Neurotensin receptor levels as a function of brain aging and cognitive performance in the Morris water maze task in the rat.

The present study evaluated whether neurotensin (NT) binding sites were altered in the aged rat brain and if these alterations were related to the cognitive status of the animal. Aged (24-25 months old) Long-Evans rats were behaviorally screened using the Morris water maze task and were classified as either aged, cognitively impaired (AI) or cognitively unimpaired (AU) based on their relative performances in the task compared to young control (Y) animals. Decreases in specific [125I]NT binding were observed in the hippocampal formation, namely the dentate gyrus (DG), as well as in the septum and hypothalamus. Both aged groups also showed significant reductions in specific [125I]NT binding levels compared to the Y animals in the hippocampal CA3 sub-field, with the AI animals exhibiting the lowest levels. In the Substantia Nigra Zona Compacta (SNc) and the ventral tegmental area (VTA), specific [125I]NT binding was decreased as a function of age while binding in the paraventricular nucleus of the hypothalamus (PVNh) was decreased as a function of age and cognitive status. These alterations in the level of specific [125I] NT binding in the aged animals suggest decreases in NT receptor signaling as a function of age and potential involvement of NT-ergic systems in the etiology of age-related cognitive deficits.

Long-term effects of BIBN-99, a selective muscarinic M2 receptor antagonist, on improving spatial memory performance in aged cognitively impaired rats.

Aged Long-Evans rats were screened for spatial memory deficits using the Morris water maze task. Rats found to have impaired performance on the task (aged-impaired, AI) were then treated with a selective muscarinic M2 receptor antagonist, 5,11-dihydro-8-chloro-11-[[4-[3-[(2,2-dimethyl-1-oxopentyl)ethylamino]propyl]-1-piperidinyl]acetyl]-6H-pyrido[2,3-b][1,4]benzodiazepin-6-one (BIBN-99; 0.5 mg/kg, s.c.), for 3 successive days while receiving additional water maze training. BIBN-99 significantly improved performance in the task during the 3 days of drug treatment. Treatment was then ceased for the remainder of the study and rats were tested again in the water maze on days 10, 17, and 24. Compared to vehicle-treated rats, enhanced performance was observed in the AI rats that had previously been treated with BIBN-99. These results indicate that BIBN-99 enhances spatial learning in AI animals and that enhanced (or long-term) memory persists in the absence of the drug. In a second experiment, a 2-month delay was imposed in between the original water maze screening and the drug treatment regime. Again, BIBN-99 significantly improved performance in AI rats. This latter study suggests that reference memory does not decay, even in an AI animal that had displayed poor learning following original water maze screening. Together, these studies help provide further insight into possible mechanism(s) of reference memory and its potential clinical usefulness.

Effects of hippocampal lesions on patterned motor learning in the rat.

Motor skill learning in rats has been linked to cerebellar function as well as to cortical and striatal influences. The present study evaluated the contribution of the hippocampus to motor learning. Adult male rats received electrolytic lesions designed to selectively destroy the hippocampus; a sham-lesioned group of animals served as a control. The animals with hippocampal lesions acquired a patterned motor learning task as well as sham controls. In contrast, rats with hippocampal lesions were impaired in spatial, but not cued, learning in the Morris water maze. In addition, lesioned rats showed profound impairment in the novel object recognition memory task, when a 1-h delay was used between training and testing. Taken together, these results suggest that the hippocampus is not necessary during acquisition of the motor learning task.

A phase I/II evaluation of oral L-2-oxothiazolidine-4-carboxylic acid in asymptomatic patients infected with human immunodeficiency virus.

