Warren C. Stern

The effects of protein malnutrition on the developing central nervous system in the rat

Abstract We have carried out a multi-disciplinary study of the effects of prenatal protein malnutrition on the developing rat brain. These experiments, involving anatomical, physiological, biochemical, and behavioral approaches, have revealed that malnutrition induced prenatally can affect various parameters of brain growth and development. Some of these effects can be reversed depending on when dietary restitutions are carried out. However, if protein malnutrition is maintained during the brain growth spurt or critical growth periods there are many permanent sequelae that cannot be reversed by subsequent restitution of high protein diets. We have reviewed the concept of critical periods of brain growth relative to the various aspects of neural morphogenesis in the rat, that is, the birth of neurons, migration of neurons, differentiation of neurons, and synapse formation. We have also discussed the rapid phases of brain growth in the rat as compared to similar phases in other species as a basis for determining whether the rat model can provide time-tables for brain growth in other species, including man. Different components of the brain, both morphological and chemical, have their own cycles of rapid development so that insults to the brain at particular periods affect particular aspects of brain chemistry and neuronal systems. Development of chemical circuits in the brain, such as the aminergic neurons, and their eventual adequate functioning, depends on development of the neurotransmitters themselves. These latter are markedly affected by protein malnutrition. Major physiological-behavioral states, such as the sleep-waking continuum, are markedly affected by protein malnutrition as are many behaviors. Some of these latter are merely late or retarded in development but others remain permanently altered. By approaching the problem of protein malnutrition from multiple points of view we have been able to pinpoint several brain areas showing the most drastic residua of early protein malnutrition and are beginning, by use of morphometric, electro-ontogenetic, biochemical development and behavioral studies, to define brain locales and basic mechanisms by which these insults produce their effects.

Ontogeny of the levels of biogenic amines in various parts of the brain and in peripheral tissues in normal and protein malnourished rats.

Abstract The ontogenetic development of serotonin, 5-hydroxyindoleacetic acid, and norepinephrine in brain regions and in peripheral tissues was examined in normal and protein malnourished rats from birth to age 300 days. The malnourished rats, which received a diet low in protein starting 5 weeks prior to conception, showed significantly elevated brain and peripheral tissue levels of the biogenic amines and 5-hydroxyindoleacetic acid at birth. This is one of the earliest ages at which protein malnutrition has been reported to affect a major biochemical measure in the brain. In malnourished rats, brain concentrations of serotonin and 5-hydroxydoleacetic acid remained elevated at older ages, up to 300 days, with the largest effects (up to 200% increase) occurring in subtelencephalic brain regions. These changes in brain indole levels probably represent a general metabolic alteration of indoleamine metabolism since elevated indole concentrations were also observed in the heart, lung, and stomach. At most ages the increase in brain norepinephrine levels in malnourished rats was less pronounced than for the indoles. Also, no increase in norepinephrine concentration in the peripheral tissues were observed. With respect to norepinephrine concentrations, the brain appears to be more sensitive to the insult of protein malnutrition than do peripheral tissues. The present results demonstrate that rearing rats on a diet low in protein, but adequate in all other respects, significantly elevates the brain amine content at most ages from birth through 300 days of age.

Seizure susceptibility and brain amine levels following protein malnutrition during development in the rat

Abstract This report examines the change in seizure susceptibility in adult rats which were reared on a diet containing either normal or inadequate levels of protein. A significantly greater percentage of the protein malnourished rats convulsed at low to moderate intensities of electroconvulsive shock (ECS) than normals. Also, seizure duration in the malnourished subjects teneed to be longer than in the normals. Switching adult rats to the opposite diet, i.e. rats reared on normal diets now receive low protein diet and vice versa, had a moderating effect on seizure susceptibility, but full reversal of the effects of protein malnutrition was not achieved. In sum, protein malnutrition during development led to enhanced seizure susceptibility in adulthood, an effect which was only partially ameliorated by restoration of adequate dietary protein levels in adulthood. Prior studies have shown a strong inverse relationship between brain biogenic amine levels and seizure activity. In a second study we therefore investigated whether decreased regional brain levels of norepinephrine and serotonin were produced by chronic protein malnutrition. Suprrisingly, levels of these two amines were elevated in the brains of the protein malnourished rats and, therefore, changes in the levels of these neurochemicals cannot simply account for the increased seizure activity seen in rats reared on inadequate amounts of protein.

