Mice undernourished before, but not after, weaning perform better in motor coordination and spatial learning tasks than well-fed controls.

Undernutrition of rodents has been claimed to result in long-term behavioural deficits in motor coordination and spatial learning ability, although the literature on this is somewhat conflicting. We have recently been engaged in a study of the effects of either pre- or post-weaning undernutrition on longevity in mice. As part of this ageing study, we have also assessed the effects of such nutritional regimes on motor coordination and spatial learning ability of mice. Motor coordination was tested in 21-week-old control and previously undernourished mice by assessing their ability to remain on a revolving drum. We have found that mice previously undernourished either during the pre- or post-weaning period performed better than controls during some, but not all, of the test days. Spatial learning was tested in 50-58-week-old mice using the Morris water maze. In this instance we found that mice previously undernourished during the pre-weaning period performed better at this task than either controls or mice undernourished for a period after weaning. It seems that undernutrition during the pre-weaning period may, paradoxically, improve the performance of mice in these behavioural tasks compared to controls. Undernutrition after weaning had little or no effect on these behavioural measures. The exact mechanisms involved in causing the observed long-term changes in functional capacity due to a period of undernutrition from conception until weaning age of the mice in our study remain unknown.

Undernutrition during the gestation and suckling periods does not cause any loss of pyramidal neurons in the CA2-CA3 region of the rat hippocampus.

The total number of hippocampal pyramidal cells in the CA2-CA3 region are reported to be unaffected by undernutrition during the gestation period. We hypothesised that this may not be the case in animals subjected to a lengthier period of undernutrition. Wistar rats were undernourished from conception until 21 post-natal days-of-age and killed for examination at 21 and 62 days-of-age. There were between 180-212 thousand pyramidal cells in control animals at both 21 and 62 days of age. Twenty-one-day-old undernourished rats had about 152 thousand such cells and this increased to about 206 thousand by 62 days-of-age. Analysis of variance tests on these data revealed a significant main effect of age but no group or interaction effects. Our experiments, therefore, confirm that these hippocampal pyramidal neurons are relatively spared the adverse effects of undernutrition during early life, even when this is extended to include both the gestation and suckling periods.

Undernutrition during either the pre- or immediate post-weaning period does not affect longevity in Quackenbush mice.

Diet restriction of rodents during a lengthy period of adult life, can lead to a marked increase in their life-span. However, undernutrition during gestation and/or the suckling period is, paradoxically, known to cause long-lasting 'deleterious' deficits in body and brain structure. It remains uncertain whether or not such undernourished rodents also have an altered life-span. We have now investigated whether a short period of undernutrition of mice either before or immediately after the weaning period could modulate their life-span. Female out-bred Quackenbush mice were undernourished for 40 days by standardised procedures either from conception until weaning or from weaning (day 19) till 60-days-of-age and compared to control mice that had been well-nourished throughout their lives. During the course of their life-span, some mice in each group became seriously ill and, because of ethical considerations, were required to be killed before their 'natural' death. The median age of mice at which they were required to be euthanized due to illness was significantly younger in the well-fed control group compared to the two previously undernourished groups. Of those mice that died of natural causes, it was found that about 90% died between about 300-700 (average, 552-570; median, 556-595) days-of-age irrespective of group. Any differences between groups were not statistically significant. There were no significant differences between groups in the numbers of mice that survived beyond the 90th percentile of maximum life-span. Our results provided no evidence that a short period of undernutrition of Quackenbush mice either before or immediately after weaning has significant effects on their life-span. However, there was some evidence that, if it occurred, serious illness happened at a younger age in the well-nourished mice than those in the two diet-restricted groups.

Short-term exposure to ethanol causes a differential response between nerve growth factor and brain-derived neurotrophic factor ligand/receptor systems in the mouse cerebellum.

Alcohol ingestion affects both neuropsychological and motor functions. We hypothesized that one of the key factors involved in such functions are neurotrophins and their receptors. We have therefore examined the effects of short-term ethanol exposure on the mRNA expression and protein levels of neurotrophin ligands and receptors in the cerebellum using real-time RT-PCR and Western blotting techniques. Male BALB/C mice were fed a liquid diet containing 5% (v/v) ethanol. The pair-fed control mice were fed an identical liquid diet except that sucrose was substituted isocalorically for ethanol. The cerebellum of mice exhibiting intoxication signs of stage 1 or 2 were used in the present study. We found that exposure to ethanol resulted in elevated levels of nerve growth factor (NGF) and TrkA mRNA expression but a decreased level of brain-derived neurotrophic factor (BDNF) mRNA expression. The expression of TrkB and p73 mRNA was unchanged. Changes in the level of these proteins were found to mirror these mRNA expression levels. We conclude that exposure to ethanol for a short period can cause a differential responsive in the various neurotrophin ligand/receptor systems. The functional consequences of these changes are unknown at present.

