The present study was undertaken to determine whether release of glutamate is capable of influencing dendritic morphology in a developing network of rat hippocampal neurons in vitro. Control cultures developed a dense network of fibers and evinced spontaneous electrical activity from the third day in vitro. Dendrites were examined in cultures maintained for 2 weeks in vitro: the experimental group grown in medium containing the glutamate receptor antagonists AP-5 and DNQX. Dendritic extensions were analyzed as a function of time (days in vitro) using a number of morphometric parameters, vis. the number of processes, the number and length of intermediate and terminal segments, as well as the total length of all segments. We found that the effect of age and treatment was most prominently reflected in the length of the terminal segments. Chronic addition of ionotropic glutamate receptor antagonists from day 2 in culture arrested all dendrite parameters at the prefunctional level. The results suggest that glutamate release is crucial for the onset of dendritic morphological development in hippocampal neurons.

Chronic suppression of spontaneous bioelectric activity in spinal cord explants in the presence of tetrodotoxin (TTX) during network formation caused a large reduction in cell number (lowered DNA levels). The addition of gangliosides failed to protect against this cell loss. Conversely, the omission of galactose from the growth medium had no effect on DNA levels. It was concluded that the presence or absence of afferent selectivity is unlikely to require the survival of a regionally specific subpopulation of preferred dorsal root ganglion target cells. Neocortical explants also showed a large reduction in DNA levels following chronic TTX treatment, and morphometric analysis confirmed that neuronal survival was affected to the same degree. Chronic ganglioside supplementation failed to influence DNA and cell counts in either control or TTX-treated explants, but one of the added gangliosides (GD1a) stimulated extensive neuritic outgrowth in electrically silenced cultures. Particular ganglioside species, therefore, may exert a growth stimulating influence that can partially compensate for the absence of bioelectric self-stimulation during early development.

Dissociated and non-dissociated cerebral cortex of fetal rat was successfully cultured in a serum-free, chemically defined medium for at least 18 days without any preincubation in serum-supplemented medium. Neurite outgrowth, synapse formation and spontaneous bioelectric activity, in its qualitative and quantitative aspects, were essentially the same as was observed in medium containing 20% heat-inactivated horse serum.

Using antibodies against AVT, α-MSH, LHRH and somatostatin, immunoreactive cells were detected in the rat pineal gland. All of these antibodies stain the same cells, which also react immunocytochemically when an antibody against the UMO5R sheep pineal fraction, a fraction that presents antigonadotropic properties in vivo, is used. Relatively more immunoreactive cells are present in the pineals of young rats than in the pineals of adult animals. Comparison of the results obtained with different potent antibodies against each of the peptides, and a study of the staining properties of the antibodies in the pineal after solid phase adsorption to different peptides or to different sheep pineal fractions, led to the proposal that the immunoreactivity found in the rat pineal is not due to the presence of AVT, α-MSH, LHRH or somatostatin, but to a cross-reaction of each of these antibodies with (an) unidentified compound(s). This compound is synthetized in the pineal gland, as was demonstrated using cultured pineals. The UMO5R and the Prot. 4 fractions of the sheep pineal seem to be chemically related to this unknown compound, the possible endocrine nature of which is discussed.

Regulation of the developing nervous system involves attraction, guidance and modification of innervating neurons by target cells through diffusible and membrane-related factors. The trophic effects from specific cell types remain to be investigated and characterized. In a series of experiments in which human fetal mesencephalic dopaminergic cells were co-cultured with target or non-target neurons or glial cells in direct or contiguous contact, we demonstrate that striatal glial cells (target-derived glia) can enhance dopaminergic neuron survival by up to 400% compared to either non-target cell co-cultures or mesencephalic controls. When in direct contact with striatal neurons, a greater proportion of dopaminergic neurons had a more differentiated morphology. The enhancement of dopaminergic neuron survival by target-derived glia appears to be mediated both by direct contact, possibly through target membrane-specific phenomena, and by diffusible substances, whereas non-target glia appear to exert the trophic effects predominantly through the latter mechanism.The finding that target neurons influence mainly dopaminergic neuron differentiation and target glia their survival indicates multiple, target cell type-specific regulation of innervating neuron development. These findings also have relevance to the establishment of neuronal cultures for neural transplantation.

In order to localize pineal indoleamine synthesis at the ultrastructural level, an electron microscopic analysis was carried out on rabbit pineal tissue, cultured for several days in a medium containing the inhibitor p-chlorophenylalanine. Furthermore, electron microscopic autoradiography was applied to rabbit pineal tissue cultured in a medium containing the tritium labeled precursor 5-hydroxytryptophan. p-Chlorophenylalanine altered the ultrastructure of the mitochondria in the light pinealocytes only. Incubation with 3H-5-hydroxytryptophan caused a moderate and uniform labeling of the pinealocytes. These data suggest that the synthesis and storage of indoleamines in the rabbit pineal gland do not take place within the conventional membrane-limited cell organelles but more diffusely in the cytosol of the light pinealocytes.

Electron microscopic evidence is given that the small dark granules in the rabbit dark pinealocytes represent glycogen. The conception is put forward that this glycogen, synthesized at the membranes of the rough endoplasmic reticulum, is taken up by lysosomes to participate then in the conversion of the lysosomes into pigment bodies.

Evidence is given of diurnal rhythms in the mean number of dense-core vesicles surrounding a Golgi complex in the perikaryon of light rabbit pinealocytes and of those present per terminal of light pinealocyte processes. These rhythms show their top at noon and in the evening, respectively.