The early intracellular signaling pathway for the insulin/insulin-like growth factor receptor family in the mammalian central nervous system.

Several studies support the idea that the polypeptides belonging to the family of insulin and insulin-like growth factors (IGFs) play an important role in brain development and continue to be produced in discrete areas of the adult brain. In numerous neuronal populations within the olfactory bulb, the cerebral and cerebellar cortex, the hippocampus, some diencephalic and brainstem nuclei, the spinal cord and the retina, specific insulin and IGF receptors, as well as crucial components of the intracellular receptor signaling pathway have been demonstrated. Thus, mature neurons are endowed with the cellular machinery to respond to insulin and IGF stimulation. Studies in vitro and in vivo, using normal and transgenic animals, have led to the hypothesis that, in the adult brain, IGF-I not only acts as a trophic factor, but also as a neuromodulator of some higher brain functions, such as long-term potentiation and depression. Furthermore, a trophic effect on certain neuronal populations becomes clearly evident in the ischemic brain or neurodegenerative disorders. Thus, the analysis of the early intracellular signaling pathway for the insulin/IGF receptor family in the brain is providing us with new intriguing findings on the way the mammalian brain is sculpted and operates.

Distribution of protein gene product 9.5 (PGP 9.5) in the vertebrate retina: evidence that immunoreactivity is restricted to mammalian horizontal and ganglion cells.

Using light microscopic immunocytochemistry, we have studied the distribution of protein gene product 9.5 (PGP 9.5), a neuron-specific protein first extracted from human brain (Doran et al., '83:J. Neurochem. 40:1542-1547), in the vertebrate retina. Retinas were obtained from frog, chicken, rat, rabbit, cow, cat, dog, and human. No immunoreactivity was observed in frog and only a faint staining was present in chicken. In mammalian retinas, a strong positive reaction was restricted to horizontal and ganglion cells, with minor interspecies variations. Immunostaining was present throughout the cell body and the dendritic tree in horizontal cells. At the level of retinal ganglion cells, immunolabel was particularly abundant in cell bodies and axons forming the optic nerve. Only the main dendrites were stained, the remainder of the dendritic tree giving rise to a diffuse punctate reaction in the inner plexiform layer. In rats, displaced amacrine cells, which are known to contribute largely (40-50%) to the total neuronal population within the ganglion cell layer (Perry, '81: Neuroscience 6:931-944) were not immunoreactive, as demonstrated from (i) analysis of the morphology, cell size and cell density of immunoreactive neurons in wholemounts; (ii) colocalization of retrograde label and PGP 9.5 immunoreactivity in about 80% of ganglion cells after injection of peroxidase into the optic nerve; and (iii) reduction of immunoreactivity in the inner plexiform and ganglion cell layers following optic nerve transection. Western blot analysis of extracts from rabbit retinas indicated that the immunoreactive species is PGP 9.5 or a closely related molecule. Recent studies have demonstrated that PGP 9.5 is a ubiquitin carboxyl-terminal hydrolase (Wilkinson et al., '89:Science 246:670-673). The present results, therefore, suggest that differences in the ubiquitination process exist between retinal neurons.

The immunocytochemical distribution of seven peptides in the spinal cord and dorsal root ganglia of horse and pig.