Early life undernutrition alters the level of reduced glutathione but not the activity levels of reactive oxygen species enzymes or lipid peroxidation in the mouse forebrain.

Diet restriction of rodents during adult life is known to cause an increased life span. It has been hypothesised that this increase may be related to effects on the anti-oxidant defence systems. However, it has been suggested that undernutrition during the gestation and pre-weaning may reduce their life span as it is known to have other deleterious effects on a rodent's growth and development. We have now examined the activity levels of some anti-oxidant defence system enzymes and other markers of oxidative stress in mice that have been undernourished from conception until 21 postnatal days of age, followed in some cases by a period of nutritional rehabilitation until 61 days of age. We found that such undernutrition exerted only minimal effects on oxidative stress markers under investigation (ROS enzyme activities, GSH levels, and lipid peroxidation). Only GSH levels were significantly affected by pre-weaning undernutrition. In conclusion, pre-weaning undernutrition may regulate anti-oxidant enzymes at the transcriptional level differently from that at the post-transcriptional, translational, or post-translational levels. The possible effects that these changes at the cellular level, may have on the longevity of the animals remain of great interest and importance.

Pre-weaning undernutrition alters the expression levels of reactive oxygen species enzymes but not their activity levels or lipid peroxidation in the rat brain.

It has been hypothesised that the increased life span commonly observed in rodents that have had their diet restricted after weaning may be related to its effects on the anti-oxidant defence systems. However, undernutrition during the gestation and pre-weaning period is known to have long-term deleterious effects on a rodent's growth and development, and it has been suggested that this may reduce their life span. We have now examined some of the anti-oxidant defence system in rats that have been undernourished from conception until 21 postnatal days-of-age, followed in some cases by a period of nutritional rehabilitation until 62 days of age. We found that such undernutrition could modulate the mRNA expression of Cu/ZnSOD and catalase in some brain regions. However, only catalase showed any undernutrition-induced change of enzyme activity level. There was some evidence that undernourished (but not control) rats had an age-related increase in the level of lipid peroxidation between 21 and 62 days of age, although the group x age interaction was not statistically significant. There was no significant change in the level of reduced glutathione induced by the pre-weaning period of undernutrition. If ROS and the extent of oxidative damage are truly implicated in the determination of life span, our results indicate that this is unlikely to be markedly affected by the relatively small changes we have observed in the anti-oxidant defence systems induced by undernutrition of rats from conception until weaning.

The effects of pre-weaning undernutrition on the expression levels of free radical deactivating enzymes in the mouse brain.

A mild degree of undernutrition brought about by restricting the amount of food in the diet is known to alter the life span of an animal. It has been hypothesised that this may be related to the effects of undernutrition on an animals anti-oxidant defense system. We have therefore, used real-time PCR (rt-PCR) techniques to determine the levels of mRNA expression for manganese superoxide dismutase (MnSOD), copper/zinc superoxide dismutase (Cu/ZnSOD), glutathione peroxidase 1 (GPx 1) and catalase in the brains of Quackenbush mice undernourished from conception until 21-post-natal days of age. It was found that 21- and 61-day-old undernourished mice had a deficit in the expression of Cu/ZnSOD in both the cerebellum and forebrain regions compared to age-matched controls. The expression of MnSOD was found to be greater in the cerebellum, but not the forebrain region, of 21-day-old undernourished mice. There were no significant differences in the expression of GPx 1 and catalase between control and undernourished or previously undernourished mice. Our results confirm that undernutrition during the early life of a mouse may disrupt some of the enzymes involved in the anti-oxidant defense systems.

Nutritional effects on neuron numbers.

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.

Exposure of rats to a high but not low dose of ethanol during early postnatal life increases the rate of loss of optic nerve axons and decreases the rate of myelination.

