Developmental sex differences in amino acid neurotransmitter levels in hypothalamic and limbic areas of rat brain

GABA, glutamate and aspartate are the predominant amino acid neurotransmitters in the mammalian brain. We have previously reported a developmental sex difference in messenger RNA levels of glutamate decarboxylase, the rate-limiting enzyme in GABA synthesis [Davis A. M. et al. (1996) Horm. Behav. 30, 538–552]. Males were found to have significantly higher levels of messenger RNA in many steroid-concentrating regions of the hypothalamus and limbic system on day 1 of life. Therefore, in this study, we have examined levels of amino acid neurotransmitters during early postnatal development in many of the same or related brain areas. We found that levels of all three transmitters change as animals age. While both GABA and aspartate concentrations increase, glutamate levels decrease. In addition, there are sex differences in neurotransmitter levels in several areas examined, including the ventromedial and arcuate nuclei of the hypothalamus, and the CA1 region of the hippocampus. Sex differences for GABA occur only on postnatal days 1 and 5. However, sex differences in aspartate occur later in development (postnatal day 20). The CA1 region of males has a significantly greater concentration of GABA, glutamate and aspartate than females on postnatal day 1. In addition, treatment of females with testosterone propionate on the day of birth results in increased GABA levels, suggesting that these sex differences may be the result of hormone exposure during development.

We hypothesize that these hormonally mediated sex differences in amino acid transmitters early in development contribute to the establishment of sexually dimorphic neuronal architecture in the adult.     

Stimulated amino acid imbalance and histidine transport in rat brain slices.

Histidine concentration in the brain decreases rapidly when rats are fed a low protein diet in which an amino acid imbalance is created by addition of an amino acid mixture devoid of histidine. Competition for histidine transport into the brain was suggested as an explanation for this effect. Therefore, animo acid mixtures simulating composition of plasma from rats fed basal or histidine-imbalanced diets were added to media to evaluate their effects on uptake of histidine by brain slices during a 60-min incubation period. At the concentrations actually found in plasma, the unbalanced mixture decreased histidine uptake significantly more than did the basal mixture. Two distinct inhibition patterns were observed with different groups of amino acids: a linear decrease in histidine uptake with a mixture of the small neutral, hydroxyl, basic, and acidic amino acids, and a hyperbolic decrease with a mixture of large neutral amino acids, and a hyperbolic decrease with a mixture of large neutral amino acids. Inhibition of histidine transport by the complete mixtures reflected these two effects. Plasma patterns and concentrations of competitive amino acids as well as the concentration of histidine appear to be factors involved in decreasing histidine transport into the brain.     

Basolateral and central amygdaloid lesions leave aversion to dietary amino acid imbalance intact

Expression of c-fos is increased in the central amygdaloid nucleus (CE) of rats ingesting a diet with a severely imbalanced essential amino acid profile (IMB), at a time associated with development of a conditioned taste aversion (CTA). The CE and the basolateral amygdaloid nucleus (BL) both are reported to be involved in the development of CTA. Large amygdaloid lesions involving CE and BL mitigate the normal decrease in intake of IMB; this treatment also impairs CTA to a flavor cue associated with gastrointestinal discomfort. To differentiate their potential roles in aversive responses to IMB, we electrolytically lesioned CE and BL separately. Neither lesion attenuated IMB-induced anorexia, or prevented the avoidance of flavored solutions previously paired with IMB. In contrast, after saccharin-LiCl pairing, CE-lesioned animals showed attenuated CTA to saccharin solution in a two-bottle test. We conclude that neither the CE nor the BL is essential for the reduction of IMB intake, or for CTA associated with IMB. Furthermore, these results suggest that the aversive consequences of IMB intake do not involve gastrointestinal malaise-evoked neurotransmission involving the CE.     