The distribution of calcitonin gene-related peptide (CGRP), enkephalin, galanin, neuropeptide Y (NPY), somatostatin, tachykinins and vasoactive intestinal polypeptide (VIP) was compared in cervical, thoracic, lumbar and sacral segmental levels of spinal cord and dorsal root ganglia of horse and pig. In both species, immunoreactivity for the peptides under study was observed at all segmental levels of the spinal cord. Peptide-immunoreactive fibres were generally concentrated in laminae I-III, the region around the central canal, and in the autonomic nuclei. A general increase in the number of immunoreactive nerve fibres was noted in the lumbosacral segments of the spinal cord, which was particularly exaggerated in the case of VIP immunoreactivity. In the horse, some CGRP-, somatostatin- or tachykinin-immunoreactive cell bodies were present in the dorsal horn. In the pig, cells immunoreactive for somatostatin, enkephalin or NPY were noted in a similar location. In the ventral horn most motoneurones were CGRP-immunoreactive in both species. However, in pig many other cell types were CGRP-immunoreactive not only in the ventral horn, but also in laminae V-VI of the dorsal horn. With the exception of enkephalin and NPY immunoreactivity, which was not seen in pig dorsal root ganglia, all peptides studied were localised to neuronal cell bodies and/or fibres in the dorsal root ganglia. In both species, immunolabeled cell bodies were observed in ganglia from cervical, thoracic, lumbar and sacral levels, with the exception of VIP-immunoreactive cells that were detected only in the lumbosacral ganglia. Numerous CGRP- and tachykinin-immunoreactive cell bodies were visualised in both species, while the cells immunolabeled with other peptide antisera were much lower in number. In both species, immunostaining of serial sections revealed that a subset of CGRP-immunoreactive cells co-expressed tachykinin, galanin or somatostatin immunoreactivity. In the horse some enkephalin-immunoreactive cells were also CGRP positive and occasionally combinations of three peptides, e.g. CGRP, tachykinin and galanin or CGRP, tachykinin and enkephalin were identified. The results obtained suggest that the overall pattern of distribution of peptide immunoreactivities is in general agreement with that so far described in other mammals, although some species variations have been observed, particularly regarding the presence of immunoreactive cell bodies in the dorsal horn of the spinal cord.

The neurochemical maturation of the rabbit cerebellum.

The immunocytochemical distribution of several neuronal and glial antigens was investigated in the cerebellum of the developing and adult rabbit. Neurofilament positive neurons appeared at embryonic day (E) 25. Purkinje cells transiently expressed neurofilament polypeptides from postnatal day (P) 0 to 15. At later postnatal ages, staining was localised to the parallel fibres, the axonal arbors of the basket cells and fibres of the white matter. Neuron specific enolase (NSE) immunoreactivity was first detected at E25. At P0 Purkinje cells were positive and their staining intensity increased up to P25. From P30 to adulthood virtually all cells in the molecular and Purkinje cell layers were stained. Scattered PGP 9.5-immunoreactive neurons appeared in the cerebellar anlage at P25. Purkinje and Golgi cells were labelled by P0. Synaptophysin immunoreactivity was first observed at P0 in the form of a fine punctate reaction surrounding the perikarya and proximal dendrites of Purkinje cells. By P10, it became particularly intense within the cerebellar glomeruli of the granular layer. Neurons of the deep cerebellar nuclei expressed NSE and PGP 9.5 starting from E25. GFAP and S-100 immunoreactivities were first detected at P10. GFAP-immunopositive astrocytes progressively increased in number up to adulthood. S-100-immunoreactive glial cells were detected throughout the white and grey matter. Bergmann glial cells and their fibres were strongly immunoreactive. Vimentin positive glial cells and fibres were first observed at E15 and persisted up to adulthood. Double labelling experiments using a monoclonal antibody against the proliferating cell nuclear antigen (PCNA), a cyclin synthesised by mitotic cells, showed that neuronal and/or glial polypeptides are expressed only by fully differentiated postmitotic cells. These results indicate that major events in the neurochemical maturation of the rabbit cerebellum occur during the first month after birth, when the same pattern of the adult animal is attained.

In vitro expression of n-cadherin adhesion molecule during early cardiac morphogenesis.

The aim of the present study was to investigate, "in vitro", the degree of organogenetic potentiality of the cells of the cardiogenic area during the early developmental stages of the chick embryo. Embryos from between the end of the presomitic stage to the 8 somite stage were studied. The subcephalic fold was cultured in liquid medium for up to 7 days. After 24 hs of culturing, an extended migration ring was observed. In the explants, from 3 somite stage, onwards, beating masses were noted, the shape and size of which suggested a vascular-like structure. Sections of the cultures were processed for the detection of the N-Cadherin adhesion molecule. The observations stated that the diffusion and intensity of expression of this receptor is related to the stage od development of the embryo. Cultures from the presomitic stage to 3 somite stage did not express the molecule. Instead, expression took place in those cultures of embryos at the 3 somite stage, onwards. In the cultures to which the antiserum against N-Cadherin had been to the medium, the formation of vascular-like structures was affected. The changes depended on the age of the embryos. These observations suggest that the expression of the N-Cadherin is related to the potentiality of the presumptive myocardic cells to organize themselves, at least "in vitro", to form a well-defined tridimensional structure. The expression of the adhesion molecule and the potentiality of the cells to build tubular structures were transient features, "in vitro" in our cultures. This suggests that "in vivo" the expression of the N-Cadherin must be aided by factors which, at present, are unidentified.

