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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.

The numerical density of synapses in the molecular layer of the parietal cortex of normal 28-day-old rats has been estimated by using two stereological methods, namely an unfolding technique and the disector method. For the unfolding technique it was assumed that the synapses formed a polydispersed population of disc-shaped particles. The technique also relies upon various assumptions regarding the size, shape and projection of synapses in the plane of section, these assumptions producing a result that is biased to an unknown extent. Such simplifying assumptions are unnecessary for the disector method. The mean +/- S.E.M. numerical density of synapses was found to be 2.16 +/- 0.14 x 10(9)/mm3 using the disector method. This was not significantly different (paired t-test; P less than 0.05) from the value of 2.41 +/- 0.22 x 10(9)/mm3 obtained using the unfolding technique with correction for truncation ('lost caps'). When the unfolding method was used without a correction for lost caps the numerical density was 1.98 +/- 0.10 x 10(9)/mm3, a value also not significantly different from that obtained using the disector method. The mean projected height (Hs) of the synaptic particles was also determined using both procedures; the results were not significantly different. When the variances of the final estimates and the time taken to carry out the procedures described were taken into account, it was estimated that the disector method was about 86% more efficient than the unfolding method with capping correction. Although the unfolding method can give essentially similar values for the numerical density of synapses to those obtained by the disector method, it is biased and much less efficient. It is concluded, therefore, that the disector method is the superior method for the estimation of the number and size of particles such as synapses, particularly when they have complex spatial configurations.

Rats whose right eyes were enucleated on day 1 after birth and nonenucleated rats were raised in either "light" or "dark" (red light) conditions from birth until 39 days of age. This resulted in 4 groups of animals: light-reared nonenucleated, light-reared enucleated, dark-reared nonenucleated, and dark-reared enucleated. At 39 days of age, the animals were killed by perfusion with 2.5% sodium cacodylate buffered glutaraldehyde. The superior colliculi were dissected out and processed for embedding in resin. Stereological procedures at the light and electron microscopical levels were used to estimate the synapse-to-neuron ratios in the superficial layers of these colliculi. Light-reared, nonenucleated rats had about 1,850 synapses-per-neuron in both the right and left superior colliculi. Rearing nonenucleated rats in the dark reduced this value to about 1,200. Enucleated rats reared in the light showed a differential response in the 2 colliculi. Thus, the contralateral (to the enucleated eye) colliculi showed a decrease, whereas the ipsilateral colliculi showed an increase in the synapse-to-neuron ratio compared with light-reared, nonenucleated rats. When enucleated rats were reared in the red light, there was a decrease in the ratio in both colliculi, although the extent of this decrease was more marked in the contralateral than the ipsilateral colliculi. However, the decrease in the contralateral colliculi was not significantly greater than that observed in the corresponding colliculi from dark-reared, nonenucleated rats. These results provide useful information on the combined and separate effects of unilateral enucleation at around birth and dark (red light) rearing during early life on the interneuronal connectivity of both the ipsi- and contralateral superior colliculi of rats. They also show the vast importance of visual stimulation for the normal development of the subcortical visual centers.

It is possible that the reported effects of early life undernutrition on brain morphology may be due to alterations in mother-infant interactions and not directly to undernutrition. We have investigated this possibility by comparing artificially reared with mother-reared rats. Four groups of black-and-white hooded male rats were reared. These consisted of mother reared control (MRC), mother reared undernourished (MRU), artificially reared control (ARC) and artificially reared undernourished (ARU). Artificially reared rats were raised in isolation away from their mothers from 5 to 21 days of postnatal age. They were fitted with a gastric cannula through which 'milk' was infused automatically. The period of undernutrition lasted from 5 to 25 postnatal days, following which the animals were fed ad libitum until 312 days of age. Rats from each group were then killed by perfusion with buffered 2.5% glutaraldehyde. Pieces of visual cortex from each rat were postfixed in osmium tetroxide and embedded in resin. Stereological procedures at the light and electron microscopical levels were used to estimate the synapse-to-neuron ratios in cortical layers II to IV. Both MRC and ARC rats had about 7000 synapses per neuron. However, this ratio was about 8300 in MRU rats whilst it was only about 5000 in ARU animals. The rearing x nutrition interaction was statistically significant at the 0.1% level. These changes in the synapse-to-neuron ratio were mainly due to alterations in the numerical densities of the synapses rather than that of neurons. These results demonstrate that environmental isolation, as a result of artificial rearing procedures, and concurrent undernutrition during the first three weeks of postnatal life, interact with one another to produce marked morphological changes in the adult rat brain. However, environmental isolation was not, by itself, sufficient to cause permanent changes in interneuronal connectivity.

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.