The neural basis of attentional alterations in prenatally protein malnourished rats.

Protein malnutrition during gestation alters brain development and produces specific behavioral and cognitive changes that persist into adulthood and increase the risks of neuropsychiatric disorders. Given evidence for the role of the prefrontal cortex in such diseases, it is significant that studies in humans and animal models have shown that prenatal protein malnutrition specifically affects functions associated with prefrontal cortex. However, the neural basis underlying these changes is unclear. In the current study, prenatally malnourished and control rats performed a sustained attention task with an unpredictable distractor, a task that depends on intact prefrontal cortical function. Radiolabeled 2-deoxyglucose was used to measure neural and brain network activity during the task. Results confirmed that adult prenatally malnourished rats were more distractible than controls and exhibited lower functional activity in prefrontal cortices. Thus, prefrontal activity was a predictor of task performance in controls but not prenatally malnourished animals. Instead, prenatally malnourished animals relied on different brain networks involving limbic structures such as the hippocampus. These results provide evidence that protein reduction during brain development has more wide-reaching effects on brain networks than previously appreciated, resulting in the formation of brain networks that may reflect compensatory responses in prenatally malnourished brains.

Prenatal protein malnourished rats show changes in sleep/wake behavior as adults.

Prenatal protein malnutrition significantly elevates brain levels of serotonin in rats, and these levels remain elevated throughout their lives. This biogenic amine is involved in the regulation of many physiological functions, including the normal sleep/wake cycle. The present study examined the effects of prenatal protein malnutrition on the sleep/wake cycle of freely moving adult rats. Six prenatally protein malnourished (6% casein) and 10 well-nourished (25% casein) male rats (90-120-day-old) were chronically implanted with a standard set of electrodes (to record cortical electroencephalogram, neck muscle electromyogram, electrooculogram, and hippocampal theta wave) to objectively measure states of sleep and wakefulness. Six-hour polygraphic recordings were made between 10.00 and 16.00 h; a time when the rats normally sleep. Prenatally malnourished rats spent 20% more time in slow wave sleep (SWS) compared to the well-nourished rats. The total percentage of time spent in rapid eye movement (REM) sleep was 61% less in prenatally malnourished rats compared to well-nourished control rats. These findings demonstrate the adverse consequences of prenatal protein malnutrition on the quality and quantity of adult sleep in rats. These sleep changes are potentially detrimental to normal social behavior and cognitive functions. Prenatally malnourished rats are an excellent animal model to study the role of endogenous serotonin in the regulation of the normal sleep/wake cycle.

Modulation of paired-pulse responses in the dentate gyrus: effects of prenatal protein malnutrition.

Since our major hypothesis is that prenatal protein malnutrition significantly affects hippocampal neuroplasticity, this study examined the effects of prenatal protein malnutrition on the modulation of dentate granule cell excitability in freely moving rats at 15, 30 and 90 days of age across the vigilance states of quiet waking (QW), slow-wave sleep (SWS) and rapid eye movement (REM) sleep. Using paired-pulse stimulation, the paired-pulse index (PPI), a measure of the type and degree of modulation of dentate granule cell excitability elicited by stimulation of the medial perforant path, was obtained for each vigilance state at each stage of development. Four specific measures of granule cell excitability were computed, namely, PPI using both population spike amplitude (PSA) and EPSP slope measures, absolute values of PSA(1) and EPSP(1) slope. PPI values obtained at 15, 30 and 90 days of age, however, were altered during normal ontogenetic development, but not by vigilance state. At 15 days of age, the malnourished group exhibits greater early inhibition of the PPI using the PSA measure at IPIs between 20 and 30 ms regardless of vigilance state, while at 30 days of age, the malnourished group exhibits greater facilitation at IPIs between 50 and 70 ms during QW and SWS, but not during REM sleep. In the control adult (PND90) and juvenile (PND30) animal, PSA(1) values are significantly higher during SWS than in QW or REM sleep. However, for the younger malnourished animals (PND15 and PND30), PSA(1) values were found to be significantly greater during REM sleep rather than SWS. Therefore, as the animal matures, there appears to be a shift in vigilance state dependent synaptic transmission through the hippocampal trisynaptic circuit from REM sleep to SWS in both control and malnourished animals, with the change occurring later in malnourished animals when compared to control ones. Furthermore, our findings suggests that prenatal protein malnutrition significantly alters modulation of dentate granule cell excitability (i.e., PPI values using the PSA measure) during the earlier stages of development but not in adulthood.

