Ultrastructural characteristics of the cerebellar cortex in the postnatal development of animals with protein-calorie malnutrition and in subsequent rehabilitation

An electron microscopical investigation of the cerebellar cortex has been carried out in mice, kept during the 10th-40th days of their life at a diet with protein insufficiency and a subsequent food rehabilitation and food rehabilitation with carnitine addition to the diet on the 41st-70th days of their life. A sharp protein limitation in the diet results in the most essential changes in the neuropil, while the ultrastructure of the piriform neurons changes slightly. Amount of the synaptic vesicles in small axonal terminals decreases and their localization in the area of the synaptic contacts changes. Simultaneously, degeneratively changed axonal terminals often occur, they demonstrate a rather electron opaque axoplasma. The food rehabilitation mainly normalizes the ultrastructure of the cerebellar cortex elements, however, in the neuropil altered axonal terminals with a high electron opaque axoplasma occur. After the food rehabilitation with carnitine addition to the diet, the cerebellar cortex ultrastructure not only normalizes, but even demonstrates certain signs of hypertrophy.     

The ratio of the types of gliocytes in the cerebellar cortex of mice during malnutrition and subsequent nutritional rehabilitation

Different types of glial cells were counted on the semithin sections on the area 0.5 mkm2 of the cerebellar cortex of mice and percent relation of glyocytes-satellites of glyocyte types was determined. The malnutrition in early postnatal life has been found to cause the aberration in the process of glyocytes differentiation, especially in the population of oligodendrocytes. After the food rehabilitatin the processes of differentiation normalize that expresses in the increase of proportion of olygodendrocytes and olygodendrocytes satellities up to the control.     

Differential expression of isoforms of the regulatory subunit of type II cAMP-dependent protein kinase in rat neurons, astrocytes, and oligodendrocytes

The RII-B isoform of the regulatory subunit (R) of cAMP-dependent protein kinase II is abundantly and selectively expressed in cerebral cortex (Erlichman, J., Sarkar, D., Fleischer, N., and Rubin, C. S. (1980) J. Biol. Chem. 255, 8179-8184). In contrast to the cytosolic RII-H isoform from heart and other non-neural tissues, a substantial fraction of cerebral cortex RII-B is tightly associated with cell organelles. In order to study the cellular basis for the localization and abundance of RII-B in this complex and heterogeneous tissue, rat cerebral cortex was fractionated into highly purified populations of neurons, astrocytes, and oligodendrocytes. In neurons and astrocytes more than 80% of the total cAMP-binding activity is contributed by RII subunits, whereas the myelin-producing oligodendrocytes contain nearly equal proportions of RI (from protein kinase I) and RII. Approximately 70% of RII and RI subunits are associated with the particulate fraction in each of the three types of brain cells. The nature of the RII isoforms expressed in the cytosolic and particulate fractions of the purified brain cells was established by performing Western immunoblot and indirect immunoprecipitation analyses with selective and sensitive polyclonal antibodies directed against RII-B. Astrocytes and neurons exhibit high levels of RII-B, whereas oligodendrocytes contain the RII-H isoform. Thus, the expression of RII isoforms is not uniform among brain cells that are anatomically and developmentally related. Rather, it appears that RII-B and RII-H are expressed in a cell-specific fashion within cerebral cortex and this might reflect an RII-mediated adaptation of protein kinase II to the specialized metabolic and functional roles of neurons, astrocytes, and oligodendrocytes.     

Quantitative characteristics of cells formed during early postnatal ontogenesis in the cytoarchitectonic layers of the parietal region of the cortex in normal mice and following brain injuries]