Theoretical view of REM sleep function: Maintenance of catecholamine systems in the central nervous system

We hypothesize that rapid eye movements (REM) sleep may play a role in maintaining the functioning of catecholamine-containing neurons in the central nervous system. The main lines of evidence supporting this view are (a) following REM deprivation the responsiveness of catecholamine systems is depressed; (b) administration of drugs which enhance catecholamine activity can reverse some of the behavioral deficits which occur after REM deprivation; and (c) acute administration of pharmacological agents which depress catecholamine activity (alpha methyltyrosine, reserpine) produce a “compensatory≓ increase in REM time whereas increasing central catecholamine availability at the synapse (electroconvulsive shock, imipramine, monoamine oxidase inhibitors) decreases REM sleep. It is suggested that the mechanism of REM is primarily cholinergic and that the locus coeruleus may be the site of interaction between the catecholamine-REM function and the cholinergic-REM mechanism. Evidence is discussed relating to the possibility that REM sleep may have a more general function in the central nervous system, i.e., the modulation of protein synthesis in the brain.

Effects of growth hormone on sleep-waking patterns in cats.

Abstract Cats given growth hormone in doses from 50–1000 μg, i.p., showed a selective elevation of REM sleep in the first 3 hr postinjection. Bovine thyrotropin control injections did not alter sleep patterns. When the effect of growth hormone on sleep was blocked by REM deprivation for the first 3 hr, the REM elevating effect of growth hormone still occurred in the subsequent sleep period. These results suggest that growth hormone affects the central nervous system, either directly or indirectly. Also, the greatly increased secretion of growth hormone, which has been reported during slow-wave sleep in man, may play a role in the occurrence of REM sleep.

Tryptophan availability: relation to elevated brain serotonin in developmentally protein-malnourished rats.

Abstract Developmental changes in tryptophan, serotonin, and 5-hydroxyindoleacetic acid in many brain regions were examined in normal and protein-malnourished rats from birth to age 30 days. The malnourished rats, whose dams received a diet low in protein starting 5 weeks prior to conception, showed significantly elevated brain tryptophan, serotonin, and 5-hydroxyindoleacetic acid at most ages examined. Brain tryptophan concentrations for both groups of animals showed a positive correlation with their respective unbound plasma tryptophan concentrations. Although the malnourished animals showed lower total plasma tryptophan concentrations than the control group, the amount of free plasma tryptophan available for brain metabolism was significantly higher in the malnourished rats. This was due, in part, to a decrease in the molar ratio of bound tryptophan to albumin in the malnourished animals. In addition, those malnourished rats had lower albumin levels and higher concentrations of nonesterified fatty acids as compared to the normal animals, causing more tryptophan to be available as the free form in plasma. Overall, the present results demonstrate that rearing rats on a diet low in protein but adequate in all other respects significantly elevates brain tryptophan and amine concentrations, probably as a consequence of developmental alterations in plasma tryptophan availability.

