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Adult neural progenitors have been isolated from diverse regions of the CNS using methods which primarily involve the enzymatic digestion of tissue pieces; however, interpretation of these experiments can be complicated by the loss of anatomical resolution during the isolation procedures. We have developed a novel, explant-based technique for the isolation of neural progenitors. Living CNS regions were sectioned using a vibratome and small, well-defined discs of tissue punched out. When cultured, explants from the cortex, hippocampus, cerebellum, spinal cord, hypothalamus, and caudate nucleus all robustly gave rise to proliferating progenitors. These progenitors were similar in behaviour and morphology to previously characterised multipotent hippocampal progenitor lines. Clones from all regions examined could proliferate from single cells and give rise to secondary neurospheres at a low but consistent frequency. Immunostaining demonstrated that clonal cortical progenitors were able to differentiate into both neurons and glial cells, indicating their multipotent characteristics. These results demonstrate it is possible to isolate anatomically resolved adult neural progenitors from small amounts of tissue throughout the CNS, thus, providing a tool for investigating the frequency and characteristics of progenitor cells from different regions.

Undernutrition during early life is known to cause deficits and distortions of brain structure although it has remained uncertain whether or not this includes a diminution of the total numbers of neurons. Estimates of numerical density (e.g. number of cells per microscopic field, or number of cells per unit area of section, or number of cells per unit volume of tissue) are extremely difficult to interpret and do not provide estimates of total numbers of cells. However, advances in stereological techniques have made it possible to obtain unbiased estimates of total numbers of cells in well defined biological structures. These methods have been utilised in studies to determine the effects of varying periods of undernutrition during early life on the numbers of neurons in various regions of the rat brain. The regions examined so far have included the cerebellum, the dentate gyrus, the olfactory bulbs and the cerebral cortex. The only region to show, unequivocally, that a period of undernutrition during early life causes a deficit in the number of neurons was the dentate gyrus. These findings are discussed in the context of other morphological and functional deficits present in undernourished animals.

We have previously shown that exposing rats to a relatively high dose of ethanol during early postnatal life resulted in a deficit in spatial learning ability. This ability is controlled, at least in part, by the hippocampal formation. The purpose of the present study was to determine whether exposure of rats to ethanol during early postnatal life affected the number of specific neurons in the hippocampus. Wistar rats were exposed to a relatively high daily dose of ethanol between postnatal days 10 and 15 by placing them for 3 h each day in a chamber containing ethanol vapor. The blood ethanol concentration was about 430 mg/dl at the end of the exposure period. Groups of ethanol-treated (ET) rats, separation controls (SC), and mother-reared controls (MRC) were anesthetized and killed at 16 days of age by perfusion with phosphate-buffered glutaraldehyde (2.5%). The Cavalieri principle was used to determine the volume of various subdivisions of the hippocampal formation (CA1, CA2+CA3, hilus, and granule cell layer), and the physical disector method was used to estimate the numerical densities of neurons within each subdivision. The total number of neurons was calculated by multiplying estimates of the numerical density with the volume. There were, on average, about 441,000 granule cells in the granule cell layer and 153,000 to 177,000 pyramidal cells in both the CA1 and CA2+CA3 regions in all three treatment groups. In the hilus region, ET rats had about 27,000 neuronal cells. This was significantly fewer than the average of 38,000 such neurons estimated to be present in both MRC and SC animals. Thus, neurons in the hilus region may be particularly vulnerable to the effects of a high dose of ethanol exposure during early postnatal life.

In the developing nervous system, cell death is an important component of refining axonal projections. In the developing rat inferior olive, previous studies have demonstrated cell death as temporally incongruent with both initial axon-target interactions and subsequent axon collateral regression. Furthermore, these studies identified a late rise in neuron numbers that is concurrent with climbing fibre regression. As axonal regression has not previously been associated with increasing neuron numbers, and since immature neurons and glia have similar morphological characteristics, it was decided to reassess the timing of cell death within the inferior olive in animals in which neurons and glia had been differentially stained. Glia were identified by the presence of glial cytoskeletal proteins, S100, or glial fibrillary acidic protein, and stereological counts were made of both neurons and glia in the inferior olive from rats of ages 0, 5, 10, 15, and 30 days. The number of inferior olivary neurons was approximately 22,000 between birth and day 10, which decreased to about 17,500 by day 30 (P<0.05). In contrast, the number of glia rose from about 5,000 at birth to approximately 15,000 by day 10 (P<0.001), after which there was no further increase. The changes in neurons and glia caused the neuron-to-glia ratio to fall to approximately 1.5 by the time of functional maturation within the olive. These results confirm that there is neuronal death in the inferior olive but that it is temporally correlated with both climbing fibre regression and functional maturation of the olivocerebellar projection.