Visual system abnormalities are commonly encountered in the fetal alcohol syndrome although the level of exposure at which they become manifest is uncertain. In this study we have examined the effects of either low (ETLD) or high dose (ETHD) ethanol, given between postnatal days 4-9, on the axons of the rat optic nerve. Rats were exposed to ethanol vapour in a special chamber for a period of 3 h per day during the treatment period. The blood alcohol concentration in the ETLD animals averaged approximately 171 mg/dl and in the ETHD animals approximately 430 mg/dl at the end of the treatment on any given day. Groups of 10 and 30-d-old mother-reared control (MRC), separation control (SC), ETLD and ETHD rats were anaesthetised with an intraperitoneal injection of ketamine and xylazine, and killed by intracardiac perfusion with phosphate-buffered glutaraldehyde. In the 10-d-old rat optic nerves there was a total of approximately 145,000-165,000 axons in MRC, SC and ETLD animals. About 4% of these fibres were myelinated. The differences between these groups were not statistically significant. However, the 10-d-old ETHD animals had only about 75,000 optic nerve axons (P < 0.05) of which about 2.8 % were myelinated. By 30 d of age there was a total of between 75,000-90,000 optic nerve axons, irrespective of the group examined. The proportion of axons which were myelinated at this age was still significantly lower (P < 0.001) in the ETHD animals (approximately 77 %) than in the other groups (about 98 %). It is concluded that the normal stages of development and maturation of the rat optic nerve axons, as assessed in this study, can be severely compromised by exposure to a relatively high (but not low) dose of ethanol between postnatal d 4 and 9.

Neurons in the hilus region of the rat hippocampus are depleted in number by exposure to alcohol during early postnatal life.

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.

Cryosections of pre-irradiated adult rat spinal cord tissue support axonal regeneration in vitro.

Neonatal X-irradiation of central nervous system (CNS) tissue markedly reduces the glial population in the irradiated area. Previous in vivo studies have demonstrated regenerative success of adult dorsal root ganglion (DRG) neurons into the neonatally-irradiated spinal cord. The present study was undertaken to determine whether these results could be replicated in an in vitro environment. The lumbosacral spinal cord of anaesthetised Wistar rat pups, aged between 1 and 5 days, was subjected to a single dose (40 Gray) of X-irradiation. A sham-irradiated group acted as controls. Rats were allowed to reach adulthood before being killed. Their lumbosacral spinal cords were dissected out and processed for sectioning in a cryostat. Cryosections (10 microm-thick) of the spinal cord tissue were picked up on sterile glass coverslips and used as substrates for culturing dissociated adult DRG neurons. After an appropriate incubation period, cultures were fixed in 2% paraformaldehyde and immunolabelled to visualise both the spinal cord substrate using anti-glial fibrillary acidic protein (GFAP) and the growing DRG neurons using anti-growth associated protein (GAP-43). Successful growth of DRG neurites was observed on irradiated, but not on non-irradiated, sections of spinal cord. Thus, neonatal X-irradiation of spinal cord tissue appears to alter its environment such that it can later support, rather than inhibit, axonal regeneration. It is suggested that this alteration may be due, at least in part, to depletion in the number of and/or a change in the characteristics of the glial cells.

The effect of the timing of ethanol exposure during early postnatal life on total number of Purkinje cells in rat cerebellum.

We have previously shown that exposing rats to a high dose of ethanol on postnatal d 5 can affect Purkinje cell numbers in the cerebellum whilst similar exposure on d 10 had no such effect. The question arose whether a longer period of ethanol exposure after d 10 could produce loss of Purkinje cells. We have examined this question by exposing young rats to a relatively high dose (approximately 420-430 mg/dl) of ethanol for 6 d periods between the ages of either 4 and 9 d or 10 and 15 d of age. Exposure was carried out by placing the rats in an ethanol vapour chamber for 3 h per day during the exposure period. Groups of ethanol-treated (ET), separation controls (SC) and mother-reared controls (MRC) were anaesthetised and killed when aged 30 d by perfusion with buffered 2.5% glutaraldehyde. Stereological methods were used to determine the numbers of Purkinje cells in the cerebellum of each rat. MRC, SC and rats treated with ethanol between 10-15 d of age each had, on average, about 254-258 thousand cerebellar Purkinje cells; the differences between these various groups were not statistically significant. However, the rats treated with ethanol vapour between 4-9 d of age had an average of only about 128000+/-20000 Purkinje cells per cerebellum. This value was significantly different from both the MRC and group-matched SC animals. It is concluded that the period between 4 and 9 d of age is an extremely vulnerable period during which the rat cerebellar Purkinje cells are particularly susceptible to the effects of a high dose of ethanol. However, a similar level and duration of ethanol exposure commencing after 10 d of age has no significant effect on Purkinje cell numbers.

Changes in the numbers of neurons and astrocytes during the postnatal development of the rat inferior olive.

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.

