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In 14-day-old thyroid-deficient rats, qualitative and quantitative ultrastructural study of the cerebellar molecular layer shows that the retardation in synaptogenesis between Purkinje cell dendritic spines or excrescences and synaptic segments of parallel fibres is related to the marked hypoplasia of Purkinje cell dendritic arborizations and to the retarded development of parallel fibres rather than to the delay in the deposition of granule cells. Synaptogenesis inhibition is more marked in the inner parts of the molecular layer so that the normal "march" of synaptogenesis from the bottom of the layer upwards is distorted. This study also indicates that thyroid hormone deprivation affects synaptogenesis by interacting with growth and branching of neuronal processes and with the organization of their microtubular apparatus rather than with the differentiation of the structural specializations forming the synapses. The first corrective effect of a very low dose of thyroxine administered 24 h before sacrifice to the hypothyroid animal is observed at the level of parallel fibres and their varicosities. Purkinje cells take longer to respond to the hormone. However, their directing role in the histogenesis of the cerebellar cortex is strongly suggested. The significance of these observations with regard to the mechanisms underlying the effects of thyroid hormone on synaptogenesis and synaptic organization between Purkinje cells and parallel fibres as well as on histogenetic granule cell death is discussed.

In 35-day-old rats which were undernourished by quantitative restriction of the mother's diet from the 6th day of gestation, the wet weight and the DNA content of the cerebellum were slightly more decreased than those of the cerebrum. Cell growth (estimated from the DNA concentration and form the ratios of RNA and protein to DNA) was significantly affected by food deprivation only in the cerebellum. In the cerebullar cortex, the number of Purkinje, Golgi and stellate cells were unchanged. The numbers of other cell tyes were affected to various extents: there were significantly less granules and basket cells per Purkinje cell, and a still more marked hypoplasia of glia involving the glial cells of the molecular layer, as well as the astrocytes of the internal granular layer and the Bergmann cells of the Purkinje cell layer. Finally, the total number of glial cells within the cortex was decreased by 44% against 13% for neurones. These effects of undernutrition on cell acquisition within the brain, and on the cellular composition of the cerebellum, contrast with those of thyroid deficiency.

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.

The formation and morphological maturation of Bergmann glia cells, which might play an important role in the histogenesis of the cerebellar cortex, was studied by histological and electron microscopic techniques in the cerebellum of normal young rats and animals made hypothyroid since the 18th day of gestation or hyperthyroid since birth. In comparison with controls, an acceleration in the morphological development of Bergmann glia cells was observed in the hyperthyroid animals; on the contrary, in the thyroid-deficient rats, the formation and morphological maturation of these cells were retarded and their final number was increased.

The cerebellar circuitry and the corticonuclear relationships were studied in the cerebellum of adult rats rendered agranular through 7 successive exposures to X-ray radiations during infancy. Data were obtained through examination of electrical responses induced in Purkinje cells (PC) and in neurons of the lateral vestibular nucleus (LVN) by cerebellar and spinal stimulations. In irradiated rats, PC exhibited antidromic activation with a high axonal threshold and 70% of them also presented typical climbing fiber responses (CFRs). By contrast, they exceptionnally exhibited responses via the mossy fiber (MF)-granule cell pathway, but two other classes of responses were identified: i) short latency single spike responses attributed to a direct excitatory impingement of MF onto PC; ii) atypical CFRs formed of high frequency bursts of simple spikes which were seen in 76% of PC tested. Furthermore, 53% of these cells also presented typical CFRs, strongly suggesting these PC were innervated by more than one CF, thus confirming previous data on the same type of agranular cerebellum. In the LVN neurons of control and irradiated rats, spinal and cerebellar stimulations evoked clear cut IPSPs. On the basis of their shape, latency, and occurrence in animals with or without cerebellum and with or without lesion of the CF pathway, they were interpreted as mediated through direct or synaptic activation of PC or through an extracerebellar pathway. In irradiated rats, the quantitative study of these IPSPs gave further arguments in favor of a multiinnervation of PC by CF and of an important reafferentation of MF onto PC. However, the functional efficiency of this reafferentation appeared very low, as tested by activation of MF originating in the spinal cord. Finally, the intracellular recording of LVN neurons showed that a large majority of PC axons retained normal synaptic connections with nuclear cells in treated animals, indicating that corticonuclear relationships do not markedly depend upon granule cells and normal CF input.

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

Effects of neonatal hyperthyroidism on cell formation in the developing rat cerebellum were reinvestigated. Administration at birth of excessive doses of thyroxine or triiodothyronine led to an early stimulation of cell acquisition, followed by a permanent deficit of cells in the cerebellum. The corrective effects of physiological doses of thyroxine on the troubles of the histological and biochemical development of the cerebellum in thyroid-deficient animals were also studied. As early as 6 days, cell maturation and formation were already retarded in animals treated with propylthiouracil, but, as previously reported, cell formation was prolonged and the final number of cells was normal. Administration to thyroid-deficient animals of progressively increasing doses of thyroxine, nearly equal to the amounts of hormone secreted by the thyroid gland of the developing normal rat, returned the evolution of the cerebellar wet weight and of the cerebellar DNA to normal, as well as the histological maturation of the cerebellum, even if it did not entirely correct the retardation of body growth. These results are consistent with the view that thyroid hormone early stimulates maturation of the cerebellar germinative cells and subsequently interacts with cell formation in the cerebellum, and that this action is physiological.

Young rats made hypothyroid by propylthiouracil (PTU) received a daily physiological dose of thyroxine (T4) from day 0, 4, 6, 8, 10, 11, 12 or 13 and their cerebella were studied on day 14. With the very low doses of T4 used and when the treatment was started at birth, cerebellar development was nearly normal in terms of the parameters studied (cell formation, migration, maturation and death). The effect of T4 on cell formation appeared after two days. With the same latency, T4 induced migration of the newly-formed granule cells. The effects on those processes requiring cell movements over long distances, e.g. the number of cells in the internal granular layer or the thickness of the molecular layer, were longer to appear. The most rapidly affected parameter was the pyknotic index in the internal granular layer. This index was half. The increased cell death in the cerebellum of hypothyroid rats is probably related to the decreased synaptogenetic competence of Purkinje cells. The rapidity of the effect of T4 on the pyknotic index may be related to an important effect of this hormone on the formation of synapses and, more generally, on the mechanisms of neuronal maturation.

The developmental pattern of S100 protein, a specific marker of astroglia, was studied by radioimmunoassay in the cerebellum of thyroid-deficient, thyroxine-treated and undernourished rats during development. In the control animals, the S100 protein content of the cerebellum increased maximally after the 3rd postnatal week, i.e., after cell multiplication had stopped and when the cerebellum had acquired more than 70% of its adult weight and protein content. This developmental pattern of S100 protein reflected essentially the maturation of astroglia. In the thyroxine-treated rats the total amount and the concentration of S100 protein were higher than in controls during the first 3 weeks of postnatal life and returned to normal values thereafter. In the thyroid-deficient rats both the amount and concentration were lower than in controls throughout development. In the undernourished animals the amount of S100 protein per organ was also lower than in controls during the whole experimental period studied; the S100 protein concentration was higher during the first 2 postnatal weeks and became lower thereafter. These results are discussed taking into consideration previous histological and ultrastructural observations on the effects of altered thyroid state and undernutrition on the formation and maturation of cerebellar astrocytes.