Several studies have reported the morphological changes induced by unilateral enucleation during early neonatal life on the developing visual system. This study has examined cellular changes in the superior colliculi by removal of a single eye in adult rats. Anaesthetised male hooded rats aged 90 d had their right eyes removed. Groups of nonenucleated control and enucleated rats were killed when aged either 150 or 390 d. The brains were removed and both the right and left superior colliculi dissected out. The volume of the stratum griseum superficiale (SGS) within these colliculi was estimated stereologically by light microscopy, as well as the numerical density and total number of neurons within this cell layer. The volume of the cell layer was reduced by about 40% on the side contralateral to the enucleated eye but not on the ipsilateral side at both survival periods examined. The numerical density of neurons within the SGS was unaffected by the enucleation so that the colliculi contralateral to the enucleated eye showed a substantial loss of neurons within this cells layer. This study demonstrates the importance of the retinal ganglion cell input, even in adult animals, for maintaining the viability of neurons in the SGS layer of the superior colliculus.

Undernutrition during early life is known to cause deficits and distortions in brain structure. However, it remains uncertain whether this includes a diminution of the total numbers of neurons. Recent advances in stereological techniques have made it possible to obtain unbiased estimates of total numbers of cells in well-defined biological structures. Rats were undernourished from day 16 of gestation to 30 postnatal days of age by standardized procedures. These rats and well-fed control rats were anaesthetized and killed by intracardiac perfusion with fixatives at 70 days of age. The left cerebral hemisphere from each animal was embedded in Paraplast and serially sectioned. The sections were analyzed via the Cavalieri principle to obtain the total cortical volume and by the "disector" method to estimate the numerical density of neurons in the cortex. These values were later used to compute estimates of the total number of cortical neurons for each animal. Well-fed control rats had 26.9 million cortical neurons, while the previously undernourished animals had 24.8 million. The difference between these two groups was not statistically significant. It therefore appears that undernutrition of rats during early postnatal life does not affect the total numbers of neurons in the cerebral cortex.

The failure of axon regeneration in the injured mammalian central nervous system has been ascribed, in part, to the inhibitory effects of myelin proteins. To investigate the influence of myelination on neurite growth and regeneration by both central nervous system and peripheral nervous system neurons, isolated rat neonatal retinal ganglion cells and adult and neonatal dorsal root ganglion neurons were cultured on cryostat sections of both immature unmyelinated and mature fully myelinated adult rat optic nerve. In agreement with earlier studies using neonatal peripheral neurons, the adult optic nerve failed to support neurite outgrowth from any of the neurons tested. A new finding was that tissue sections from unmyelinated optic nerve (aged embryonic days 18 and 20, and postnatal days 1-3), also failed to support the growth of neurites from neonatal retinal ganglion cells and both neonatal and adult dorsal root ganglion neurons. Neonatal retinal ganglion cells also failed to extend neurites on sections of pre-degenerated sciatic nerve, a tissue shown in our previous work to be a good substratum for supporting neurite growth for both neonatal and adult DRG neurons. These results suggest that cells in the immature optic nerve either express widely acting axon growth inhibitory molecules unrelated to previously described myelin proteins, or do not synthesize appropriate axon growth promoting molecules. They also reveal that, for axon regeneration, central nervous system and peripheral sensory neurons require distinct substratum interactions.

Undernutrition during early life is known to affect the morphology of the hippocampal formation. Recent advances in stereological techniques have made it possible to make relatively unbiased estimates of total cell numbers in well-defined brain regions. It was decided to use these methods to determine the effects of different levels of undernutrition during early postnatal life on the granule cells of the rat dentate gyrus. Male hooded Long Evans rats were undernourished between the 16th day of gestation and 30 postnatal days of age to two different levels. The daily food intake of level-1 and level-2 rats represented about 60 and 40%, respectively, of that eaten by well-fed, age-matched controls. Nutritional rehabilitation of the rats was commenced when they had reached 30 days of age by placing them on an ad libitum diet. Groups of control and experimental rats were killed at 70 and 212 days of age. The Cavalieri principle was used to determine the granule cell layer volume within the dentate gyrus, and the "dissector" method was used to determine numerical densities of these granule cells. These estimates were used to calculate the total numbers of granule cells. There were between 260,000 and 320,000 granule cells within the dentate gyrus of 70-day-old control and experimental rats. By 212 days of age, well-fed controls had an average of about 834,000 granule cells. The level-1 and level-2 previously undernourished rats had about 515,000 and 595,000 granule cells, respectively. Two-way analysis of variance procedures showed significant main effects of nutrition and age as well as a significant interaction between them.(ABSTRACT TRUNCATED AT 250 WORDS)

Black and white hooded Lister rats were undernourished for various times up to 150 days of age; some of them were nutritionally rehabilitated from 75 days. Undernourished rats weighed significantly less than well-fed controls at all ages studied. After embedding in resin, sections of cerebellar cortex were cut and examined at the light and electron microscopical levels using traditional morphometric methods. Undernourished rats showed significant deficits in synapse-to-neuron ratio, compared with controls, at 21 days of age. This deficit disappeared by 75 days despite continued undernutrition. Indeed, there was no alteration in this ratio even when undernutrition was extended up to 150 days even though the ratio for the controls decreased after that period. Rats undernourished from birth to 75 days and subsequently rehabilitated to 150 days had significantly more synapses per neuron than controls. The functional sequelae of these morphological changes remain unknown.