By means of computer analysis, size distribution of neurons and glial cells together with the label of 3H-thymidin labelled cells were studied in cytoarchitectonic layers of the field 1 in the mouse parietal cortex injected with the isotope on the 5th day of life and killed 1 month later. In some animals, 3 days before 3H-thymidin injection, the parietal region in the right hemisphere was perforated. The labelled cells were subdivided into 2 groups according to their size: astrocytes (A) and small gliocytes (mg). Labelled A and mg were stated to be rather evenly distributed along the cytoarchitectonic layers in the field 1. The trauma performed resulted in increasing amount of the labelled A and MG in the operated hemisphere. Uniformity in distribution of the labelled A and mg, occupying the position of satellites, did not change considerably at the trauma, in spite of general quantitative increase of the labelled glial cells. One month after the trauma, body dimentions of the mice labelled with A and mg did not considerably differ from those of intact animals, while the size of nerve cells increased. Thus, neurons in the parietal cortex of a growing brain respond to brain trauma with hypertrophy of cell bodies, and glial populations--with increase of their number.     

Angiogenic protection from focal ischemia with angiotensin II type 1 receptor blockade in the rat

Angiogenesis within an ischemic region of the brain may increase tissue viability and act to limit the extent of an infarct. The ANG II pathway can both stimulate and inhibit angiogenesis depending on the tissue and the activated receptors. Previous work showed that 2-wk losartan administration (ANG II type 1 receptor blockade) initiates a significant cerebral angiogenic response. We hypothesized that administration of losartan in the drinking water of rats for 2 wk before initiation of focal ischemia would decrease the extent of the resulting infarct. Adult male Sprague-Dawley rats were given losartan (50 mg/day) in drinking water for 2 wk before initiation of cerebral focal ischemia produced by cauterization of cortical surface vessels. Controls received normal drinking water. In control animals, three main vessels feeding the whisker barrel cortex were cauterized, resulting in cessation of blood flow. The same protocol was followed for losartan-treated animals but did not result in cessation of blood flow in the whisker barrel cortex. Another group of losartan-treated animals received between 8 and 14 cauterizations of surface vessels feeding the whisker barrel cortex, and cessation of blood flow was verified. Rats were killed 72 h after surgery. Morphological examination revealed angiogenesis, maintained vascular delivery, and significantly decreased infarct size in losartan-treated animals compared with controls. These results demonstrate that pretreatment with losartan reduces infarct size after cerebral focal ischemia and support the hypothesis that cerebral angiogenesis may be one of the mechanisms responsible.     

Single gene deletions of orexin, leptin, neuropeptide Y, and ghrelin do not appreciably alter food anticipatory activity in mice

Timing activity to match resource availability is a widely conserved ability in nature. Scheduled feeding of a limited amount of food induces increased activity prior to feeding time in animals as diverse as fish and rodents. Typically, food anticipatory activity (FAA) involves temporally restricting unlimited food access (RF) to several hours in the middle of the light cycle, which is a time of day when rodents are not normally active. We compared this model to calorie restriction (CR), giving the mice 60% of their normal daily calorie intake at the same time each day. Measurement of body temperature and home cage behaviors suggests that the RF and CR models are very similar but CR has the advantage of a clearly defined food intake and more stable mean body temperature. Using the CR model, we then attempted to verify the published result that orexin deletion diminishes food anticipatory activity (FAA) but observed little to no diminution in the response to CR and, surprisingly, that orexin KO mice are refractory to body weight loss on a CR diet. Next we tested the orexigenic neuropeptide Y (NPY) and ghrelin and the anorexigenic hormone, leptin, using mouse mutants. NPY deletion did not alter the behavior or physiological response to CR. Leptin deletion impaired FAA in terms of some activity measures, such as walking and rearing, but did not substantially diminish hanging behavior preceding feeding time, suggesting that leptin knockout mice do anticipate daily meal time but do not manifest the full spectrum of activities that typify FAA. Ghrelin knockout mice do not have impaired FAA on a CR diet. Collectively, these results suggest that the individual hormones and neuropepetides tested do not regulate FAA by acting individually but this does not rule out the possibility of their concerted action in mediating FAA.     