A randomized double-blind, placebo-controlled study was conducted in 37 asymptomatic HIV-infected individuals (mean CD4 count 707 cells/mm3) to characterize the safety, pharmacokinetics, and effect on blood thiols of three dosage levels of a cysteine prodrug, L-2-oxothiazolidine-4-carboxylic acid (OTC; Procysteine; Clintec Technologies, Deerfield, IL). Single-dose administration of OTC resulted in measurable plasma levels at all dosages, with a mean peak plasma concentration of 734 +/- 234 nmol/mL at the highest dosage studied. After 4 weeks of administration three times daily, a statistically significant increase was seen in whole blood glutathione in the 1,500 mg and 3,000 mg dose groups compared with the placebo group. A significant increase in whole blood cysteine and peripheral blood mononuclear cell (PBMC) glutathione was not seen during the study period.

Reactivity to novelty in cognitively-impaired and cognitively-unimpaired aged rats and young rats.

Two distinct populations of aged, Long-Evans rats can be identified on the basis of performance in the Morris water maze task. Aged (24 month) unimpaired rats perform similarly to young (six month) animals. Aged, impaired rats display latencies to find the submerged platform greater than two standard deviations from the mean of the young animals. A hallmark of efficient cognitive processing is the ability to cope with environmental change. Consequently, the present studies were conducted to assess if aged, impaired animals display differential reactivity to repeated exposure to novel stimuli. Reactivity was assessed by examining the degree of (i) consumption of a novel gustatory/olfactory stimulus (sweetened milk), (ii) pain inhibition induced by exposure to a novel hot-plate (48.5 degrees C) apparatus and (iii) exploratory behaviour in an elevated plus maze and a novel open field. Aged, impaired rats exhibited lower milk consumption on day one and protracted reactivity (lower consumption over days two to eight) in comparison to aged, unimpaired and young animals. Aged, impaired rats were more reactive to novelty on the hot plate test (as indicated by longer paw lick latencies); this novelty-induced pain inhibition did not habituate in aged, impaired rats following repeated plate exposures. The degree of exploratory behaviour in both the plus maze and the open field was reduced in aged, impaired rats. This effect was not entirely a consequence of deficient affective mechanisms, as measures of anxiety (e.g., time in open arms, time in inner squares) were not different among aged impaired, aged unimpaired and young animals. These results are the first to demonstrate that behavioural deficits observed in aged, impaired animals extend beyond the impairments observed in the water maze. This behavioural profile is attributed, in part, to heightened anxiety. In addition, the impairments observed in aged, impaired animals may also reflect a reduced sensitivity to the positive incentive properties of novel stimuli.

Central administration of the neurotensin receptor antagonist, SR48692, modulates diurnal and stress-related hypothalamic-pituitary-adrenal activity.

Previous studies in our laboratory suggest that neurotensin (NT) acts centrally to modulate adrenocorticotropin hormone (ACTH) and corticosterone release. In the present studies, we examined hypothalamic-pituitary-adrenal (HPA) function under basal conditions and during restraint stress following central administration of the highly specific NT receptor antagonist, SR48692. Chronic delivery of SR48692 to the paraventricular nucleus (PVN) of the hypothalamus via indwelling central cannulae attenuated both the diurnal- and stress-induced elevations in HPA activity. Thus, SR48692 decreased the diurnal increase in plasma ACTH and corticosterone during the evening phase of the cycle, but did not affect morning levels. Restraint-induced increases in plasma ACTH and corticosterone levels were also significantly reduced in the SR48692-implanted animals. This suggests that the inhibitory effects of SR48692 were restricted to periods of stimulated HPA activity. A decrease in corticotropin-releasing hormone (CRH)-like immunoreactivity was observed within the PVN following chronic SR48692, and parallel decreases in CRH-like immunoreactivity were observed within the external zone of the median eminence. These findings suggest that endogenous NT serves to increase HPA activity during periods of enhanced stimulation.

Endogenous neurotensin regulates hypothalamic-pituitary-adrenal axis activity and peptidergic neurons in the rat hypothalamic paraventricular nucleus.

