Ch. Legrand

Calcium-binding protein in the developing rat cerebellum

SummarySpecific antibodies raised against a human 28 000 dalton cerebellar calcium-binding protein (CaBP) were used in an immunocytochemical study during development of the rat cerebellum. Both light and electron microscopy showed (1) that labelling was entirely restricted to the Purkinje cells, (2) that it appeared very early in Purkinje cell development, (3) that the entire cell was labelled from the tip of the smallest dendrites to the axonal terminals, and (4) that with increasing age, the immunoreaction appeared to be progressively restricted to the cell and organelle membranes.

Immunocytochemical detection of calcium-binding protein in the cochlear and vestibular hair cells of the rat.

SummarySpecific antibodies raised against human cerebellar calciumbinding protein (CaBP) intensely labelled the cochlear hair cells of the rat. The vestibular hair cells also stained weakly. In both inner and outer cochlear hair cells, the cuticular plate was the most stained area. These results suggest that CaBP may prevent excessive concentrations of intracellular calcium and thus modulate some Ca2+-mediated biochemical processes, especially at the level of the cuticular plate and stereocilia; CaBP could be involved in the mechanochemical coupling of hearing or vestibular function.

Effects of thyroid deficiency and corrective effects of thyroxine on microtubules and mitochondria in cerebellar Purkinje cell dendrites of developing rats

Abstract A quantitative ultrastructural study of microtubules and mitochondria was performed in cross-sections of Purkinje cell dendrites in 14-old-day normal and thyroid-deficient rats. The corrective effects of thyroxine were also studied. In normal rats, the density of microtubules was found nearly constant whatever the size of the dendrite might be. In the hypothyroid animals, the density of microtubules was about reduced by half in the largest dendritic tranks but remained nearly normal in the smallest ones. This severe reduction in the number of microtubules in primary and secondary dendrites might reduce considerably the possibilities of branching and might explain the striking hypoplasia of the Purkinje cell dendritic arborization characteristic of thyroid deficiency. In the thyroid-deficient animals the corrective effects of thyroxine on the development of the microtubular apparatus were obvious after only one day and complete after 4 days. The effects of hypothyroidism on mitochondria were relatively weak when compared to those on microtubules. On the other hand, mitochondria responded more rapidly than microtubules to thyroxine treatment. It is proposed that, besides more general effects on neurones, thyroid hormone might control growth and branching of dendrites by an action on microtubules mediated through a local stimulation of mitochondria.

An interaction between thyroid hormone and nerve growth factor promotes the development of hippocampus, olfactory bulbs and cerebellum: a comparative biochemical study of normal and hypothyroid rats.

The effects of treatment with L-thyroxine (T4;20 ng/g body weight, given subcutaneously on days 1, 3, 5, 7 and 9), 2.5 S nerve growth factor (NGF; 2 ng/mg brain weight, given intracerebroventricularly on days 1, 3, 5, 7 and 9), monoclonal anti-NGF (2 ng/mg wet weight, given intracerebroventricularly on days 1, 3, 5, 7 and 9), and monoclonal anti-NGF receptor (192 IgG; 2 ng/mg wet weight, injected daily from day 1 to day 9) antibodies, separately or together, were studied on the biochemical development of hippocampal formation, olfactory bulbs and cerebellum in 10-day-old and 15-day-old normal and hypothyroid rats. The results provide the following information: (1) CNS structures other than the basal forebrain are sensitive to NGF during early development. (2) Both normal and hypothyroid rats are more sensitive to NGF deprivation than NGF supplementation. (3) The effects of anti-NGF antibodies in normal rats are similar to those induced by anti-NGFr antibodies. (4) NGF alone had little or no effect, but interacts with T4 in promoting cell maturation, especially in hypothyroid rats. (5) Hypothyroid rats are more sensitive to T4 and to T4 plus NGF than are normal ones. (6) The synergistic action of both trophic factors, but not that of T4, tend to disappear at long term in hypothyroid rats. (7) The differential sensitivity of the brain areas to T4, NGF, or both trophic factors correlates with their cell acquisition rate, especially in hypothyroid rats. (8) T4 and NGF together act more markedly (but not exclusively) on the cholinergic structures in both normal and hypothyroid rats. (9) RNA appears to be very sensitive to NGF, especially in hypothyroid rats. In close correlation with preliminary morphological observations, the results clearly demonstrate that an interaction between T4 and NGF regulates the ontogeny of a number of neuronal structures in CNS independently of their neurotransmitter phenotype, but with a regional specificity. The possibilities of accounting for this interaction, in particular the major role of thyroxine, are discussed.