The effects of median raphé electrical stimulation on serotonin release in the dorsal hippocampal formation of prenatally protein malnourished rats.

Our previous work had shown an enhanced inhibition in the hippocampal formation of prenatally protein malnourished rats. We have also found a diminishment in 5-hydroxytryptamine (5-HT) fibers in the hippocampal formation of malnourished rats as well as increased levels of 5-HT in the brain. The purpose of the present study was to determine 5-HT release in the dorsal hippocampal formation following electrical stimulation of the median raphé nucleus (MRN) in unanesthetized prenatally malnourished rats. Stimulation of this nucleus at 20 Hz in malnourished rats resulted in a significantly diminished release of 5-HT compared to well-nourished rats. The latter group showed a lesser, though still significant, decrease in 5-HT release following raphé stimulation. Basal release of 5-HT prior to stimulation was significantly higher in malnourished rats as compared to well-nourished controls. This may be the result of a decreased density of 5-HT neurons leading to a diminished control of release. Stimulation of the MRN in behaving malnourished animals may markedly affect the recurrent negative feedback collaterals onto somatodendritic 5-HT(1A) and 5-HT(1D) autoreceptors thus enhancing the inhibitory effects of stimulation of the median raphé on 5-HT release. Studies are underway to examine the sensitivity of both the somatodendritic and terminal 5-HT autoreceptors in malnourished animals, in order to understand possible mechanisms for our findings.

Dentate granule cell modulation in freely moving rats: vigilance state effects.

Dentate granule cell population responses to paired-pulse stimulation applied to the perforant pathway across a range of interpulse intervals (IPIs) were examined during different vigilance states-quiet waking (QW), slow-wave sleep (SWS), and rapid-eye movement (REM) sleep-in freely moving rats at 15, 30 and 90 days of age. Using these evoked field potentials, the paired-pulse index (PPI), a measure of the type and degree of modulation of dentate granule cell excitability, was computed and shown to be altered as a function of age. Animals, 15 days old, showed significantly lower levels of early inhibition (20-40 ms IPIs), i.e., greater PPI values, during all three vigilance states when compared to both the 30- and 90-day old animals. Adult, i.e, 90-day old animals, on the other hand, showed significantly greater levels of late inhibition (300-1000 ms IPIs), i.e., lower PPI values, than the younger animals (15- and 30-day old) during QW and SWS. These results indicate that as the dentate field of the hippocampal formation matures there are significant alterations in the modulation of dentate granule cell activity.

Hippocampal neurochemical and electrophysiological measures from freely moving rats.

This paper describes surgical and recording procedures that have been developed which permit the simultaneous monitoring of levels of select neurochemicals (via microdialysis) and measures of dentate-evoked field potentials within the hippocampal formation of freely moving adult rats. To test and evaluate these procedures, they were employed to examine changes in hippocampal neurochemistry and neuronal excitability associated with the establishment and maintenance of hippocampal long-term potentiation (LTP). Measures of hippocampal norepinephrine (NE) and glutamate levels along with measures of the dentate granule cell population spike amplitude (PSA) were obtained before, during, and after tetanization of the medial perforant path using two separate tetanization paradigms. Results obtained using these new procedures in several animals indicated that changes in NE and glutamate levels were strongly correlated with increases in the dentate granule cell PSA measure obtained following tetanization. The results indicate that this newly developed procedure can be effectively used to directly examine the relationship between neurochemical and neurophysiological changes associated with hippocampal neuroplasticity.

Comparative analysis of calcium-binding protein-immunoreactive neuronal populations in the auditory and visual systems of the bottlenose dolphin (Tursiops truncatus) and the macaque monkey (Macaca fascicularis).