Change in the staining properties of pyriform neurocytes in the mouse cerebellum during protein-calorie deficiency and subsequent rehabilitation

Quantitative analysis has been carried out on semithin sections of cerebellum cortex to investigate the relation between Purkinje cells with different dyeing properties. The number of dark Purkinje cells was found to increase after a month-long food rehabilitation of ill-fed mice. At the same time addition of carnitine to the mouse food has resulted in a significant decline in the number of dark Purkinje cells, as compared to control animals. The data obtained suggest that the rising number of dark Purkinje cells in the cerebellum cortex under conditions of malnutrition is probably due to the increased intracellular accumulation of free fatty acids.     

Development of the various glial cell types in the cerebral cortex of postnatal rats

The present quantitative study in the postnatal rats showed the rapid growth of the various glial cell types in the cerebral cortex. Among them, the increase of microglia was most dramatic. The increase was about 15 times, covering a period of 15 days extending from 5 days of age to 20 days. The majority of the microglia observed were in the outer third of the cortex. During the same period, the number of oligodendrocytes and astrocytes also showed a steady but moderate increase. The increase of oligodendrocytes was most significant between 5 and 10 days. Their density was greater in the inner third of the cortex. Astrocytes were distributed uniformly throughout. Examination of the cerebral cortex in 1- to 3-day-old rats by electron microscopy showed sporadic ameboid microglia cells and glioblasts. The possibility that they served as the precursor cells of microglia and macroglia (astrocytes and oligodendrocytes), respectively, was considered.     

Acute food restriction increases collagen breakdown and phagocytosis by mature decidual cells of mice

An ultrastructural study was undertaken on antimesometrial mature decidual tissue of fed and food-restricted mice, on day 9 of pregnancy. The mean ad libitum food intake was established on mice from the 8th till the 9th day of pregnancy. Fed mice were used as controls. Experimental animals were divided into two groups: one was allowed to feed 25% of normal diet and the other 50%. Extracellular collagen fibrils were scarce in fed animals and conspicuous in food restriction. Granular electron-dense deposits and filamentous aggregates of disintegrating collagen fibrils were observed in all food-deprived mice but were rarely noted in fed animals. Intracellular vacuolar structures exhibited other typical cross-banded collagen immersed in finely granular electron-translucent material (clear vacuole) or electron-dense material containing collagen fibrils with a faint periodicity (dark vacuole). The clear and dark vacuoles were scarce in fed animals and evident in food-restricted mice, mainly in those 25% food restricted. Although collagen breakdown may be part of the normal process of decidual tissue remodelling our results suggest that it is enhanced in food-restricted animals. Thus it seems that collagen breakdown is a normal mechanism that may be regulated by the food intake of the pregnant animal.     

Effect of carbohydrate-enriched diet and subsequent food restriction on life prolongation in Fischer 344 male rats

Increased proportion of carbohydrates (dextrin) in the diet has a life prolonging effect upon male Fischer 344 rats; however, the effect of this diet appears only when the rats aged from 6 weeks to 6 months are on diet, after this treatment median survival of experimental animals increases by 96 days and the 10th percentile increases on the average by 10 days (n-60). Further maintenance of animals on the same diet has minimum effect: animals being on this diet throughout the whole life exhibit a median lifespan increase by 120 days and an increase in the 10th percentile by 41 days. However, if such animals aged 6 months are transferred to a restricted (60%) food intake regimen (control diet, not enriched with carbohydrate) a further increase in median and 10th percentile lifespan prolongation can be observed reaching 328 and 396 days, respectively as compared to controls. The effects of this early feeding (from 6 weeks to 6 months) with a carbohydrate-enriched diet available ad libitum and food restricted (60% controls) regimen fed from the age of 6 months onwards are additive, the final results being identical as if the animals are kept on the 60% food restricted intake throughout the whole life.     

