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.

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.

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.

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.

Postnatal proliferation of DRG non-neuronal cells in vitamin E-deficient rats.

Changes in the number of satellite cells in neuron body sheaths in dorsal root ganglia (DRGs) were studied from 1 to 5 months of age in control and in vitamin E-deficient rats; furthermore, the satellite cell proliferation rate was detected in the same groups of animals with immunohistochemistry for 5-bromo-2'-deoxyuridine (BrdU). The number of satellite cells in sheaths of DRG neurons increased in the period of life considered both in control and in vitamin E-deficient rats. Satellite cell proliferation was observed in both groups, but its rate was found to be higher in vitamin E-deficient rats. The results obtained in control rats confirm that mitotic ability is retained by satellite cells in adulthood and show that at least some of newborn satellite cells add to the pre-existing population. The results obtained in vitamin E-deficient rats suggest that a faster turnover in satellite cell population takes place in these animals and support the idea that vitamin E could be an exogenous factor controlling cell proliferation.

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.

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.

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.

Quantitative changes in neuron and glial cells of neocortex following prenatal exposure to methylazoxymethanol.

The quantitative changes in neuron and glial cell density were analyzed in different cortical layers from frontal cortex of 3 and 12 months old mice exposed to Methylazoxymethanol (MAM) on embryonic day 15 (MAM 15) or 17 (MAM 17). An increase was found in glial cell density in all cortical layers from 3 months old MAM 17 compared to the age-matched controls. In particular, electron microscopic analysis showed that the only cells to increase in all cortical layers were the microglia cells. When the young and adult mice were compared it was noted that the glia increased significantly in all cortical layers of controls, whereas it did not change the older MAM 17. In the older MAM 15 the increase in glial density was similar to that observed in controls. No loss of cortical neurons was found between young and adult animals. In addition, the different trend of glial cell density between control and MAM 17 mice during aging was accompanied by a decline in the mean nuclear area in neurons of treated mice with respect to the controls. The hypothesis that MAM treatment seems to produce events similar to those normally occurring in aging finds support in the data of glial density and neuron size.

Cell death and cell number in the developing cerebral cortex of MAM treated mice.

The postnatal cell death decline in microencephalic mice produced after methylazoxymethanol-acetate injection during embryonic development may adjust the final cell number of treated animals in the mature neocortex. Quantitative observations of cortical cell number in normal and treated mice, from 5 days to adult age were carried out. The proportion of cortical dead cells per 1000 live cells in a standardized cortical column and the cumulative cell death across time were studied in mice aged 5-10-20-30 days. Two main results were obtained. First, the mature neocortex of treated animals shows a number of cells which is significantly lower with respect to the controls but the difference is less accentuated than in the developing neocortex. Secondly, the proportion of cortical dead cells per 1000 live cells at every date studied and the cumulative cell death across time decreased in cerebral cortex of treated mice when compared with age-matched controls. Our data show that decreasing initial cell number by prenatal MAM injection is correlated to an increase in the survival of cortical cells.

Changes in the number of primary sensory neurons in normal and vitamin-E-deficient rats during aging.

In the dorsal root ganglia (DRGs) of vitamin-E-deficient rats, we previously found an increase in the number of neurons during the first 5 months of life (Cecchini et al., 1993, 1994). This neurogenetic event seems to bring forward in time the increase in the number of primary sensory neurons that Devor et al. (1985) found in normal rats aged more than 1 year, but that other authors have not confirmed. The present study had two aims: first, to verify whether neurogenesis spontaneously occurs in DRGs of 14-month-old Sprague-Dawley rats; and, second, to determine whether the neurogenesis enhanced by vitamin E deficiency continues further in the long run, or whether it stops or reverses into neuron loss. A quantitative and morphometric analysis was performed on neurons of L3-L6 DRGs in 14-month-old normal and vitamin-E-deficient rats: the results obtained were compared to those previously obtained in 1-month-old and 5-month-old animals of both dietetic treatment groups, in order to observe the effects of aging on these neuronal populations. The total number of DRG neurons in the control group was higher in older than in younger animals, whereas the value in the vitamin-E-deficient group was lower in older than in younger animals. The present data confirm that neurogenesis occurs in DRGs of normal rats during adult life. Moreover, they show that once the premature neurogenesis in the deficient rats is completed, no further increase in the number of neurons takes place.

Regeneration of unmyelinated peripheral axons in vitamin E-deficient rats.