Neuropharmacology of the afferent projections from the lateral habenula and substantia nigra to the anterior raphe in the rat

Abstract Previous neuroanatomical studies have demonstrated the existence of major afferent projections to the nucleus raphe dorsalis originating in the lateral habenula and substantia nigra. These projections have a predominantly suppressive effect on raphe unit activity. The present acute electrophysiological study in anesthetized rats evaluated: (a) the effects of pharmacological blockade of GABAergic transmission on the suppression of activity of units in the nuclei raphe dorsalis and raphe medianus by stimulation of the lateral habenula, and (b) the effects of pharmacological blockade of GABA, dopamine, acetylcholine, glycine, opiates, serotonin, norepinephrine and histamine on the suppression of raphe activity by electrical stimulation of the substantia nigra. Results showed that the GABA bloeker, picrotoxin, attenuated the effects of lateral habenula stimulation on unit activity in the raphe dorsalis and medianus and that GABA may exert a tonic inhibitory influence on spontaneous unit activity in the raphe. The effects of stimulation in the nigra on raphe dorsalis and medianus neuronal activity were not blocked by any of the 10 drugs tested. Indirect evidence was obtained, however, that norepinephrine and possibly histamine exerted an excitatory effect on raphe units since drugs which block these transmitters tended to enhance the suppressive effects of nigral stimulation. The present results do not permit identification of the neurotransmitter which mediates the marked suppression of unit activity in the anterior raphe produced by electrical stimulation of the substantia nigra.

Noradrenergic pathways and sleep-waking states in cats ☆

Abstract Four female cats were surgically prepared with an array of recording electrodes (cerebral cortex, hippocampus, lateral geniculate nuclei, neck and eye muscles) for electrophysiological recording of sleep-waking patterns. Polygraphic recordings were obtained before and after administration of 6-hydroxydopamine into the region of the ventral noradrenergic pathway at the mesencephalic level. Records were scored in five categories: active awake, quiet awake, light slow wave sleep, deep slow wave sleep and REM (rapid eye movement) sleep. The acute effect of 6-hydroxydopamine was a shift toward electrocortical arousal; this probably reflects release of endogenous amines within ascending noradrenergic systems. The chronic effect was a small increase in deep slow wave sleep and REM sleep. Biochemical analyses of regional norepinephrine and serotonin levels indicated significant depletion of norepinephrine in all parts sampled (occipital cortex, temporal cortex, pyriform lobe, basal forebrain region, striatum, hypothalamus, cerebellum, and brain stem). Serotonin was depleted to a lesser extent in occipital cortex, temporal cortex, pyriform lobe, cerebellum, and brain stem. Since the greatest depletion of norepinephrine was found in the basal forebrain (to 24% of control levels), the ventral noradrenergic pathway was selectively influenced to some extent. The data thus appear to indicate that activation of the ventral pathway induces electrocortical arousal, while depletion of amines within this pathway may induce somnolence.

Absence of ponto-geniculo-occipital (PGO) spikes in rats

Abstract In contrast to the cat, EEG recordings from macroelectrodes in the lateral geniculate nucleus and occipital neocortex of rats (a) failed to show spiking activity during REM sleep, and (b) failed to show spiking activity after reserpine, i.p., during wakefulness or sleep. Other phasic activities during REM sleep, such as eye movements or muscle twitches, were present in rats. The absence of the ponto-geniculo-occipital spikes in rats, at least in the latter two regions, suggests that these monophasic events are not essential or fundamental characteristics of mammalian REM sleep.

Effects of reserpine on sleep and brain biogenic amine levels in the cat

Cats receiving 0.15 mg/kg of reserpine, i.p., had rapid eye movement (REM) sleep suppressed for two days and showed increased ponto-geniculo-occipital (PGO) spikes in waking and slow-wave sleep; these effects were not reversed by administration of dopa. Reserpine (0.125 mg) given intraventricularly, however, significantly increased REM sleep and did not change the frequency of PGO spikes in waking and slow-wave sleep. The differences in sleep characteristics seen between i.p. and central administration of reserpine are probably due to (a) the marked peripheral autonomic side effects accompanying reserpine, i.p., and (b) the much smaller central depletion of brain monoamines following intraventricular reserpine. Also, the correspondance between the regional levels of brain biogenic amines and the occurrence of the sleep states is poor since REM and slow-wave were normal on the third day after reserpine, i.p., while norepinephrine and serotonin levels were depressed by about 50% at that time.