The frontal cortex and granular layer of the cerebellum have been examined in 30-day-old rats undernourished from birth. Quantitative stereological procedures at the light microscopical level have been used to estimate the volume proportion and numerical densities of neuronal nuclei. Similar methods at the electron microscopical level were employed to calculate the numerical densities of synapses. Hence, synapse-to-neuron ratios have been calculated in these brain regions. In the frontal cortex, the undernourished group of rats showed a 37% deficit (P less than 0.05) in the synapse-to-neuron ratio. This was due to a combination of an increase in the numberical density of neurons and a decrease in the numerical density of synapses, although, individually, neither of these reached statistical significance at the 5% level. In the granular layer of the cerebellum there was a 31% (P less than 0.01) deficit in the synapse-to-neuron ratio. This was a function of the reduced numerical density of synapses, with no difference in the numerical density of granule cells between groups. For the frontal cortex, the volume proportion of neuronal nuclei was significantly greater in the undernourished group of rats. There were no significant differences between control and undernourished rats in the volume of the 'forebrain' occupied by cortex. The mean diameters of neuronal nuclei and synaptic discs did not differ in any given region between treatment groups. These observations are discussed in context with the previously published results on synapses and neurons in undernourished animals.

Recent evidence showing alterations in spatial memory due to a period of undernutrition during early life has implicated the hippocampus as one of the brain centres that may be particularly adversely affected. However, there are very few quantitative morphological studies that have examined the neuronal and synaptic populations of the hippocampi from undernourished animals. We decided to carry out such experiments, paying particular attention to the granule cell of the dentate gyrus. Male rats were undernourished from the 18th day of gestation until 21, 75, or 150 days of age. Some of these previously undernourished rats were nutritionally rehabilitated between 150 and 250 days of age. Groups of well-fed control and experimental rats were killed by intracardiac perfusion with 2.5% sodium-cacodylate-buffered glutaraldehyde. The right hippocampus from each rat was dissected out and processed for electron microscopy. Stereological procedures at the light and electron microscopical levels were used to estimate the numerical densities of granular cell neurons and molecular layer synapses in the dorsal lip of the dentate gyrus. These estimates were used to calculate synapse: neuron ratios. There were 5,056 +/- 347 (mean +/- SE) and 5,002 +/- 190 synapses per neuron in 21-day-old control and undernourished rats, respectively. By 75 days these values had increased to 9,215 +/- 588 and 6,683 +/- 237. The difference was statistically significant. By 150 days of age the value for control animals had fallen once again to 6,518 +/- 209 whereas undernourished rats had increased slightly to 7,689 +/- 288 (P less than .01); 250-day-old rats, previously undernourished from birth to 150 days of age, showed a substantial increase in the synapse: neuron ratio during the period of nutritional rehabilitation. Thus these nutritionally rehabilitated rats had 9,407 +/- 365 synapses per neuron whereas age-matched controls had only 6,323 +/- 239 (P less than .01). These results indicate that the rat dentate gyrus is vulnerable to undernutrition even during the postweaning period and that a lengthy period of undernutrition can alter the developmental growth curve for synapse: neuron ratios.

Male rats undernourished from the 18th day of gestation till 100 days of age were nutritionally rehabilitated until 200 days of age. Six control and six experimental rats at each of 25, 50, 100, and 200 days of age were killed by perfusion with buffered 2.5% glutaraldehyde. Pieces of optic nerve from just behind the left eye of each rat were postfixed in osmium tetroxide and embedded in resin. Estimates of the total number of fibres and their mean diameter and size-frequency distributions were made for each nerve. Rats undernourished until 25 days of age had a mean +/- SE of 57,464 +/- 6,778 fibres per optic nerve. This represented a 36% deficit (P less than 0.01) compared with the 25-day-old control value of 89,778 +/- 6,625. In addition, these undernourished rats had proportionately more small fibres than the age-matched controls. This resulted in a significant deficit in mean fibre diameter at this age. These deficits and distortions disappeared in all the older rats studied, despite the continued undernourishment of some animals up to 100 days of age. It appears that even the lengthy period of undernutrition imposed in the present experiments could not produce a permanent deficit in optic nerve fibre number, size, and size distribution. It is suggested that the deficits seen initially at 25 days of age may have been due to a temporary delay in the growth and development of these nerve fibres.

30-day-old rats undernourished from birth are known to have large deficits in the synapse-to-neuron ratio in certain brain regions. It has not been possible to demonstrate any statistically significant deficits in this ratio in animals undernourished from birth to 30 days but then provided with an ad libitum amount of food til 6 months of age.