Ethanol exposure during the third trimester equivalent results in long-lasting decreased synaptic efficacy but not plasticity in the CA1 region of the rat hippocampus.

Fetal alcohol syndrome is a major cause of mental retardation. We investigated possible long-lasting effects of alcohol on the hippocampus using a model for human third trimester brain development. Treatment of neonatal rats with an ethanol vapor atmosphere of 39.4+/-2.6 mg ethanol/liter of air for 3 h a day from postnatal day 4 through 9 produced daily blood ethanol levels of 351+/-14 mg/dL. Separation control animals were removed from their mothers in parallel with the ethanol vapor treatment, while suckle controls were left to develop normally. We prepared hippocampal slices from these animals between postnatal days 45 and 60 and recorded extracellular responses to Schaffer collateral stimulation. The maximum population spike in the CA1 pyramidal region and population excitatory postsynaptic potentials in the stratum radiatum did not differ significantly between groups. However, slices prepared from ethanol-treated rats as opposed to separation and suckle controls required larger stimulus currents to produce normal postsynaptic responses. In addition, the ratio of the population excitatory postsynaptic potential (pEPSP) slope to the presynaptic volley was significantly reduced in ethanol-treated rats. Ethanol vapor-treated rats and separation control rats did not exhibit any significant changes in long-term potentiation or paired-pulse potentiation compared with normal suckle controls. These results suggest that early postnatal ethanol treatment produces a long-lasting reduction in synaptic efficacy but not plasticity.

Spatial learning ability of rats following differing levels of exposure to alcohol during early postnatal life.

Rats exposed to a relatively high dose (7.5 g/kg body weight) of alcohol on either the fifth or tenth postnatal day of age have been reported to have long-lasting deficits in spatial learning ability as tested on the Morris water maze task. The question arises concerning the level of alcohol required to achieve this effect. Wistar rats were exposed to either 2, 4 or 6 g/kg body weight of ethanol administered as a 10% solution. This ethanol was given over an 8-h period on the fifth postnatal day of age by means of an intragastric cannula. Gastrostomy controls received a 5% sucrose solution substituted isocalorically for the ethanol. Another set of pups raised by their mother were used as suckle controls. All surgical procedures were carried out under halothane vapour anaesthesia. After the artificial feeding regimes all pups were returned to lactating dams and weaned at 21 days of age. The spatial learning ability of these rats was tested in the Morris water maze when they were between 61-64 days of age. This task requires the rats to swim in a pool containing water made opaque and locate and climb onto a submerged platform. The time taken to accomplish this is known as the escape latency. Each rat was subjected to 24 trials over 3 days of the test period. Statistical analysis of the escape latency data revealed that the rats given 6 g/kg body weight of ethanol had significant deficits in their spatial learning ability compared with their control groups. However, there was no significant difference in spatial learning ability for the rats given either 2 or 4 g/kg body weight of ethanol compared with their respective gastrostomy or suckle control animals. We concluded that ethanol exposure greater than 4 g/kg over an 8-h period to 5-day-old rats is required for them to develop long-term deficits in spatial learning behaviour.

Unilateral enucleation of adult rats does not effect the synapse-to-neuron ratio within the stratum griseum superficiale of the superior colliculi.

Ninety-day-old hooded male rats were anaesthetised with an intraperitoneal injection of a mixture of xylazine and ketamine and had their right eyes removed. Groups of non-enucleated control and enucleated rats were killed at either 150 or 390 days of age by intracardiac perfusion with fixatives. Stereological methods were used to estimate the synapse-to-neuron ratios within the stratum griseum superficiale (SGS) layers of both the ipsi- and contra-lateral superior colliculi. The enucleation had no significant effects on this ratio irrespective of the side or age of the brains examined. This experiment shows that a constant synapse-to-neuron ratio may be maintained within the SGS layer of the rat superior colliculus despite the inevitable loss of synaptic contacts due to the anterograde transneuronal degeneration initiated by the enucleation.

Unilateral eye enucleation in adult rats causes neuronal loss in the contralateral superior colliculus.

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.

The influence of predegenerated nerve grafts on axonal regeneration from prelesioned peripheral nerves.