Effect of amino acid imbalance and deficiency on dietary choice patterns of rats

Detailed dietary choice patterns were determined with a computerized feeding monitoring system in groups of Sprague Dawley rats kept on a 12:12 hr light-dark cycle and offered in sequence a series of dietary choice regimens involving amino acid-imbalanced or deficient diets with threonine as the most limiting amino acid. Animals established their preference for a threonine-basal diet over a threonine-imbalanced or a threonine-devoid (devoid of threonine) diet shortly (within 2-3 hr) after the consumption of small quantities of either diet in the beginning of the first dark-cycle. An intensive sampling process characterized by frequent small bouts was evident throughout the light period. Both the meal size and the meal frequency of the imbalanced or devoid diet were curtailed after prolonged choices. Animals preferred the threonine-corrected (imbalance corrected by threonine supplementation) over the threonine-basal diet initially with an increase in meal frequency. But no clear choice for either diet was observed thereafter. Animals did not establish their preference for the threonine-corrected diet when paired with the threonine-devoid diet until after 5 days with a steady decrease in the meal size of the devoid diet but not the meal frequency. When the protein-free diet was introduced as an alternative for the threonine-imbalanced diet, animals selected the protein-free diet during the first dark-cycle after consuming a small amount of the imbalanced diet. Initially there was a drastic reduction in meal size of the imbalanced diet and subsequently a decrease in meal frequency as well. Nevertheless, animals immediately rejected the protein-free diet and chose the threonine-basal diet when it replaced the imbalanced diet as an alternative. The almost exclusive preference for the basal diet occurred in the beginning of the first dark-cycle with an increase in meal size but no change in meal frequency. The sampling bouts of small quantities, which followed the first introduction of the diets in the choice regimens, may be an inherent investigative behavior whereby the physical or oropharyngeal properties of the diets are recognized. The establishment of the choices for the alternative diets in the present experiments provides additional information about the rapid time course of the food intake control mechanisms in rats fed amino acid-imbalanced or deficient diets.     

Dietary regulation of liver and muscle transport of amino acid.

Distribution ratios (intracellular concn/extracellular concn) of alpha-amino isobutyric acid (AIB) after intravenous injection were determined in fed, 12-h, 1-day, and 5-day starved rats. Progressive increases (over fourfold) in the distribution ratios of AIB in the liver and progressive decreases (over threefold) in the gastrocnemius muscle occurred within these periods. On full day of protein deprivation was without effect on AIB distribution ratios, but after 5 days it produced an increased distribution ratio of AIB in the liver (twofold), without affecting that of the muscle. A sudden increase in hepatic glucose output, induced by phlorizin, was followed by an increase in the liver distribution ratio of AIB. In starvation the increase in plasma concentration of glucagon and decrease in insulin level preceded the changes in AIB distribution ratios; in protein deprivation there was no change in plasma concentrations of these hormones. It is concluded that caloric restriction profoundly affects amino acid transport by the liver and by the skeletal muscle. These transport changes would enhance the availability of substrates for increased gluconeogenesis during starvation.     

Neuronal and neurochemical mechanisms of hypothalamic inhibition of the nociceptive reflex

Using cats anesthetized with chloralose-Nembutal we have studied the effect of high frequency stimulation of the medial and lateral structures of the posterior, tuberal, and anterior hypothalamus, and also of the central gray matter of the midbrain on the nociceptive jaw-opening reflex induced by tooth pulp stimulation. We recorded the EMG response of the digastric muscle as the index of the nociceptive reflex. We have shown that the EMG response of the nociceptive reflex is effectively suppressed on stimulation of all hypothalamic structures and the central gray matter, the threshold of the suppressive action being lower on stimulation of the central gray matter. The effects of the hypothalamic supression of the nociceptive reflex were eliminated almost completely after naloxone administration, with the exception of the central gray matter, which is slightly more resistant to the action of this agent. After bilateral electrolytic destruction of the central gray matter the antinociceptive effect of the hypothalamus was retained, decreasing only insignificantly, The effect of complete supression of the amplitude of the EMG response, similar to the effect of stimulation of the hypothalamus and the central gray matter, was also observed after intravenous administration of phenapidine, an opiate agonist with a marked central analgesic action. The neuronal and neurochemical mechanisms of hypothalamic suppression of the nociceptive jaw-opening reflex are discussed.Translated from Fiziologicheskii Zhurnal SSSR imeni I. M. Sechenova, Vol. 73, No. 9, pp. 1160–1169, September, 1987.     