"In vitro" expression of the cardiogenic potentiality of chick embryo heart-forming cells.

Chick embryos between final presomitic and 4 somites stage were studied. The subcephalic fold was handly severed from the embryos and cultivated in liquid medium for 7 days. Because of the embryo age, no heart anlage was observed at the moment of dissection. After 4 hours of culture the cells began to migrate from the explants. After 20 hours a very extended migration ring was observed in all of the cultures; in the explants, one or more newformed tubular or spherical masses of cells throbbed rhythmically. Their size and shape were related to the embryos age: from presomitic embryos, irregular clusters appeared, while starting from two somites embryos tubular, vascular-like structures were formed. The cells of the throbbing areas at submicroscopic observation showed organizing myofibrillar apparatus into the cytoplasm; junctional complexes between the cells and gap junctions in course of organization were present in the vascular-like structures. This suggests that very early, in the lateral mesoderm are the presumptive cardiac cells which can develop "in vitro" as myocardic elements even in absence of the interactions that occur during the development "in vitro"; the observed vascular-like structures may be considered as an attempt to form a sort of cardiac primordium "in vitro", and a further step in the expression of the cardiogenic potentiality, involving cell-cell communications. The serial sections of the embryos enhanced that into the cultivated areas, vessels from yolk sac are always present; this suggests that the vascular structures, i.e. the endothelium may be involved in the determination of the myocardic elements.

Distribution of five peptides, three general neuroendocrine markers, and two synaptic-vesicle-associated proteins in the spinal cord and dorsal root ganglia of the adult and newborn dog: an immunocytochemical study.

This study describes the immunocytochemical distribution of five neuropeptides (calcitonin gene-related peptide [CGRP], enkephalin, galanin, somatostatin, and substance P), three neuronal markers (neurofilament triplet proteins, neuron-specific enolase [NSE], and protein gene product 9.5), and two synaptic-vesicle-associated proteins (synapsin I and synaptophysin) in the spinal cord and dorsal root ganglia of adult and newborn dogs. CGRP and substance P were the only peptides detectable at birth in the spinal cord; they were present within a small number of immunoreactive fibers concentrated in laminae I-II. CGRP immunoreactivity was also observed in motoneurons and in dorsal root ganglion cells. In adult animals, all peptides under study were localized to varicose fibers forming rich plexuses within laminae I-III and, to a lesser extent, lamina X and the intermediolateral cell columns. Some dorsal root ganglion neurons were CGRP- and/or substance P-immunoreactive. The other antigens were present in the spinal cord and dorsal root ganglia of both adult and newborn animals, with the exception of NSE, which, at birth, was not detectable in spinal cord neurons. Moreover, synapsin I/synaptophysin immunoreactivity, at birth, was restricted to laminae I-II, while in adult dogs, immunostaining was observed in terminal-like elements throughout the spinal neuropil. These results suggest that in the dog spinal cord and dorsal root ganglia, peptide-containing pathways complete their development during postnatal life, together with the full expression of NSE and synapsin I/synaptophysin immunoreactivities. In adulthood, peptide distribution is similar to that described in other mammals, although a relative absence of immunoreactive cell bodies was observed in the spinal cord.

Cell proliferation in the post-natal and adult mammalian central nervous system.

In the mammalian central nervous system cell proliferation is generally linked to developmental processes that are ultimated in the perinatal period. Few exceptions to this rule are known in certain regions of the mammalian brain, namely the post-natal cerebellar cortex and the adult subependymal layer. We report here the results of our studies about cell proliferation and related phenomena in these regions. Cell proliferation was visualised after bromodeoxyuridine incorporation and labeling of the proliferating cell nuclear antigen (PCNA), an endogenous protein expressed during the cell cycle. The occurrence of programmed cell death in the post-natal cerebellar cortex and the persistence of the embryonic isoform of neural cell adhesion molecule (NCAM) associated with proliferating cells in the adult subependymal layer were also investigated.