This study compares the distribution of three calcium-binding protein-immunoreactive (CaBP-immunoreactive) neuronal populations (calretinin-, calbindin- and parvalbumin-immunoreactive) in the visual and auditory systems in two mammalian species which are fundamentally different in their evolutionary traits and ecology, the aquatic toothed whale Tursiops truncatus (bottlenose dolphin) and the terrestrial Old World primate, Macaca fascicularis (long-tailed macaque). Immunocytochemical analyses, combined with computerized morphometry revealed that in the visual and auditory systems of the bottlenose dolphin, calretinin and calbindin are the prevalent calcium-binding proteins, whereas parvalbumin is present in very few neurons. The prevalence of calretinin and calbindin-immunoreactive neurons is especially obvious in the auditory system of this species. In both auditory and visual systems of the macaque monkey, the parvalbumin-immunoreactive neurons are present in comparable or higher densities than the calretinin and calbindin-immunoreactive neurons. In some structures of the visual and auditory systems of the macaque monkey, the calretinin- and calbindin-immunoreactive neurons are nearly absent. The prevalence of parvalbumin-immunoreactive over calretinin- and calbindin-immunoreactive neurons is particularly prominent in the visual system of primates. Thus, the dominant sensory systems in both aquatic and terrestrial mammals are enriched in specific phenotypes of calcium-binding protein-immunoreactive neurons.

Serotonin neuronal release from dorsal hippocampus following electrical stimulation of the dorsal and median raphé nuclei in conscious rats.

We have studied 5-hydroxytryptamine (5-HT) release in the hippocampal formation following electrical stimulation of the dorsal and median raphé nuclei in the behaving rat. The primary finding in this study is a decrease in neuronal release of serotonin in the dorsal hippocampal formation following electrical stimulation of either the dorsal or median raphé nucleus in conscious rats. At no time did electrical stimulation of either raphé nucleus result in behavioral, including vigilance state, changes. The amount of 5-HT released was found to be frequency dependent with higher frequencies (20 Hz) producing larger decreases in release of 5-HT. However, the pattern of release differs between the two raphé nuclei. Extracellular levels of 5-HT decrease during stimulation of the dorsal raphé, whereas levels decrease only following cessation of stimulation of the median raphé nucleus. This may relate to the patterns of innervation of the dorsal hippocampal formation by these two midbrain raphé nuclei and also may reflect an inhibition of median raphé cell firing during stimulation of the dorsal raphé. Electrical stimulation of the dorsal raphé in anesthetized animals resulted in an enhanced release of 5-HT. The suppression of 5-HT release in the dorsal hippocampal formation in behaving animals was long-lasting (over 2 h), suggesting that the control mechanisms that regulate 5-HT release operate over a long time-course. This difference in release between non-anesthetized and anesthetized animals may relate to anesthesia blocking long- and/or short-loop serotonin recurrent axonal collaterals negatively feeding back onto 5-HT1A and 5-HT1D somatodendritic autoreceptors on raphé neurons. Further, the anesthetized animal has diminished monoaminergic "gating" influences on the hippocampal formation, whereas the behaving animal is more complex with behavioral (vigilance) states associated with different patterns of gating of information flow through the hippocampal formation.

Effects of prenatal protein malnutrition on hippocampal long-term potentiation in freely moving rats.

It has been demonstrated that prenatal protein malnutrition significantly affects hippocampal plasticity, as measured by long-term potentiation, throughout development. This paper focuses on the hippocampal dentate granule cell population response to two separate paradigms of tetanization of the medial perforant pathway in prenatally protein-malnourished and normally nourished adult male rats. The 100-pulse paradigm consisted of the application of ten 25-ms-duration bursts of 400 Hz stimulation with an interburst interval of 10 s. The 1000-pulse paradigm consisted of the application of five 500-ms bursts of 400 Hz stimulation with an interburst interval of 5 s. No between-group differences were obtained for input/output response measures prior to tetanization. No between-group, nor between-paradigm, differences were obtained in the degree of population EPSP slope enhancement. However, in response to both paradigms, prenatally malnourished animals showed significantly less enhancement of the population spike amplitude (PSA) measure than normally nourished animals. Normally nourished animals showed a significantly greater level of PSA enhancement in response to the 100-pulse paradigm than the 1000-pulse paradigm. Prenatally malnourished animals showed no significant differences in the degree of PSA enhancement between the two paradigms. Results indicate that short duration bursts (< or = 25 ms) are more effective in inducing maximal PSA enhancement in normally nourished rats than longer duration stimulus bursts. The apparent inability of prenatally malnourished rats to transfer enhanced cellular activation (population EPSP slope enhancement) into enhanced cellular discharge (PSA enhancement) suggests that a preferential enhancement of GABAergic inhibitory modulation of granule cell excitability may result from the prenatal dietary insult. Such potentiation of inhibitory activity would significantly lower the probability of granule cell population discharge, resulting in the significantly lower level of PSA enhancement obtained from these animals.