Adrenocorticotropin (ACTH) secretion depends primarily on hypophysiotrophic factors released from neurons of the paraventricular nucleus of the hypothalamus. However, the neurochemical factors controlling these neurons, in particular neuropeptides, have had little investigation. In this study, we have investigated the role of neurotensin in the regulation of the different components of the hypothalamo-pituitary-adrenal (HPA) axis under basal and stress conditions in rats. For this purpose, animals were implanted with bilateral cannulae filled with crystals of the neurotensin antagonist, SR 48692, and which were located above the paraventricular nucleus. Five days after surgery, the effects of SR 48692 implants were studied on basal and stress-induced secretion of ACTH and corticosterone. Such treatment did not modify plasma levels of ACTH and corticosterone in basal conditions but reduced ACTH but not corticosterone levels after tail cut procedure. After an exposure to a novel environment for 30 min, both ACTH and corticosterone plasma levels were reduced in the SR 48692-treated group. In situ hybridization studies revealed that chronic administration of SR 48692 induced a significant reduction of CRF mRNA levels in the parvocellular division of the paraventricular nucleus of the hypothalamus. In addition, a 2-fold increase in basal levels of plasma vasopressin associated with an increase in vasopressin mRNA levels in the magnocellular neurons of the paraventricular nucleus was also detected. Finally, the basal plasma levels of oxytocin were not affected by the same treatment. Taken together, these findings strongly suggest that endogenous neurotensin in the paraventricular nucleus plays a tonic stimulatory role on HPA axis activity and an inhibitory effect on vasopressin secretion.

Neonatal handling alters serotonin (5-HT) turnover and 5-HT2 receptor binding in selected brain regions: relationship to the handling effect on glucocorticoid receptor expression.

Neonatal handling permanently alters hypothalamic-pituitary-adrenal responses to stress. This effect is, in part, mediated by a handling-induced increase in forebrain glucocorticoid receptor gene expression. The effect of postnatal handling on glucocorticoid receptor expression appears to be mediated by an increase in serotonin (5-HT) activity, acting via a 5-HT2 receptor with a high affinity for 5-HT (i.e. the 5-HT2H receptor). In the present study we examined the nature of the effects of handling on the relevant 5-HT systems. We found that: (1) handling increases 5-HT turnover in regions of the neonatal rat brain where glucocorticoid receptor expression is altered (i.e. the hippocampus and frontal cortex), but not in regions where glucocorticoid receptor expression in unaffected (e.g. hypothalamus and amygdala); (2) handling has no long-term effects on hippocampal or frontal cortex 5-HT turnover, and is actually associated with a decrease in 5-HT concentrations; and (3) handling does not alter 5-HT2 receptor density in the hippocampus or frontal cortex in neonates (although there are surprising effects on 5-HT2 receptor density in the frontal cortex of adult animals). Taken together these data provide further evidence for the importance of 5-HT in mediating the effects of handling on the development of glucocorticoid receptor expression, but suggest that the role of 5-HT is unique to early development; differences in glucocorticoid receptor expression in adult handled and non-handled animals are not associated with long-term differences in either 5-HT levels or 5-HT2 receptors.

Toxicity evaluations of L-cysteine and Procysteine, a cysteine prodrug, given once intravenously to neonatal rats.

Decreased enzymatic production of cysteine in premature and newborn infants may limit the synthesis of glutathione. Unfortunately, cysteine supplementation is limited by associated toxicity and product instability. Procysteine (L-2-oxothiazolidine-4-carboxylate) is a prodrug of cysteine that is inert until metabolized to cysteine intracellularly, thus stimulating glutathione synthesis. The potential toxicities of cysteine and Procysteine were compared in two studies with neonatal rats (10 per group; 3 +/- 1 days of age) after a single intravenous administration. In one study, acute high dosage survivorship was compared for approximately equimolar cysteine dosages of L-cysteine and Procysteine. Mortality at 7 days after single intravenous dosages of L-cysteine at 1.52 or 1.14 g/kg or Procysteine at 1.80 or 1.35 g/kg was 80, 50, 10 and 0%, respectively. Clinical pathology parameters and body and organ weights were compared in a second study, following a moderate dosage of Procysteine or equimolar or lower dosages of L-cysteine. No differences were observed in clinical pathology parameters nor body or organ weights at 14 days following single intravenous dosages of L-cysteine at 369, 185 or 37 mg/kg or Procysteine at 450 mg/kg. Also, Procysteine solutions were considerably more stable than L-cysteine solutions (months vs. hours, respectively). These studies indicated that cysteine supplementation in infants may be enhanced by Procysteine administration.

