in cell bodies. Twenty-four hours after the colchicine injection we observed a re-distribution of fluorescent label from the cell body of neurons to the dendrites. Seventy-two hours after the colchicine injection the fluorescent label had returned from the dendrites to the cell body. A similar pattern was obtained by following the effect of colchicine on the distribution of acid phosphatase reaction product. We conclude that the reappearance of fluorescent label in cell bodies following colchicine treatment is the result of the retrograde transport of dendritic lysosomes.