The microtubular apparatus of cerebellar purkinje cell dendrites during postnatal development of the rat: The density and cold-stability of microtubules increase with age and are sensitive to thyroid hormone deficiency

A quantitative ultrastructural study of microtubules in Purkinje cell dendrites of normal and hypothyroid developing rats was performed after fixation either at room or at low temperature (4°C). In normal animals, the density of microtubules and their fold‐stability increased with age, more especially during the period of intense dendritic growth. Thyroid deficiency delayed the appearance of microtubules and still more the acquisition of their fold‐stability. These effects might explain the defects in Purkinje cell dendritic growth and branching observed in hypothyroid animals.

LOCALIZATION OF S100 PROTEIN IN THE RAT CEREBELLUM: AN IMMUNOELECTRON MICROSCOPE STUDY

Immunoelectron microscopy has shown that, in adult rat cerebellum, S100 protein is localized exclusively in the astrocytes of both the cortex and the white matter. The labelling pattern was unaffected by the inclusion of glutaraldehyde in the primary paraformaldehyde fixative. The immunoperox‐idase reaction product is observed over both the perikaryal cytoplasm of astrocytes and their processes. S100 protein was not found in neuronal structures nor in oligodendrocytes.

Con A-binding glycoproteins in the developing cerebellum of control and hypothyroid rats

Concanavalin A (Con A)-binding glycoproteins were studied during the postnatal development of the cerebellum of control and hypothyroid rats. Only 4 glycoprotein bands have a transient behavior in control animals. They progressively increase until the 13th day and markedly decline between the 15th and the 18th postnatal day. In the cerebellum of hypothyroid rats, the level of these compounds is greatly reduced and the previous decrease observed in controls is not found again. This defect of Con A-binding glycoproteins mainly localized on the plasma membrane of parallel fibers might be related to the reduced synaptogenesis observed in the molecular layer of hypothyroid rats between parallel fibers and Purkinje cell dendritic spines.

HISTOCHEMICAL AND BIOCHEMICAL STUDIES OF BUTYRYLCHOLINESTERASE ACTIVITY IN ADULT AND DEVELOPING CEREBELLUM. EFFECTS OF ABNORMAL THYROID STATE AND UNDERNUTRITION

The cellular and subcellular localization of BuchE activity (EC.3.1.1.8) was studied in the developing and adult rat cerebellum at light and electron microscope levels. In the adult cerebellum, BuchE activity was exclusively localized to glial cells, myelin and endothelial cells. In the immature cerebellum, BuchE activity was additionally found transiently localized to the neuroblasts of the external germinative layer and in Purkinje cells of the nodulus. In both the immature and the adult animals, the main part of the activity seemed to be membrane‐bound. The developmental pattern of cerebellar BuchE activity was assayed in developing normal, hypothyroid, thyroxine‐treated and undernourished rats. In normal newborn rats, the specific activity was higher than in adults and it showed one characteristic peak at 6 days (1–8 times the adult value reached at 30 days). At the age of 5 days, the ratio of BuchE‐containing astrocytes (numbered in the ganglionic layer) to Purkinje cells was the same as the ratio of Bergmann astrocytes to Purkinje cells determined at 35 days in Nissl preparations; their nucleus size already represented 80% of the adult value and their processes were well developed. The three experimental conditions modified the timing of BuchE development. During the early post‐natal period, it was accelerated in the thyroxine‐treated and undernourished animals, while in the hypothyroid rats it was delayed. During the same period, the number of labelled astrocytes per Purkinje cell was modified only by hypothyroidism and undernourishment. On the basis of these histochemical and biochemical results, BuchE can be considered as a good marker for the study of Bergmann glia development in the early post‐natal period.

Thyroid state and cholecalcin (calcium-binding protein) in cerebellum of the developing rat

Cholecalcin (28,000 Da, vitamin D-dependent calcium-binding protein) is a marker of Purkinje cell development in the rat cerebellum from embryonic day 17 when these cells can first be distinguished. Specific antibodies raised against human cerebellar or rat renal cholecalcin were used in an immunocytochemical and quantitative study in altered thyroid states. The immunocytochemical staining was qualitatively similar in both normal and hypothyroid animals but clearly demonstrated the slowing of Purkinje cell development resulting from the lack of thyroxine. This effect was also reflected in quantitative studies which showed that the total cholecalcin per cerebellum was lower in thyroid-deficient rats. However, there was, in these animals, no specific reduction in cholecalcin level. Moreover, the response to thyroxine treatment indicated that the synthesis of cholecalcin occurred later and slower than that of the majority of cerebellar proteins and even after other more complex mechanisms of cerebellar cortex development (such as neurite outgrowth) have been induced. Thus, cholecalcin synthesis does not appear particularly sensitive to thyroid hormone level but might rather follow the increase in cell size induced by the hormone.