Medium-chain-triglyceride lipid emulsion: metabolism and tissue distribution.

The utilization and distribution of radioactively labeled lipid emulsions were evaluated in Sprague-Dawley rats. Animals received one of three lipid emulsions. Group 1 received [14C]medium-chain-triglyceride (MCT) lipid emulsion, group 2 received a 75%:25% (vol:vol) admixture of [14C]MCT: unlabeled long-chain-triglyceride (LCT) lipid emulsion, and group 3 received only [14C]LCT. The radioactive dose appearing in expired carbon dioxide and various body tissues was monitored over a 24-h period. Results indicate that MCT is oxidized more rapidly and completely than in LCT; approximately 90% of the MCT is converted to carbon dioxide with in 24 h compared with 45% for LCT. When MCT and LCT are administered together, the metabolism of MCT is slowed but remains more rapid than that of LCT. Removal of MCT from the blood was more rapid than was removal of LCT, and tissue radioactivity was lower.

Competitive effects of long-chain-triglyceride emulsion on the metabolism of medium-chain-triglyceride emulsions.

This study was conducted to assess the potential metabolic competitive interactions of intravenous medium-chain-triglyceride (MCT) and long-chain-triglyceride (LCT) lipid emulsions. To assess this competition increasing concentrations of LCT emulsion were added to an intravenous dose of MCT emulsion of 3.0 g/kg body wt up to a maximum dose of 3.0 g LCTs/kg body wt. Blood samples were assessed for competitive interactions by analyzing the following metabolites: glucose, insulin, lactate, pyruvate, ketones (acetoacetate, beta-hydroxybutyrate), elimination of triglycerides, and free fatty acids. Evaluation of the data showed a strong competitive interaction between the MCT and LCT emulsions. This competition was evident as soon as LCTs were added to the MCT infusions and appeared to favor LCTs for removal and metabolism over MCTs. This appears to indicate that there is a peripheral, strong affinity site for LCT removal and metabolism and a shared peripheral site and specific visceral site for MCT removal and metabolism.

Fatty acid profiles in response to soybean oil lipid emulsion infusions in essential fatty acid-deficient miniature swine.

The ability of soybean oil lipid emulsions to affect essential fatty acid deficiency (EFAD) and plasma fatty acid distribution was studied in neonatal pigs. The test animals were maintained on a fat-free diet prior to administration of lipid emulsion. Plasma and red blood cell (RBC) membrane levels of essential [linoleic (C-18:2 omega 6) and arachidonic (C-20:4 omega 6)] and nonessential [palmitic (C-16, palmitoleic (C-16:1 omega 7), stearic (C-18), and oleic (C-18:1 omega 9)] fatty acids and the triene:tetraene ratio [5,8,11-eicosatrienoic acid (C-20:3 omega 9):arachidonic acid (C-20:4 omega 6)] were monitored to ascertain the establishment of EFAD and its correction. Nonessential fatty acids were studied, as these components of lipid therapy have received little attention. Results indicate that soybean oil emulsions are effective in reversing fatty acid profiles found in EFAD, and both essential and nonessential fatty acids are under strict metabolic control.

A metabolic comparison of a pure long-chain triglyceride lipid emulsion (LCT) and various medium-chain triglyceride (MCT)-LCT combination emulsions in dogs.