The paired-pulse index: a measure of hippocampal dentate granule cell modulation.

This study was undertaken to assess whether the paired-pulse index (PPI) is an effective measure of the modulation of dentate granule cell excitability during normal development. Paired-pulse stimulations of the perforant path were, therefore, used to construct a PPI for 15-, 30-, and 90-day old, freely moving male rats. Significant age-dependent differences in the PPI were obtained. Fifteen-day old rats showed significantly less inhibition at short interpulse intervals [interpulse interval (IPI): 20 to 30 msec), a lack of facilitation at intermediate IPIs (50 to 150 msec), and significantly less inhibition at longer IPIs (300 to 1,000 msec) than adults.

Studies of dentate granule cell modulation: paired-pulse responses in freely moving rats at three ages.

Dentate granule cell population responses to paired-pulse stimulations applied to the perforant pathway across a range of interpulse intervals (IPI) were examined in freely moving rats at 15, 30, and 90 days of age. The profile of the paired-pulse index (PPI), a measure of the type and degree of modulation of dentate granule cell excitability, was shown to change significantly as a function of age.

Diet-induced alterations in the ontogeny of long-term potentiation.

The ability of prenatally malnourished rats to establish and maintain long-term potentiation (LTP) of the perforant path/dentate granule cell synapse was examined in freely moving rats at 15, 30, and 90 days of age. Measures of the population EPSP slope and population spike amplitude (PSA) were calculated from dentate field potential recordings obtained prior to and at various times following tetanization of the perforant pathway. Significant enhancement of both population EPSP slope and PSA measures was obtained from all animals of both malnourished and well-nourished diet groups at 15 days of age. However, the magnitude of enhancement obtained from 15-day-old prenatally malnourished animals was significantly less than that of age-matched, well-nourished controls. At 30 days of age, PSA measures obtained from approximately 50% of prenatally malnourished 30-day-old rats showed no significant effect of tetanization, while measures obtained from the remaining 50% of these animals did not differ significantly from controls. EPSP slope measures for this age group followed much the same pattern, i.e., malnourished animals showing no significant enhancement of PSA measures exhibited only slight increases in EPSP slope beginning 1 h after tetanization and returned to baseline by 18 h post-tetanization. EPSP slope measures obtained from PSA-enhanced malnourished animals did not differ significantly from controls. At 90 days of age, PSA measures obtained from 50% of malnourished animals declined from pretetanization levels immediately following tetanization. Three hours after tetanization, however, this measure had increased to a level which did not differ significantly from that of the control group. PSA measures obtained from the remaining 50% of 90-day-old malnourished animals showed initial and sustained enhancement which did not differ significantly from those obtained from well-nourished age-matched controls. These results indicate that gestational protein malnutrition significantly affects the magnitude of tetanization-induced enhancement of dentate granule cell response in preweanling rats (15-day-old animals) and significantly alters the time-course and magnitude of potentiation in approximately half of prenatally malnourished animals tested at 30 and 90 days of age. Given the primarily postnatal development of the dentate granule cells, these results may reflect malnutrition-induced delays in the neurogenesis and functional development of granule cells previously reported by our group. Most striking is the fact that significant impairments in LTP establishment were obtained from prenatally malnourished animals at 90 days of age, implying that dietary rehabilitation commencing at birth is an intervention strategy incapable of ameliorating the effects of the gestational insult.

Effects of an every other day rapid kindling procedure in prenatally protein malnourished rats.

