Peter J. Morgane

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.

Effects of prenatal protein malnutrition on the hippocampal formation.

In this review we have assessed the effects of prenatal protein malnutrition on the hippocampal formation of the developing brain. In investigating this insult in the hippocampal neuronal model we have concentrated on aspects of enhanced inhibition we have shown in our earlier studies. Since this involves particular attention to the GABAergic interneurons we have examined the complex interneuronal networks of the hippocampal formation and their neurotransmitter afferent inputs, particularly the serotonergic system from the midbrain raphé nuclei. A variety of combinations of specialized interneurons are discussed in terms of how malnutrition insults perturb function in these inhibitory and disinhibitory networks. Pathological enhancement of inhibition manifests itself by diminished plasticity, alterations in theta activity and deficits in long-term learning behaviors. Long-term inhibition in select GABA interneuron systems may form a major derangement seen following prenatal protein malnutrition. The focus of this study is to relate enhanced inhibition to the several forms of inhibitory systems present in the hippocampal formation and develop hypotheses as to the primary derangements that may account for pathological inhibition in prenatal malnutrition.

Dorsal raphe, substantia nigra and locus coeruleus: Interconnections with each other and the neostriatum ☆

Using a retrograde axonal transport method, direct projections to the neostriatum were demonstrated from the dorsal raphe nucleus, a large area of the ventral midbrain tegmentum (including the ventral tegmental area of Tsai, the substantia nigra pars compacta, reticulata and suboculomotoria), and the tegmentum ventral to the caudal red nucleus. A direct projection was also found from the mediodorsal part of the substantia nigra to the rostral part of the dorsal raphe nucleus. Projections from the entopeduncular nucleus (pallidum) and the lateral hypothalamic area to the lateral habenular nucleus, and from the latter to the dorsal raphe nucleus were also found. This habenular projection arises primarily from large neurons in the medial part of the lateral habenula and also from another group of small cells immediately adjacent to the medial habenular nucleus. A non-reciprocal connection of the dorsal raphe nucleus to the locus coernuleus was also found. On the basis of these results and the data available in the literature on the possible neurotransmitters used by these various structures, it is suggested that the dorsal raphe nucleus may play an important role in brain stem modulation of neostriatal function.

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.

The anatomy of the brain of the bottlenose dolphin (Tursiops truncatus). Rhinic lobe (rhinencephalon): The archicortex

The hippocampal formation or archicortical division of the rhinecephalon of the bottlenose dolphin, Tursiops truncatus, is described from the standpoint of its gross topographic relations and cytoarchitecture. A feature of the dolphin brain, which lacks olfactory bulbs and peduncles, is the striking reduction of the archicortical relative to the paleocortical formations. The small, poorly developed archicortex covered by massive epihippocampal portions of the hemispheres (parietal and temporal lobes), appears greatly reduced relative to the large, well developed olfactory lobes which are covered by small epistriatal portions of the hemispheres (orbital lobes). The archicortex exhibits three junctional zones with the paleocortex, two laterally in the unci and one anteriorly in the septal area. Despite the small size of the hippocampal formations, the general topographic disposition of its cytoarchitectonic areas and their cellular organization in Tursiops have many features that are similar to those in other placental mammals. The archicortex is subdivisible into four major sectors: temporal, retrosplenial, supracallosal and subcallosal. With the exception of the temporal sector, cytoarchitectonic areas of the other sectors are variously attenuated and poorly differentiated, particularly the dentate area and the hippocampal areas H5 and H4. Here, the dentate area and hippocampal areas H5 and H4 which are present along the paradentate bank of the hippocampal sulcus, extend to the level of the oblique sulcus of the parahippocampal gyrus and then disappear. Hippocampal areas H3, H2 and H1 are also clear in the floor and along the parahippocampal bank of the hippocampal sulcus in the temporal sector. These areas are less definable as they extend beyond the oblique sulcus into the retrosplenial sector and are difficult to recognize as distinct areas in the supracallosal and subcallosal sectors of the archicortex. The archicortex is demarcated bilaterally from limbic formations in the border of the hemisphere by segments of the rhinic cleft which are very clear. Equally clear is the cytoarchitectonic demarcation of the archicortex from the neocortex in the border (limbus) of each hemisphere, i.e., where the subiculum abuts against the presubiculum. The subicular area, best expressed in the temporal sector, extends anteriorly over the corpus callosum to the subcallosal gyrus and, throughout its extent from the uncal to the septal junction, is clearly demarcated from limbic neocortex by a transition zone characterized by archicortical cells merging with cells in the deep layer of the bordering neocortex. Overall, the archicortical formations of the dolphin and other whale brains we have examined exhibit many regional peculiarities that we have described, both grossly and architectonically, with emphasis on the comparative anatomical approach.

