Neurogenesis in the adult rat dentate gyrus is enhanced by vitamin E deficiency.

Neurogenesis occurs throughout adult life in rat dentate gyrus. Factors and mechanisms of adult neurogenesis regulation are not well known. Vitamin E deficiency has been found to deliver a neurogenetic potential in rat dorsal root ganglia. To determine whether the role of tocopherols in adult neurogenesis may be generalized to the central nervous system, changes in adult rat dentate gyrus neurogenesis were investigated in vitamin E deficiency. Neurogenesis was quantitatively studied by determination of the density of 5-bromo-2'-deoxyuridine (BrdU)-labeled cells and by determination of the total number of cells in the granule cell layer. The BrdU-labeled cells were immunocytochemically characterized by demonstration of neuronal marker calbindin D28K. The following results were found: (1) the volume of the granule layer increased in controls from 1 to 5 months of age, mainly due to cell density decrease; (2) the volume increased by a similar amount in vitamin E-deficient rats, mainly because of an increase in cell number; (3) BrdU-positive cells were more numerous in vitamin E-deficient rats in comparison to age-matched controls; (4) the increase in proliferated cells was located in the hilus and in the plexiform layer. This study confirms that neurogenesis occurs within adult dentate gyrus and demonstrates that this process is enhanced in vitamin E deficiency. This finding indicates that vitamin E may be an exogenous factor regulating adult neurogenesis.

Neuron and glial cells in neocortex after methylazoxymethanol treatment in early development.

The quantitative changes were investigated in neuron and glia density in the different cortical layers of the frontal cortex of 3 and 12 month old mice, exposed to methylazoxymethanol on embryonic day 13 (MAM13). No loss of cortical neurons was found between young and adult animals. MAM exposure on the 13th day of development induced a neuron density decrease throughout on the entire cortical depth and did not produce changes in the density of glial cells with respect to the controls and to age. Consequently, at 3 months of age we observe a glia/neuron ratio greater than that of controls and at 12 months a similar value. In the neocortex of MAM-mice at this numerical uniformity of glial cell density, did not correspond to a similar proportional composition: the frequency of the astrocytes is lower, adapting to the decreased neuron density; the greater oligodendrocyte percentage may be related to disturbed layering and to the hyperinnervation of the hypoplastic cortex; the microglia shows a trend similar to that of the controls. These results, together with those of other studies, suggest that prenatal exposure to MAM causes a cortical compensatory response regulating glial cells proliferation.

Vitamin E affects quantitative age changes in lumbar motoneurons and in their peripheral projections.

Vitamin E deficiency was previously found to induce plastic changes in the number of primary sensory neurons and in motoneuron peripheral field projections. In this work, quantitative changes in motoneurons of lumbar segments, in nerve fibres constituting ventral roots and in innervating leg motor fibres were studied in normal and vitamin E deficient rats from 1 to 5 months of age. The number of lumbar motoneurons was found to decrease, while there were no changes in the number of ventral root fibres. An increase in the number of innervating leg motor fibres was observed during ageing in control rats; in vitamin E deficient rats the number of fibres in the ventral roots did not change, as occurred in controls, but the decrease in the number of motoneurons was smaller and the number of innervating leg motor fibres increased further in comparison to the controls. The findings are consistent with the idea that vitamin E deficiency causes a decrease in motoneuron death or, alternatively, that it induces some process partially compensating naturally occurring motoneuron death.

High glucose-6-phosphate dehydrogenase activity contributes to the structural plasticity of periglomerular cells in the olfactory bulb of adult rats.

Glucose-6-phosphate dehydrogenase (G6PD) activity, assayed spectrophotometrically, was found to be higher in the olfactory bulb (OB) than in other brain areas of adult rats [P. Ninfali, G. Aluigi, W. Balduini, A. Pompella, Glucose-6-phosphate dehydrogenase is higher in the olfactory bulb than into other brain areas, Brain Res. 744 (1997) 138-142]. Histochemical demonstration of G6PD activity in cryostat sections of OB, analyzed with optical microscopy, revealed a marked and well defined line of formazan deposition in the internal part of the glomerular layer (Glm), indicating that G6PD was much higher in cells distributed along the glomeruli. Electron microscope analysis showed that G6PD activity was mainly concentrated in cytoplasm and dendrites of periglomerular cells, the interneurons which span glomeruli and connect olfactory nerves with mitral/tufted cells. Since G6PD regulates the flux through the hexose monophosphate shunt (HMS) pathway, which provides NADPH for reductive biosynthesis and pentose phosphates for nucleic acid formation, it can be concluded that high G6PD activity in periglomerular neurons is functional to their differentiating capability. This result is consistent with the occurrence of structural plasticity events in the OB of adult rats.

Glucose-6-phosphate dehydrogenase supports the functioning of the synapses in rat cerebellar cortex.

This study investigates heterogeneous glucose-6-phosphate dehydrogenase (G6PD) expression in the rat cerebellar cortex. G6PD activity and its electrophoretic pattern, evaluated on the cerebellar homogenate, were found to be similar to those of other brain areas. However, histochemical and immunohistochemical analyses revealed that the highest expression of G6PD activity and protein was in Purkinje's cells, followed by the molecular and granular layers. Electron microscopy analysis showed that, in Purkinje's cells, the G6PD reaction products were concentrated in the neurites while in the basket cells in the cell body. The granules showed a weaker activity everywhere. The quantitative distribution of G6PD is discussed in the light of the neurochemical function of these cells.