Epigenetic dysregulation of the dopamine system in diet-induced obesity

Chronic intake of high-fat (HF) diet is known to alter brain neurotransmitter systems that participate in the central regulation of food intake. Dopamine (DA) system changes in response to HF diet have been observed in the hypothalamus, important in the homeostatic control of food intake, as well as within the central reward circuitry [ventral tegmental area (VTA), nucleus accumbens (NAc), and pre-frontal cortex (PFC)], critical for coding the rewarding properties of palatable food and important in hedonically driven feeding behavior. Using a mouse model of diet-induced obesity (DIO), significant alterations in the expression of DA-related genes were documented in adult animals, and the general pattern of gene expression changes was opposite within the hypothalamus versus the reward circuitry (increased vs. decreased, respectively). Differential DNA methylation was identified within the promoter regions of tyrosine hydroxylase (TH) and dopamine transporter (DAT), and the pattern of this response was consistent with the pattern of gene expression. Behaviors consistent with increased hypothalamic DA and decreased reward circuitry DA were observed. These data identify differential DNA methylation as an epigenetic mechanism linking the chronic intake of HF diet with altered DA-related gene expression, and this response varies by brain region and DNA sequence.     

Glial Promoter Selectivity following AAV-Delivery to the Immature Brain

Recombinant adeno-associated virus (AAV) vectors are versatile tools for gene transfer to the central nervous system (CNS) and proof-of-concept studies in adult rodents have shown that the use of cell type-specific promoters is sufficient to target AAV-mediated transgene expression to glia. However, neurological disorders caused by glial pathology usually have an early onset. Therefore, modelling and treatment of these conditions require expanding the concept of targeted glial transgene expression by promoter selectivity for gene delivery to the immature CNS. Here, we have investigated the AAV-mediated green fluorescent protein (GFP) expression driven by the myelin basic protein (MBP) or glial fibrillary acidic protein (GFAP) promoters in the developing mouse brain. Generally, the extent of transgene expression after infusion at immature stages was widespread and higher than in adults. The GFAP promoter-driven GFP expression was found to be highly specific for astrocytes following vector infusion to the brain of neonates and adults. In contrast, the selectivity of the MBP promoter for oligodendrocytes was poor following neonatal AAV delivery, but excellent after vector injection at postnatal day 10. To extend these findings obtained in naïve mice to a disease model, we performed P10 infusions of AAV-MBP-GFP in aspartoacylase (ASPA)-deficient mouse mutants presenting with early onset oligodendrocyte pathology. Spread of GFP expression and selectivity for oligodendrocytes in ASPA-mutants was comparable with our observations in normal animals. Our data suggest that direct AAV infusion to the developing postnatal brain, utilising cellular promoters, results in targeted and long-term transgene expression in glia. This approach will be relevant for disease modelling and gene therapy for the treatment of glial pathology.     

Expression of thrombospondin-1 and CD36 and CD47 receptors in the rat brain after exposure to damaging factors in the early postnatal period

The expression of thrombospondin-1 (TS-1) and its receptors CD47 and CD36 in the cerebral cortex and hippocampus of rats under damaging factors in the early postnatal period was studied. After hypoxia on the 7th day of postnatal development, an increase in the number of CD47-expressing cerebral endothelial cells (days of postnatal development: P28–P70) and reduction in the number of TS-1-expressing astrocytes in the cortex at P28 were observed. In animals subjected to early postnatal stress at the age of P2–P15, a decrease in TS-1-expressing astrocytes in the cortex and hippocampus was registered (predominantly at the age of P28). It was noted that these changes characterize the period of long-term effects (P28–P70) of early stress that is relevant to the processes of reparative angiogenesis and arresting of neurological deficits.     

Expression of Transferrin mRNA in the CNS of Normal and Jimpy Mice

Both the iron mobilization protein transferrin and iron itself are found predominantly in oligodendrocytes in the brain and consequently have been hypothesized to have a role in myelination. This study is designed to begin to understand the mechanism(s) that control the expression of transferrin at the gene level in the nervous system using a hypomyelinating murine mutant (jimpy mouse). With this animal model it is possible to determine if transferrin gene expression in the nervous system is dependent on the presence of a mature oligodendrocytic population. The results demonstrate that normally expression of the transferrin gene increases from postnatal day 5 to 22-25 and then levels off in the adult. In the jimpy mouse, the relative amount of transferrin gene expression is less than that of littermate controls at 5 days of age. Furthermore, transferrin gene expression does not increase with age beyond the level observed at postnatal day 5 in the jimpy mouse. It is concluded from this study that the majority of the transferrin mRNA in the mouse brain is expressed by and/or requires the presence of a mature oligodendrocytic population.     