Recent in vitro work has indicated that predegenerated segments of peripheral nerve are more capable of supporting neurite growth from adult neurons than fresh segments of nerve, whereas previous in vivo studies which investigated whether predegenerated nerve segments used as grafts are capable of enhancing axonal regeneration produced conflicting results. We have reinvestigated this question by using predegenerated nerve grafts in combination with conditioning lesions of the host nerve to determine the optimal conditions for obtaining the maximal degree of regeneration of myelinated axons. The sciatic nerve of adult Dark Agouti rats were sectioned at midthigh level, and the distal portion was allowed to predegenerate for 0, 6 or 12 d in situ. 10-15 mm lengths of these distal nerve segments were then syngenically grafted onto the central stumps of sciatic nerves which had themselves received a conditioning lesion 0, 6, and 12 d previously, making a total of 9 different donor-host combinations. The grafts were assessed histologically 3 or 8 wk after grafting. Axonal regeneration in the 9 different donor-host combinations was determined by counting the numbers of myelinated axons in transverse sections through the grafts. All grafts examined contained regenerating myelinated axons. The rats given a 3 wk postgrafting survival period had an average of between 1400 and 5300 such axons. The rats given an 8 wk postgrafting survival period had between about 13,000 and 25,000 regenerating myelinated axons. Analysis of variance revealed significant main effects for both the Donor and Host conditions as well as Weeks (i.e. survival period after grafting). These results indicate that both a conditioning lesion of the host neurons and the degree of predegeneration of peripheral nerve segments to be used as grafts are of importance in influencing the degree of axonal regeneration. Of these 2 factors the conditioning lesion of the host appears to have the greater effect on the final number of regenerating myelinated axons.

Spatial learning ability of rats following acute exposure to alcohol during early postnatal life.

Previous research has indicated that the developing brain is vulnerable to the effects of alcohol exposure. Most of this research has used an experimental design in which animals where chronically subjected to alcohol for a lengthy period of time during gestation and/or the preweaning period. Recent evidence has indicated that the morphology of the brain and the subsequent behaviour of the animal may also be susceptible to alcohol administered for a short duration during specified periods of development. Wistar rats were exposed to 7.5 g/kg body weight of ethanol administered as a 10% solution via an intragastric cannula over an 8 h period either on the 5th (PND5) or the 10th (PND10) postnatal day of age. Gastrostomy controls received a 5% sucrose solution substituted isocalorically for the ethanol. Another set of pups raised by their mother were used as 'suckle controls'. All surgical procedures were carried out under halothane vapour anaesthesia. After the artificial feeding regimes all pups were returned to lactating dams and weaned at 21 days of age. The spatial learning ability of these rats was tested in the Morris water maze when they were between 41-54 days of age. This task requires the rats to swim in a pool containing water made opaque and locate and climb onto a submerged platform. The time taken to accomplish this is known as the "escape latency." Each rat was subjected to 24 trials over three days and a further trial on each of days 4 and 11 of the test period. Statistical analysis of the escape latency data revealed that both the PND5 and PND10 ethanol treated groups had significant deficits in their spatial learning ability compared with the control groups. However, there was no significant difference in the degree of impairment between the PND5 and PND10 rats. It is concluded that even short periods of alcohol exposure during brain development can cause lasting impairment of spatial learning behaviour in rats.

Acute exposure to alcohol during early postnatal life causes a deficit in the total number of cerebellar Purkinje cells in the rat.

Alcohol taken regularly over a lengthy period of time has been claimed to cause the loss of neurons in both the adult and developing brain. However, it remains uncertain whether acute, as opposed to chronic, exposure to alcohol at specified periods can also cause disruption in the neuronal population of the developing brain. This question was investigated by exposing Wistar rat pups to 7.5 g/kg body weight of ethanol administered as a 10% solution via an intragastric cannula over an 8 hour period either on the 5th (PND5) or the 10th (PND10) postnatal day of age. Gastrostomy controls received a 5% sucrose solution substituted isocalorically for the ethanol. Another set of pups raised by their mothers was used as "suckle controls." All surgical procedures were carried out under halothane vapour anaesthesia. After the artificial feeding regimes, all pups were returned to the lactating dams and weaned at 21 days of age. Between 52 and 54 days of age, the rats were anaesthetised with an intraperitoneal injection with Nembutal and killed by intracardiac perfusion with 3% glutaraldehyde in 0.1 M phosphate buffer. The relatively unbiased stereological procedure known as the "fractionator" method was used to estimate the total number of Purkinje cells in the cerebellum of each animal. The Purkinje cell nucleolus was used as the counting unit; it was assumed that each Purkinje cell contained only one nucleolus. PND10 ethanol-treated rats and gastrostomy and suckle controls had between about 210,000-232,000 Purkinje cells in the cerebellum. However, the PND5 ethanol-treated rats had only about 137,000 Purkinje cells.(ABSTRACT TRUNCATED AT 250 WORDS)