Dietary self-selection of protein-unbalanced diets supplemented with three essential amino acids in Nile tilapia

Animals do not eat whatever food item they encounter, but choose different foods that best match their requirements. Fish exhibit such "nutritional wisdom" and adapt their feeding behaviour and food intake according to their needs and the nutritional properties of diets. In this paper, we tested the ability of Nile tilapia to select between diets with a balanced or unbalanced composition of essential amino acids. To this end, three different diets were prepared: a gelatine based diet (D(1)), a gelatine diet supplemented with three essential amino acids (EAA, l-tryptophane, l-methionine, l-threonine) (D(2)), and a diet containing only cellulose and the three crystalline EAA (D(3)). In addition, the putative role of both orosensorial factors (using pellets vs capsules) and social interactions (single vs groups of ten fish) was investigated. To this end, a total of 68 male tilapia of about 141±48 g (mean±S.D.) were challenged, individually or in groups, to select between D(1)vs D(2) using pellets dispensed by self-feeders (exp. 1). In another experiment (exp. 2), 11 individual fish were challenged to select encapsulated diets with non flavour or smell proprieties (D(1)vs D(2)), and in exp. 3 fish were challenged to self-supplementation in EAA (D(1)vs D(3)). The results showed the ability of tilapia to avoid the EAA-deficient diet, choosing 82.2% D(2) in the case of individual fish, and 80.8% D(2) in the case of fish groups. Dietary selection was not directly driven by the orosensorial characteristics of food, since tilapia sustained a higher preference for D(2) when fed with encapsulated diets. Finally, in exp. 3 tilapia self-supplemented the EAA deficiency by selecting a synchronised combination of D(1) and D(3) that matched their nutritional requirements. These findings highlighted the capacity of fish to make dietary selection based on the EAA content, which should be considered when discussing food intake regulation mechanisms, and diet formulation and supplementation with EAA.     

Amino acid imbalance in cystinuria

After oral ingestion of a free amino acid mixture by three cystinuric patients, plasma increments of lysine and arginine were lower and those of many other amino acids were significantly higher than those found in control subjects.Similar results were obtained in control subjects after amino acid imbalance had been artificially induced by the omission of cystine, lysine, and arginine from the amino acid mixture. Especially high increments of alanine and proline provided the best evidence of amino acid imbalance caused by a temporary lysine and, to a lesser extent, arginine and cystine deficit.No such amino acid imbalance was found to occur in the cystinuric patients after ingestion of whole protein, indicating that absorption of oligopeptides produced by protein digestion provided a balanced physiological serum amino acid increment. This is considered to explain the lack of any unequivocal nutritional deficit in cystinuric patients despite poor absorption of the essential free amino acid, lysine.     

Dietary effects on rhesus social behavior: altered amino acid diets

Twenty-one monkeys continuously fed one of five diets high in tyrosine, histidine, alanine, glycine, or tryptophan between the ages of 1 and 12 months were compared with 20 controls. Social behavior either (a) while on the diet and tested in familiar groups of four or (b) while on a normal diet and paired with unfamiliar monkeys showed no effects of the high amino acid diets, confirming a previous analysis of learning ability.     

Inhibition of norepinephrine release in the rat ventromedial hypothalamic nucleus in essential amino acid deficiency

Effects of dietary amino acid deficiency on interstitial levels of norepinephrine (NE) were assessed in the ventromedial hypothalamic nucleus (VMH). Microdialysates, collected from the VMH, were analyzed using high pressure liquid chromatography with electrochemical detection (HPLC-EC). Ingestion of an amino acid imbalanced diet, which causes a rapid deficiency of the limiting amino acid, induced a significant decrease in the NE concentration from the VMH. The changes in the NE concentration appeared 60 min after diet ingestion and the lowest NE level was observed at 180 min. The present results suggest that ingestion of an amino acid imbalanced diet inhibits NE release in the VMH and support the hypothesis that the VMH plays a role in the integration of signals for the feeding responses to changes in essential amino acid availability.     