Early Undernutrition and [3H]γ-Aminobutyric Acid Binding in Rat Brain

The effect of early undernutrition and dietary rehabilitation on [3H]gamma-aminobutyric acid ([3H]GABA) binding in rat brain cerebral cortex and hippocampus was examined. Undernourished animals were obtained by exposing their mothers to a protein-deficient diet during both gestation and lactation. Saturation analysis of [3H]GABA binding in the cerebral cortex and hippocampus revealed high- and low-affinity components in the undernourished group, whereas control animals possessed only a low-affinity site. The concentration of low-affinity binding sites was greater in the undernourished animals. Rehabilitation of undernourished animals completely abolished the binding site differences. Treatment of brain membranes with Triton X-100 yielded two binding components in both the undernourished and control animals, although the concentration of lower affinity sites was still greater in the undernourished group. Neither the efficacy nor the potency of GABA to activate benzodiazepine binding in cerebral cortex was modified by undernutrition. These data suggest that early undernourishment modifies the characteristics of [3H]GABA binding, perhaps by reducing the brain content of endogenous inhibitors of the higher affinity binding site. The lack of effect on GABA-activated benzodiazepine binding suggests the possibility that neither the high- nor the low-affinity GABA binding sites are coupled to this receptor component.     

Effects of food restriction and social cues on sexual maturation and growth in male musk shrews (Suncus murinus)

In the first experiment, the effects of food availability on growth and reproduction were assessed by placing male musk shrews on 3 different feeding schedules, i.e. fed ad libitum, fed 50% of the amount eaten by the ad-libitum controls or fed 25% of the amount eaten by the ad-libitum controls. After 25 days, growth and sexual maturation were significantly retarded in both of the food-restricted groups compared to the control group. In the second experiment, the combined effects of food availability and social cues were investigated in juvenile males that were either fed ad libitum (during the day) or placed on a restricted diet known to inhibit growth and reproduction. Half of these males were either housed alone or with an adult female (separated from female during the day by a wire barrier, but in full contact at night). The ad-libitum-fed males living with a female ate more food, gained more body weight and were more sexually mature than ad-libitum-fed animals living alone. Furthermore, males in the two food-restricted groups had lower body weights and were sexually immature compared with males in both of the ad-libitum-fed groups. Finally, there were little or no differences in growth and reproduction between animals in the two food-restricted groups. These results suggest that food availability is an important environmental regulator of the timing of sexual maturation, and that stimulatory social cues cannot override the inhibitory effects of an inadequate diet.     

Meal-feeding studies in mice: effects of diet on blood lipids and energy expenditure

To identify optimal study-design conditions to investigate lipid metabolism, male, C57BL/6J mice (age, 59 +/- 3 days) were allotted to eight groups, with six animals per group that were stratified by three factors: diet type (high fat [HF]: 60% of energy from fat versus that of a standard rodent diet, 14% fat, fed for 7 weeks), feeding regimen (ad libitum [ad lib] versus meal fed), and metabolic state (data collected in fasted or fed states). Serum free fatty acids (FFA) and triacylglycerols (TAG) concentrations, and energy expenditure (EE) were assessed. Mice gained 0.30 +/- 0.11 g of body weight/day when allowed ad lib access to HF diet, similar weight when meal-fed the HF or ad lib-fed the standard diet (0.10 +/- 0.03 g/day), and no weight when meal-fed the standard diet (0.01 +/- 0.02 g/day). Fed-state TAG concentration was 88 to 100% higher (P < 0.02) than that of the fasted state, except when animals were ad lib-fed the HF diet. When the standard diet was meal fed, FFA concentration was 30% higher in the fasted compared with the fed state (P = 0.003). Mice had 33% higher postprandial EE when either diet was meal fed (P = 0.01). Mice adapted to meal feeding developed transitions in metabolism consistent with known physiologic changes that occur from fasting to feeding. When fed the standard diet, a 6-h per day meal-feeding regimen was restrictive for normal growth. These data support use of a meal-feeding regimen when HF diets are used and research is focused on metabolic differences between fasted and fed states. This protocol allows study of the metabolic effects of an HF diet without the confounding effects of over-consumption of food and excess body weight gain.     