Unmyelinated axons of normal and regenerated sciatic nerve were counted in controls and vitamin E-deficient rats. No significant change in the number of unmyelinated axons of uninjured nerve was found in the vitamin E deficiency in comparison to controls (12961 +/- 1591 and 12450 +/- 1290, respectively, mean +/- SEM). In regenerated nerve of control rats the number of unmyelinated axons was higher than in uninjured nerve (16971 +/- 1854 and 20786 +/- 1574 at 1 and 2 months after crush, respectively). In vitamin E-deficient rats the increase in number of unmyelinated axons was greater than in corresponding controls (21880 +/- 662) at 1 month after lesion, but the number returned to value found in uninjured nerve at 2 months after lesion (12536 +/- 659). These results suggest that sprouting at lesion may be enhanced but some regenerated axons does not survive at long term in vitamin E-deficiency.

Increased number of sciatic sensory neurons in vitamin-E-deficient rats.

The number and morphometric characteristics of sciatic sensory neurons were studied in Vitamin-E-deficient rats. Horseradish peroxidase (HRP) was injected into the sciatic nerves of normal and vitamin-E-deficient rats of the same age, and retrogradely labeled sensory neurons were counted and measured. The study was also carried out in rats that had previously undergone sciatic nerve crush, in order to observe the effects of axotomy on primary sensory neurons. In vitamin-E-deficient rats the number of sciatic sensory neurons was significantly higher than normal, with an increase of about 30%, in agreement with a previous finding concerning total population of primary sensory neurons in lumbar dorsal root ganglia (DRGs) of vitamin-E-deficient rats. The increase involved the small cell classes in particular. Axotomy induced similar percentages of neuron loss in normal and in vitamin-E-deficient rats (about 40%). In the latter, death affected small cell classes in particular--that is, the same classes that had increased in number in vitamin-E-deficient rats by comparison with controls. These results, together with previous findings, suggest that neurogenesis may occur in DRGs of vitamin-E-deficient rats.

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.

Increased number of dorsal root ganglion neurons in vitamin-E-deficient rats.

Quantitative and morphometric observations were carried out on neurons of L3-L6 dorsal root ganglia (DRGs) in control and vitamin-E-deficient rats at different ages. Controls were fed a standard diet and sacrificed at 1 or at 5 months of age; deficient rats were fed a diet without vitamin E from 1 to 5 months of age and then sacrificed. No significant difference in total number of neurons was found, but an increase in neuron sizes, a decrease in nucleus-cytoplasm ratio, and a more circular neuron shape were found in controls with increasing age (from 1 to 5 months). In L3-L6 DRGs of vitamin-E-deficient rats (5 months of age), a higher number of neurons was found than in those of either young or adult controls. Moreover, some morphometric characteristics of neurons in the deficient rats were similar to those of neurons in 1-month-old controls. The findings suggest that vitamin E deficiency can trigger events resulting in appearance of new neurons, possibly anticipating phenomena that normally occur in aging.

[Changes due to age in the regenerating axons of the adult rat]. / Modificazioni dovute all'età negli assoni rigeneranti di ratti adulti.

Muscle reinnervation after nerve crush was observed in rats at different ages with a combined technique that simultaneously demonstrates nerve endings and endplates. At early times of reinnervation the amount of sprouting was higher in older rats than in younger rats; according to this finding an enhanced number of polyinnervated endplates was found in older rats. A similar enhancement of sprouting and polyinnervation was observed during muscle reinnervation of vitamin E deficient rats, supporting the proposed analogy between vitamin E deficiency and aging.

6-H3 thymidine uptake in the central nervous system of newt following lesion and cold shock.

The uptake of 6-H3 thymidine in the optic tectum and the forebrain of the newt following injury to one of the two nervous areas, shows a marked proliferative response in the telencephalon in the presence of a lesion on the right optic tectum, and a poor response of the optic tectum after a lesion on the right telencephalic lobe. A similar proliferative response is also obtained when the specimens received only the cold shock. In both experimental conditions maximum uptake is obtained after 7 days. It is assumed that the matrix cells of the forebrain and the undifferentiated cells of the optic tectum respond differently to the regeneration factor released by the nervous tissue following a lesion or a cold shock.

Nerve cell rearrangement in neocortical layers of MAM treated foetal mice.

In this study we examined the changes in cortical layers when Methylazoxymethanol-acetate (MAM) was injected on 13th, 15th and 17th day of gestation (MAM13, MAM15, MAM17). Prenatal MAM administration prevents neuron precursor proliferation; the subsequent cell number decrease is followed by an increase in size of surviving cells. When MAM was injected on 13th or 15th day of gestation the significant decrease in cell density, in II and III zone (MAM13) and in the upper layers (MAM15), was compensated by a proportional increase in cell size and synaptic density. When MAM was injected on 17th embryonal day it did not cause a significant change in the neuron density and soma size was not altered while the distribution of cell size among layers showed significant differences with respect to the controls. Our findings of some area and synaptic density show that the cell surface increase is proportional to the decrease in cell number and that in the surviving cells there is a rearrangement in synaptic connections that compensate for the cellular density decrease.