Implications of the “initial brain” concept for brain evolution in Cetacea

We review the evidence for the concept of the “initial” or prototype brain. We outline four possible modes of brain evolution suggested by our new findings on the evolutionary status of the dolphin brain. The four modes involve various forms of deviation from and conformity to the hypothesized initial brain type. These include examples of conservative evolution, progressive evolution, and combinations of the two in which features of one or the other become dominant. The four types of neocortical organization in extant mammals may be the result of selective pressures on sensory/motor systems resulting in divergent patterns of brain phylogenesis. A modular “modification/multiplication” hypothesis is proposed as a mechanism of neocortical evolution in eutherians. Representative models of the initial ancestral group of mammals include not only extant basal Insectivora but also Chiroptera; we have found that dolphins and large whales have also retained many features of the archetypal or initial brain. This group evolved from the initial mammalian stock and returned to the aquatic environment some 50 million years ago. This unique experiment of nature shows the effects of radical changes in environment on brain-body adaptations and specializations. Although the dolphin brain has certain quantitative characteristics of the evolutionary changes seen in the higher terrestrial mammals, it has also retained many of the conservative structural features of the initial brain. Its neocortical organization is accordingly different, largely in a quantitative sense, from that of terrestrial models of the initial brain such as the hedgehog.

Raphe projections to the locus coeruleus in the rat.

Afferent projections to the locus coeruleus from the various raphe nuclei, particularly of the midbrain (nuclei raphe dorsalis and medianus) and pons (nuclei raphe pontis and magnus), have been studied in the rat by retrograde transport methods using horseradish peroxidase (HRP). The locus coeruleus, in both its dorsomedial and ventrolateral divisions, and in its various anterior-posterior components, were injected with 0.05 microliters of horseradish peroxidase following which various structures of the brainstem, particularly the raphe nuclei, were examined for HRP reactive cells. It was found that injections in most components of the locus coeruleus were associated with HRP positive cells in varying degrees of density in the nuclei raphe dorsalis, medianus, pontis, and magnus, with considerably sparser labelling in the anterior aspects of the medullary raphe nuclei pallidus and obscurus. Labelled cells were also seen in the nuclei of the solitary tract, contralateral locus coeruleus. lateral reticular areas of the pons and midbrain, nuclei pontis oralis and caudalis, vestibular nuclei, mesencephalic nucleus of the trigeminal nerve, fastigial nuclei of cerebellum and medial parabrachial nuclei. These data, showing widespread innervation of the locus coeruleus from all raphe nuclei, as well as many other brainstem areas, in the rat support the general view of heavy innervation of the locus coeruleus from both extra-raphe and raphe nulcei. These latter raphe projections, probably serotonergic in nature, provide anatomical support for the various experiments indicating considerable regulation of locus coeruleus activities, such as phasic events of REM sleep, among other, by widespread projections from most raphe nuclei was well as several other regions of the brainstem.

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.

Developmental protein malnutrition: Influences on the central nervous system of the rat ☆

Our group has been carrying out interdisciplinary studies on the effects of prenatal and postnatal protein malnutrition on the developing rat brain. Anatomical, physiological, biochemical and behavioral approaches using the same animal model have revealed that protein malnutrition affects the brain at various levels, i.e., (1) anatomical, as revealed by Golgi findings of deranged dendritic trees on analysis of cortical and subcortical areas; (2) physiological, as revealed by delayed sleep pattern maturation, disturbances in seizure thresholds, slowing of sensory cortico-cortical and thalamocortical evoked potentials, and changed power in hippocampal theta activity; (3) biochemical, as revealed by marked increases in biogenic amines dating from birth, as well as modifications in tryptophan metabolism; and (4) behavioral, as revealed by various changes in responses to different kinds of aversive stimulation. Reversal studies have revealed that many changes are permanent and not amenable to nutritional rehabilitation even at birth, which is before the brain growth spurt in the rat. Our paradigm closely mimicks the human condition of low level, chronic protein undernutrition and thus reveals the underlying disturbances due to malnutrition. The dietary reversal studies are attempts at pin-pointing critical brain growth periods, beyond which recovery of functions is not possible.

Calretinin-immunoreactive neurons in the primary visual cortex of dolphin and human brains

A new class of gamma-aminobutyric acid (GABA)ergic neurons immunoreactive to the calcium-binding protein calretinin (CR) was demonstrated in primary visual cortices of the bottlenose dolphin (Tursiops truncatus) and humans (Homo sapiens). Comparative analysis revealed several differences between dolphin and human visual cortex in the laminar distribution of CR-positive perikarya, although general typology of the immunoreactive CR-positive neurons was similar in both species. Thus, in both human and dolphin primary visual cortex almost all CR-positive neurons are non-pyramidal, either fusiform or bipolar cells, oriented with their long axis along the radial axis of the cortex. Large multipolar stellate cells were also observed in layers I and VI. The CR-positive neurons in the dolphin visual cortex are concentrated almost exclusively in layer I and, to a lesser extent, in layer II. In all other layers (IIIa, b, IIIc/V and VI) of the dolphin visual cortex CR-positive neurons were only rarely seen. In the human primary visual cortex CR-positive neurons are located mainly in layers II, III and IVa, b, c, with considerably lower densities of these cells observed in layers V and VI. CR-positive neurons in layer I of the human visual cortex are represented by Cajal-Retzius horizontal cells, whereas no such cells were seen in layer I of the dolphin neocortex. The numerical density of CR-positive neurons in the dolphin primary visual cortex is significantly lower than in the area of cortex in humans.(ABSTRACT TRUNCATED AT 250 WORDS)