Experimental protein malnutrition in primates--cytochemistry of the nervous system

Experimental protein malnutrition was induced in groups of young juvenile squirrel monkeys by feeding them ad libitum diets very low in protein content, whereas a diet containing 25% protein content was fed to the control animals. Detailed cytochemical studies have clearly shown the sensitivity of the nervous system to dietary abuse. The motor neurons of the spinal cord and the Purkinje cells of the cerebellum are very sensitive to protein deficiency, and the number of oligodendroglia cells increases sharply. Gallocyanin stained preparations from the malnourished animals show significant decrease in the amount of RNA in the Purkinje cells of cerebellum and the anterior horn cells of the spinal cord. The larger neurons show prominent chromatolysis with concomitant increase in the number of oligodendrocytes surrounding such nerve cells. The different layers of the cerebellar cortex of the malnourished animals, as well as some of the neurons of the spinal cord, showed decreased activity of succinate dehydrogenase and increased levels of thiamine pyrophosphatase and lactate dehydrogenase. Although ATPase activity appeared to remain unchanged quantitatively, it showed profound disturbance in its in situ localization, especially in the mitochondrial ATPase located in the cytoplasm. The enzyme activity in the nuclear membrane appeared unchanged. These studies emphasize the importance of studying in situ changes in the anatomically heterogeneous nervous tissue, rather than of working only with biochemical methods using homogenized material.     

Effects of long-term intake of a fine-grained diet on the mouse masseter muscle

Muscle fibers of the masseter muscle of mice which had been fed a fine-grained diet for various periods were studied histochemically and morphometrically. The diameters of both extrafusal and intrafusal muscle fibers decreased with time in mice fed a fine-grained diet, compared with those of control mice. In animals maintained on the special diet for 160 days after weaning at the 20th postnatal day, the effects of the diet on the diameter of muscle spindles were severe, and the diameter of each type of red and white fibers was significantly smaller than those of control animals. But a significant difference was not recognized in the diameter of intermediate fibers between control and treated mice. Unexpectedly, white fibers having a smaller diameter than red fibers were observed in diet-fed mice after the 180th postnatal day, although white fibers having such small diameter were not detectable in control animals. Succinic dehydrogenase activities were decreased in both extrafusal and intrafusal fibers of experimental animals. Moreover, muscle spindles with no annulospiral endings were increased in number in mice fed the diet for 130 and 160 days after weaning, although those spindles also increased in control animals. The diameters of outer capsules and primary endings were also significantly decreased in the animals kept on the diet for a long time. These effects of the fine-grained diet on the mouse masseter muscle became severer with time.     

Functional development of oligodendrocytes and open-field behavior in developing rats: an approach using monoclonal antibody to immature oligodendrocytes.

To examine the relation between functional development of oligodendrocytes and open-field behavior during the postnatal period, a mouse monoclonal antibody termed 14F7, which predominantly labels stage-specific immature oligodendrocytes, was employed. Antibody 14F7 was administered intraperitoneally into male pups on day 3 and 4 after birth. The open-field test was performed on days 12 and 18 of the postnatal period. Horizontal activity increased remarkably with the growth of pups. On day 18, horizontal activity in the group with 14F7 was significantly higher than the control, while there was no significant difference between treatments on day 12. In contrast to the horizontal activity, the frequency of hind leg rearing, vertical activity, in the group with 14F7 was significantly lower than that in the control. On day 12, choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activities in the cerebral cortex were similar between the groups. These activities increased with the growth of pups in both groups. In the 14F7 group on day 18, ChAT activity was the same as the control, whereas AChE activity was significantly lower compared with the control. These results suggest that neonatal exposure to 14F7 induces abnormal neurotransmission by reducing the degradation of acetylcholine and alters the spontaneous activities in developing rats.