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This is the first report of the effects of malnutrition during brain development on biocytin-labelled axon terminals and histochemical pattern of NADPH-diaphorase (NADPH-d)-containing neurons in area 17 of the adult rat. Wistar rats (n = 6) were submitted early in life (from gestation up to 42 days of age) to a multideficient diet (the 'regional basic diet' (RBD) of low-income human populations of north-east Brazil, containing only 8% protein). From day 43 up to adulthood (135-212 days), they were switched to a commercial laboratory chow diet (Purina do Brasil Ltda), with 22% protein. These animals were compared to control rats (n = 11), fed the laboratory chow diet until adulthood. The brains of four adult malnourished and five controls were processed according to the indirect method of the malic enzyme to reveal NADPH-d-containing neurons. Five other adult subjects (three controls and two malnourished) received iontophoretic injections of the tracer biocytin in area 17and were processed according to the glucose oxidase-DAB-nickel protocol in order to visualize axon terminals filled with biocytin. Three other control rats were processed for both techniques. In these last brains, no double-labelled cells could be found, suggesting that the NADPH-d-containing-neurons and the biocytin-labelled ones belong to different groups of cells, in area 17. The appendages of the NADPH-d-positive cells showed minor qualitative and quantitative differences between undernourished and control rats. The soma area of these cells was reduced in the white matter of malnourished rats, as compared to the controls (468.6 ± 54.3 μm(2); n = 4 and 515.4 ± 30.5 μm(2);n = 8, respectively; p < 0.05). The cell density (cells/mm(2)) was greater in the malnourished group than in the control, both at the grey matter (16.6 ± 4.4; n = 4 and 11.3 ± 4.3; n = 8, respectively; p < 0.05) and at the white matter (55.9 ± 15.7; n = 4 and 24.4 ± 8.5; n = 8; p < 0.005). The number of potential synaptic sites in the biocytin-labelled axon terminals was reduced as compared to the control (126 ± 33 boutons/mm, n = 32 and 160 ± 37; n = 30, respectively; p < 0.01). The results indicate that the rat area 17 is affected differently by early malnutrition, regarding biocytin-labelled axon terminals, on the one hand, and NADPH-d-containing neurons, on the other. Concerning these last cells, the data also suggest that they are less sensitive to the injury represented by early malnutrition.

Cortical spreading depression (CSD) propagation was investigated in rats under dietary essential fatty acid (EFA) deficiency over two generations (F1 and F2). Wistar rat dams received diets containing 5% fat either from coconut-oil (EFA-deficient) or soybean-oil (control). F1-pups received their dams' diets until the day of CSD recording (30–40 days or 90-100 days). F2-pups were kept on their F1 dams' diet until 30–40 days. Compared to the controls, the EFA-deficient group had reduced (P < 0.05) body weights in both F1 and F2 conditions. This effect was more conspicuous (P < 0.001) in the F2-animals where brain weight was also reduced (P < 0.05). All EFA-deficient groups displayed lower CSD velocities (P < 0.001) than the corresponding controls. Within the same dietary group and generation, F1 young rats showed higher CSD velocities (P < 0.001) than adults. Data show that EFA deficiency reduces CSD propagation, and this effect is long lasting as it persists up to the second generation.

Tissue distribution of nitric oxide-synthases was investigated in the rat hippocampus and visual cortex under nutritional changes induced by modification of the litter size. Young (30-45-days-old) rats, suckled in litters formed by 3,6 or 12 pups (called small, medium and large litters, respectively), were studied by using nicotine-adenine-dinucleotide phosphate-diaphorase histochemistry (shortly, diaphorase), a simple and robust procedure to characterize tissue distribution of nitric oxide-synthases. We assessed morphometric features of the diaphorase-positive cells in visual cortex, and the neuropil histochemical activity in hippocampal CA1 and dentate gyrus using densitometry analysis. In the large-litter group, the labeled-cell density in white matter of area 17 was higher, as compared to the small-litter group. There was a clear trend, in the large-litter group, to lower values of soma area, dendritic field and branches per neuron, but the differences were not significant. Densitometry analysis of hippocampus revealed a significant increase in the relative neuropil histochemical activity of the dentate gyrus molecular layer in the larger litters, which may be associated to increased compensatory blood flow in the hippocampus. The pathophysiological mechanisms of the observed changes remain to be investigated.

We investigated the histochemical positivity to NADPH-diaphorase, which reveals nitric oxide synthase activ- ity, in area 17 of rats malnourished early in life, both in the post-weaning period (group M1), and in adulthood after nutri- tional recovering (group M2). Control pups (C1 and C2 groups) received ad libitum after weaning the same diets as their moth- ers. Rats of group M2 were nutritionally recovered by receiving the control diet from post-natal day 42 until adulthood. Alde- hyde-fixed sections (200-mm thick) through area 17 were pro- cessed for NADPH-diaphorase histochemistry following the malic enzyme indirect method. The features of NADPH-diaph- orase-containing neurons of area 17 of malnourished young (M1) and adult (M2) rats were analyzed quantitatively in com- parison to the matched groups C1 and C2. Permanent changes, represented by increase in the density and dendritic field areas of NADPH-diaphorase-positive cells, and transitory ones, rep- resented by decreased values of soma areas, were observed in area 17 of the M1 and M2 cases. However, some other features, such as dendritic branch angle and number of dendrites per cell in the gray matter, remained unchanged after malnutrition. Thus, the findings indicate a possible relationship between early malnutrition and alterations in nitric oxide synthase-con- taining cells in the visual cortex. Physiological implications of these data may be related to synaptic plasticity and refinement of developmental brain circuits. © 2000 Elsevier Science Inc.