Prenatally protein (6/25) rats have been reported to require significantly more stimulations to attain a stage 5 seizure than well-nourished controls (25/25) when using either a traditional or rapid every day, kindling procedure. In the present study, a rapid kindling procedure was utilized where both prenatally malnourished and control rats received every other day perforant path kindling (50 Hz, 10 s train) 12 times a day at 5-min intervals. Using this procedure, stage 5 seizures and a fully state were attained in both nutritional groups at approximately the same rate. It is postulated that it is the every other day component of the present procedure which overcomes seizure-induced inhibition in the 6/25 subjects, thereby allowing them to attain stage 5 seizures at the same rate as controls.

Distribution of dopaminergic fibers and neurons in visual and auditory cortices of the harbor porpoise and pilot whale.

The distribution of putative dopaminergic fibers in two sensory cortical areas in the brain of the harbor porpoise (Phocoena phocoena) and pilot whale (Globicephala melaena) was analyzed at the light and electron microscopic levels using tyrosine hydroxylase (TH) immunohistochemistry. The quantitative analysis of the distribution of labeled fibers demonstrates that the primary visual cortex located in the lateral gyrus and entolateral sulcus contains a denser dopaminergic innervation than the auditory cortex within the posterior portion of the presylvian gyrus. In both areas, TH-immunoreactive fibers are densest in layer I, while layers IIIab and VI have intermediate densities and layers II and IIIc-V have the lowest fiber counts. Layer I is characterized by the presence of very thick TH-immunoreactive fiber populations, in addition to the thin and varicose fiber plexus observed throughout the cortical layers. Electron microscopic analyses demonstrated that some of these thick fibers represent the dendrites of TH-immunoreactive neurons located in the deep portion of layer I. The patterns observed in the present study suggest that the dopaminergic projections to the neocortex in whales have a different organization than in terrestrial mammals, particularly rodents and primates. These differences may reflect the fact that during evolution, the cetacean neocortex has retained many of the cytoarchitectonic features that are usually observed only in proisocortical regions in progressive terrestrial mammals.

Quantitative analysis of long-term potentiation in the hippocampal dentate gyrus of the freely-moving 15-day-old rat.

The magnitude and duration of long-term potentiation (LTP) of perforant path/dentate granule cell synapses was examined in freely moving rats beginning at 15 days of age. Measures of dentate granule cell population EPSP slope and population spike amplitude (PSA) obtained before and after tetanization were used to evaluate the level of LTP. Tetanization resulted in significant enhancement of both the population EPSP slope (approximately +75%) and PSA (approximately +40%) measures. This enhancement was maintained without significant change for 18 h, after which both measures began a steady and continuous rise. Daily input/output response measures from age-matched nontetanized animals were used to factor out enhancement related to normal development. Under this schema, tetanization-induced enhancement of both EPSP slope and PSA measures decayed slowly, beginning 18-24 h after tetanization, returning to baseline 5 days after tetanization. Enhancement obtained from 90-day-old animals decayed to baseline 24 h after tetanization. The longer duration of LTP obtained from preweanlings is discussed with regard to the development of inhibitory systems modulating granule cell excitability.

Maturation of long-term potentiation in the hippocampal dentate gyrus of the freely moving rat.

The ability of the perforant path/dentate granule cell synapse of the hippocampal formation to establish and maintain enhanced levels of synaptic transmission in response to tetanization (long-term potentiation, LTP) was investigated in freely moving rats at 15, 30, and 90 days of age. Measures of 1) the slope of the population excitatory postsynaptic potential (EPSP), and 2) the population spike amplitude (PSA) obtained before, and at several times following tetanization, were used to evaluate the magnitude and duration of LTP as a function of age. Significant enhancement of both EPSP slope and PSA measures was obtained from animals of all three ages in response to perforant path tetanization. The initial degree of enhancement was essentially the same across the age groups, ranging from +27% to +38% of pretetanization levels for EPSP slope measures and +60% to +75% of pretetanization levels for PSA measures, obtained 15 min after tetanization. The duration of this enhancement obtained from animals of the preweaning group was significantly longer than that obtained from either 30- or 90-day-old animals. Enhanced measures of both EPSP slope and PSA decayed to baseline levels in these older animals 18 to 24 h after tetanization, while animals tetanized at 15 days of age maintained potentiated levels of both measures for a period of 5 days following tetanization. Tetanization of 15-day-old animals resulted in a significant reduction in the latency to EPSP onset without affecting the time-based relationships among the other measured parameters, which included latency of the population spike onset, population spike minimum, and population spike offset.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium-binding protein-containing neuronal populations in mammalian visual cortex: a comparative study in whales, insectivores, bats, rodents, and primates.