Muscle amino acid arylpeptidases and their serum changes with exercise

Summary Activities of serum amino acid arylpeptidases were determined in five groups of healthy male adults at rest and after various exercise types using the following substrates: Ala-, Leu-, Phe-, Tyr-, Gly- and Pro-4-Nitroanilide. The exercise-induced changes were compared to the activities of the respective enzymes in resting leg muscles. A regression function was found, showing a close correlation between the mentioned parameters in all groups. The influence of haemoconcentration and intravascular hemolysis on the postexercise measured activities was of no consequence.The authors express their thanks to G. Haralambie for his encouragement during this work     

Excitatory amino acid antagonists inhibit synaptic responses in the guinea pig hypothalamic paraventricular nucleus.

1. The effects of specific excitatory amino acid (EAA) antagonists on evoked excitatory synaptic responses were studied in the hypothalamic paraventricular nucleus (PVN) of the guinea pig, by the use of the in vitro slice preparation. Intracellular recordings were obtained from paraventricular neurons, and excitatory postsynaptic potentials (EPSPs) and currents (EPSCs) were induced by perifornical electrical stimulation. To reduce the influence of a potential gamma-aminobutyric acidA (GABAA) inhibitory component on the synaptic responses, all experiments were performed in the presence of 50 microM picrotoxin. 2. Of 20 cells tested, 13 had electrophysiological characteristics similar to magnocellular neuropeptidergic cells (MNCs) and 7 displayed low-threshold Ca2+ spikes (LTSs). No difference was detected in the effect of the antagonists on the synaptic responses of cells with or without LTS potentials. 3. The broad-spectrum EAA antagonist kynurenic acid decreased the amplitude of the EPSPs and EPSCs in a dose-dependent manner: the mean decrease was 5% for 100 microM, 43% for 300 microM, and 70% for 1 mM. 4. The quisqualate/kainate-receptor-selective antagonist 6-cyano-2,3-dihydroxy-7-nitroquinoxaline (CNQX) induced a dose-dependent decrease of the EPSPs and EPSCs: 1 microM had no detectable effect, 3 and 10 microM caused 30 and 70% decreases, respectively, and 30 microM blocked the response almost completely. This effect was not accompanied by a change in resting membrane potential or input resistance and was slowly reversible. 5. The N-methyl-D-aspartate (NMDA)-receptor-selective antagonist DL-2-amino-5-phosphonopentanoic acid (AP5), applied at 30 and 300 microM, reduced slightly the amplitude of the decay phase of the EPSP but did not significantly affect the peak amplitude. In some cells, the current-voltage relationship of the decay phase of the EPSC revealed a region of negative slope conductance between -70 and -40 mV. 6. These results suggest that 1) glutamate or a related EAA is responsible for the fast excitatory input to magnocellular and parvocellular neurons in the PVN and probably also for cells around PVN, 2) a quisqualate/kainate receptor type is responsible for the rising phase and peak amplitude of the synaptic current, and 3) an NMDA receptor contributes to the late part of the synaptic response.     

Time-dependent changes in the plasma amino acid concentration in diabetes mellitus

We investigated longitudinal change in the amino acid (AA) profile in type 1 diabetes mellitus (DM) using AKITA mice, which develop DM as a result of insulin deficiency. The plasma concentrations of valine, leucine, isoleucine, as well as the total branched chain amino acids, alanine, citrulline and proline, were significantly higher in the diabetic mice. We show that the degree and timing of the changes were different among the plasma amino acid concentrations (pAAs) during the development of type 1 DM.     

Amino-terminal amino acid sequences of structural proteins of three flaviviruses

N-terminal amino acid sequences of structural proteins of three flaviviruses, yellow fever, St. Louis encephalitis, and dengue-2 viruses, have been obtained. The glycoproteins of these three viruses are 52-60% conserved in the region sequenced, depending upon which pair of viruses are compared, and 40% of the amino acids are invariant in all three viruses. Thus, flaviviruses are closely related and have in all probability descended from a common ancestor. Furthermore, residues important in the secondary structure of proteins are conserved, suggesting that the overall conformation of the glycoproteins is the same in all three viruses while considerable variation in the primary sequence can be accommodated. The N-terminal regions of the nucleocapsid proteins of yellow fever and St. Louis encephalitis viruses show markedly less homology (25%) and this region is highly basic with one-quarter (yellow fever) or one-third (St. Louis encephalitis) of the residues being lysine or arginine. N-terminal sequences for the M protein of yellow fever and for NV2(GP19) of St. Louis encephalitis viruses are also reported.