Two 20% lipid emulsions containing mixtures of long-(LCT) and medium-chain triglycerides (MCT) were compared with a 20% LCT lipid emulsion. Beagles were infused with emulsions containing either 100% LCT, 75% LCT-25% MCT, or 50% LCT-50% MCT. The emulsions were part of a total parenteral nutrition (TPN) regimen that included 10% dextrose and 5.5% amino acids. Basic nutritional parameters as well as elimination kinetics were monitored. Plasma linoleic acid, ketone, lactate, pyruvate, insulin, glucose, and carnitine were analyzed. The 75% LCT-25% MCT emulsion offers little advantage over 100% LCT as a metabolic substrate. The 50% LCT-50% MCT combination proved to be a potentially better caloric source due to rapid elimination kinetics, increased ketone production, lack of deposition, and no interference with linoleic acid metabolism.

A bioengineering analysis of human muscle and joint forces in the lower limbs during running.

A two-dimensional, dynamic bioengineering model of the lower limbs was developed in order to estimate muscle and joint forces present during running at 4.5 m s-1. Data were collected from four subjects using a force platform and cine film. Individual X-rays and anthropometric data from the lower limbs were utilized to produce accurate bone models of the subjects' legs. Electromyographic verification of the model was undertaken while a runner was undergoing treadmill running at 4.5 m s-1. Results indicate that peak muscle forces of 22 times subject body weight (22 BW) could be present in the quadriceps muscle group and 7 BW in the gastrocnemius. The anterior shin muscles were found to be active for the first 9% of stance phase only, and compressive loads of 33 BW were found in the knee joint. The relationship between these high forces in the lower limbs and running related injuries is discussed.

Comparison of the elimination of 10 and 20% TRAVAMULSION lipid emulsion from the blood of beagle dogs.

Metabolic utilization of fat emulsions containing 20% lipid and 10% lipid were compared using beagles. The key parameter measured was elimination of the lipid from the bloodstream, which serves as an indication of the emulsion's availability for metabolism. Nonlinear kinetic analysis was used in this determination. Blood concentrations of free fatty acids, phospholipid, and cholesterol were also measured as additional ways of determining emulsion metabolism. The 10 and 20% emulsions appeared to be equivalent in elimination of the caloric substrate triglyceride from the blood stream. Results also showed an adaptation to emulsion infusion over time at both dosages administered (3 and 6 g/kg of body weight). This was indicated by increased elimination capacity and stabilization of each lipid class measured. However, blood concentrations of phospholipid and cholesterol indicate that the 20% emulsion provides a lesser lipid load for the amount of calories administered when compared to an emulsion containing 10% lipid.

Comparison of the elimination and metabolism of 10% Travamulsion and 10% Intralipid lipid emulsion in the dog.

A study was performed comparing the elimination kinetics of two soybean oil/egg phosphatide lipid emulsions (10% Travamulsion vs 10% Intralipid) from the vascular compartment of the dog. Elimination kinetics were evaluated after bolus injection and after continuous infusion studies. Evaluation of triglyceride and free fatty acid data indicates the emulsions are similar metabolic substrates. Phospholipid and cholesterol data indicate a possible difference in remnant particle removal. At the higher lipid dosages, remnant particles from Travamulsion injection were removed at a faster rate than those from Intralipid.

Nonlinear kinetic analysis of the elimination of lipid emulsion administered intravenously to dogs.

A model based on a Michaelis-Menten nonlinear kinetic approach was used to analyze the kinetics of elimination of an intravenously infused lipid emulsion in the dog. The emulsion was administered at doses of 0.3, 3, or 6 g of lipid/kg of body weight. The model was applicable to all data regardless of the kinetic order of the elimination process operating. This can be either zero (linear), mixed, or first order (exponential). Furthermore, it was also found that parameters derived with the model can be used to predict the kinetics of elimination at different dosages. However, it was also shown that elimination parameters for a single bolus dose of lipid emulsion cannot be used to predict the manner in which a continuously infused dose will be eliminated.