This study is focused on comparative analysis of gamma-aminobutyric acid-positive (GABAergic) neuronal populations in primary visual cortex of totally aquatic toothed whales and select terrestrial mammals with different evolutionary histories and various ecological adaptations. The distribution of neuronal populations containing the calcium-binding proteins calbindin and parvalbumin, which are recognized markers for the GABAergic neurons in cerebral cortex, is compared in five species of toothed whales and in representatives (one species each) of insectivores, bats, rodents, and primates. Computerized image analysis has shown that overall quantitative characteristics of GABAergic cortical neurons in toothed whales are similar to those in other mammalian orders. Thus, GABA-positive neurons represent 26% of the total population of cortical neurons in the visual cortex of whales. Some 97% of GABA-positive cells contain calcium-binding proteins, which is numerically similar to these parameters found in primates and other mammals. On the other hand, the typology and laminar distribution of calcium-binding protein-containing neurons in the primary visual cortex of five whale species (Delphinapterus leucas, Globicephala melaena, Phocoena phocoena, Stenella coeruleoalba, and Tursiops truncatus) differ significantly from those of primates (Macaca mulatta) and rodents (Rattus rattus) and are similar to those found in insectivorous bats (Eptesicus fuscus) and hedgehogs (Erinaceus europaeus). In whales, bats, and hedgehogs a significant concentration of calbindin-positive, vertically oriented bipolar and bitufted neurons was found in layers I, II, and IIIc/V with their axons arranged in a three-dimensional network. In primates and rodents they are distributed evenly across all cortical layers and are predominantly multipolar or bitufted neurons found in all cortical layers with their axons oriented along the vertical axis of the cortical plate. The parvalbumin-positive neurons in all mammalian species, including toothed whales, are represented by variously sized multipolar non-pyramidal cells. As opposed to all other mammalian species, the major concentrations of parvalbumin-positive neurons in whales are found in layers IIIc/V and VI, whereas in other cortical layers there are only scattered parvalbumin-positive neurons.(ABSTRACT TRUNCATED AT 400 WORDS)

Prenatal malnutrition and development of the brain.

In this review, we have summarized various aspects as to how prenatal protein malnutrition affects development of the brain and have attempted to integrate several broad principles, concepts, and trends in this field in relation to our findings and other studies of malnutrition insults. Nutrition is probably the single greatest environmental influence both on the fetus and neonate, and plays a necessary role in the maturation and functional development of the central nervous system. Prenatal protein malnutrition adversely affects the developing brain in numerous ways, depending largely on its timing in relation to various developmental events in the brain and, to a lesser extent, on the type and severity of the deprivation. Many of the effects of prenatal malnutrition are permanent, though some degree of amelioration may be produced by exposure to stimulating and enriched environments. Malnutrition exerts its effects during development, not only during the so-called brain growth spurt period, but also during early organizational processes such as neurogenesis, cell migration, and differentiation. Malnutrition results in a variety of minimal brain dysfunction-type syndromes and ultimately affects attentional processes and interactions of the organism with the environment, in particular producing functional isolation from the environment, often leading to various types of learning disabilities. In malnutrition insult, we are dealing with a distributed, not focal, brain pathology and various developmental failures. Quantitative assessments show distorted relations between neurons and glia, poor formation of neuronal circuits and alterations of normal regressive events, including cell death and axonal and dendritic pruning, resulting in modified patterns of brain organization. Malnutrition insult results in deviations in normal age-related sequences of brain maturation, particularly affecting coordinated development of various cell types and, ultimately, affecting the formation of neuronal circuits and the commencing of activity of neurotransmitter cell types and, ultimately, affecting the formation of neuronal circuits and the commencing of activity of neurotransmitter systems. It is obvious that such diffuse type "lesions" can be adequately assessed only by interdisciplinary studies across a broad range of approaches, including morphological, biochemical, neurophysiological, and behavioral analyses.