Are there proliferating neuronal precursors in adult rat dorsal root ganglia?

The origin of new neurons in dorsal root ganglia of adult rat was investigated using an experimental model in which postnatal neurogenesis naturally occurring is enhanced and restricted in a brief period of life. Possible mitotic origin of new neurons was investigated by means of 5-bromo-2-deoxyuridine, anti-NF 200 antibody was used to detect if proliferated cells showed a neuronal phenotype. The results suggest that postnatal neurogenesis in dorsal root ganglia could depend only in part on precursor proliferation and that normally new neurons derive from the late differentiation of postmitotic cells.

Alpha-tocopherol controls cell proliferation in the adult rat dentate gyrus.

The effect of alpha-tocopherol on cell proliferation and proliferated cell survival was investigated in the dentate gyrus of adult rats. Adult rats were supplemented with alpha-tocopherol, injected with 5-bromo-2'-deoxyuridine (BrdU), that is incorporated into DNA during the S-phase, and killed at different time after BrdU injection. The number of newborn cells decreased after alpha-tocopherol supplementation, confirming the hypothesis that alpha-tocopherol is able to depress cell proliferation in vivo. Most newborn cells die within few days; more newborn cells survive in alpha-tocopherol-treated rats, suggesting the hypothesis that alpha-tocopherol decreases cell death.

Spatial learning affects immature granule cell survival in adult rat dentate gyrus.

Neurogenesis occurs throughout life in mammalian dentate gyrus. The effect of learning on newborn cell survival was studied in rat. Rats were trained on a hippocampus-dependent spatial learning task by using Morris water maze. Neurogenesis was evaluated by 5-bromo-2'deoxyuridine administered before learning. Several newborn cells expressed the immature neuron marker TOAD-64. The main findings were as follows: (1) the survival of newborn cells was enhanced by learning at early stage of differentiation; (2) the newborn cells saved by learning were mainly located in the rostral part of external blade of granule cell layer and (3) there was a correlation between the actual individual learning and newborn cell survival.

Nodal and terminal sprouting by regenerating nerve in vitamin E-deficient rats.

The increased number of poly-innervated cells in normal and reinnervated extensor digitorum longus (edl) muscle of vitamin E-deficient rats suggests enhanced sprouting by motor neurons in conditions of decreased protection against lipid peroxidation. End-plates and terminal axons were observed by a combined technique that shows both end-plate acetylcholinesterase area and axons. Quantitative observations of nodal and terminal sprouting in normally innervated and reinnervated edl muscles of vitamin E-deficient rats were carried out. Branch points of nerve terminal within end-plates were also observed. Three main results were obtained. First, a notable increase of both terminal and nodal sprouting was found in reinnervated muscles of normal and vitamin E-deficient rats; moreover, a relative increase in the number of nodal sprouts occurs in the long run. Second, in muscles of uninjured, vitamin E-deficient rats, nodal and terminal sprouting and branching within end-plate was greater than in controls. Third, nodal sprouting by regenerating axons was more affected by vitamin E-deficiency than terminal sprouting and branching within end-plates.

Cytochemical and immunocytochemical methods for electron microscopic detection of glucose-6-phosphate dehydrogenase in brain areas.

This paper reports on protocols for the cytochemical and immunocytochemical determination of the glucose-6-phosphate dehydrogenase (G6PD) in brain areas by electron microscopy (EM). The cytochemical assay consists of a pre-embedding staining of small and flat tissue blocks, which were first mildly fixed and then floated in a staining mixture based on the reduction of tetrazolium salts by NADPH. Tissue blocks were then washed, post-fixed in OsO(4), dehydrated through graded ethanol concentrations and embedded in resin. Ultrathin sections were then obtained and observed at the EM. The immunocytochemical technique was performed on completely fixed tissues of perfused animals. After the tissue embedding in resin, ultrathin sections were obtained and treated with a primary anti-erythrocyte G6PD antibody, produced and purified in our laboratory. The immunostaining was performed with secondary gold-conjugated antibody. Gold grains were well evident by EM analysis thus revealing the G6PD protein in the subcellular compartments. These protocols are useful to detect peculiar populations of neurons which express high levels of G6PD to sustain processes of neural plasticity in some brain areas.

Effect of vitamin E-deficiency on regeneration of the sciatic nerve.

The regeneration of the sciatic nerve fibres was studied in both normal and vitamin E-deficient rats at 30 and 60 days after crush. The vitamin E is involved in one of the most important mechanisms of protection against peroxidation of plasma membrane lipids; the plasma membrane plays certainly a role in nerve regeneration. Both the diameter and the total number of myelinated nerve fibres was calculated at different times. The number of myelinated fibres in the undenervated deficient animals was lower than that found in the undenervated normals animals. Following the nerve crush, in normal animals after two months the number of myelinated fibres exceeded the number found in undenervated normal animals, whereas in the deficient rat nerves it was significantly lower than in the corresponding controls and moreover it did not even reach the number found in the nerves of undenervated deficient rats. Finally, the caliber distribution of myelinated fibres in undenervated and denervated deficient rats shows a relative percent increase in the number of greatest axons and a decrease in smaller axons. This result confirm the vitamin E to be an important factor of the normal process of nerve regeneration.