Morphine-induced effects on the hypothalamic stimulation-evoked emotional/behavioral reactions and peculiarities of abstinent syndrome in adult and old rats

Experiments were carried out on adult young (6- to-9-month-old) and old (28- to-30-month-old) rats. The effects of a single i.p. injection of morphine on self-stimulation of the lateral hypothalamic region and on active avoidance responses evoked by stimulation of the ventromedial hypothalamic nucleus were studied. In a separate series of the experiments we studied age-related specificities of the abolition syndrome after a course of intraventricular injections of morphine. In most old rats single injections of morphine resulted in intensification of self-stimulation, while in most adult rats such injections suppressed this reaction. In old animals injections of morphine resulted in more pronounced increases in the threshold and latency of the active avoidance responses, as compared with similar changes in adult rats. Natural abolition of a 15-day-long course of injections of morphine resulted in the development of a characteristic abstinent syndrome that was more intensively manifested in old rats. Our results show that “hedonic” and anti-aversive effects of morphine increase with aging; this phenomenon probably facilitates the development of morphine dependence in old organisms.     

Effect of morphine on the concentration of monoamines in the emotiogenic zones of the hypothalamus in adult and old rats

The content of monoamines in the emotiogenic hypothalamic zones has been shown to noticeably change with aging of rats. The level of noradrenaline and serotonin increased in the ventromedial nucleus of the hypothalamus, while the concentration of noradrenaline increased in the lateral hypothalamic zone. Single i.p. injections of 10 mg/kg morphine evoked qualitatively different shifts in the monoamine concentrations in the hypothalamic emotiogenic zones of the rats of different ages: the level of dopamine increased in adult animals, while the levels of noradrenaline and serotonin dropped in old rats. It is supposed that in old age the effect of morphine on dopaminergic structures in the emotiogenic hypothalamic zones becomes more moderate, whereas that on the noradrenergic and serotonergic structures is facilitated. The age-related specificities of the morphine effect on the monoaminergic regulation of the emotiogenic hypothalamic zones can determine considerable modifications of a psychotropic effect of the drug in old age.     

Effect of electrostimulation of the hypothalamus on protein biosynthesis in organs of adult and aged rats

The effect of hypothalamus electrical stimulation on total protein biosynthesis was studied in skeletal muscle, heart, liver, adrenal cortex and thyroid gland of adult rats. In adult animals hypothalamus stimulation provokes a pronounced increase in 3H-leucine incorporation into total protein of all tissues, as well as into liver chromatin proteins. No significant changes were observed in protein biosynthesis when hypothalamus of old rats was stimulated. This can serve as evidence of age-related decrease in the ability of the hypothalamus to stimulate protein synthesis in peripheral tissues.     

The Lateral Hypothalamic Area: Peculiarities of Aging and the Effect of Chronic Electrical Stimulation on the Lifespan in Rats

Age-related morphological and functional changes in the lateral hypothalamic area (LHA) were studied in experiments on young adult (6-8 months) and old (26-28 months) male Wistar rats. It was found that during aging the neuronal density in the LHA decreased, and significant qualitative destructive and dystrophic changes in the neuronal population developed. The background impulse activity of LHA neuronal units, the mass background electrical activity recorded from this structure, and the Na⁺, K⁺-ATPase activity decreased during aging. In old rats, the rate of LHA self-stimulation was lower, and the range of reinforcing current amplitudes, which provided self-stimulation intensity close to the maximum, was narrower than in adult animals. Chronic electrical LHA stimulation in old rats ensured an increase in the lifespan and maximum life expectancy in these animals. In addition, the lifespan positively correlated with the duration of LHA stimulation. It is concluded that lowering of the functional activity of the LHA neural systems is one of the substantial aspects of the aging process, and activation of this structure in old animals by its chronic electrical stimulation can exert a geroprotective effect.     

Effect of hypothalamic extracts and synthetic LH-RH on proteic synthesis by rat hypothalamus in vitro (author's transl)]

The incorporation of 14C-Leucine in pituitary proteins in rats, in vitro, has been studied. In absence of stimulation, the pituitaries of adult female rats have shown approximately twice the capacity of protein biosynthesis in vitro than the pituitaries of prepuberal female rats (21 days old). For the stimulation in vitro of the pituitaries, synthetic LH-RH or hypothalamic extracts from adult or prepuberal female rats were used. The pituitaries of adult female rats did not respond to any of the stimulation tests employed. The pituitaries of prepuberal female rats increased their biosynthetic activity significantly, when synthetic LH-RH or adult female rat hypothalamic extract was added to the culture medium. The addition of prepuberal female rat hypothalamic extract did not alter the basic response. The female prepuberal rats injected during 5 consecutive days with FSH and LH, have shown a greater sensibility to LH-RH in vitro than the ones injected with estradiol and progesterone, or with synthetic LH-RH.     

Diurnal rhythmicity of norepinephrine activity associated with the estradiol-stimulated luteinizing hormone surge: effect of age and long-term ovariectomy on hemispheric asymmetry

The age-related decline in female reproductive capacity in rats is accompanied by an inability to respond positively to estradiol (E2) treatment. This age-related change is associated with a loss in diurnal rhythmicity of norepinephrine (NE) activity in brain areas important in the control of LH. Decreased exposure to ovarian secretions during adulthood delays certain aspects of neuroendocrine aging. We tested the hypothesis that long-term ovariectomy (OVX) would delay the age-related loss of diurnal rhythmicity in NE activity in microdissected hypothalamic nuclei. Intrigued by reports of lateralization of hypothalamic function, we also assayed NE activity in the left and right sides of the hypothalamus separately. Young (2-3 mo) and middle-aged (11-12 mo) rats that exhibited regular estrous cycles were OVX. One week later (Day 0) these short-term OVX animals (Y-ST, MA-ST) plus a group of middle-aged (11-12 mo) rats that were OVX at 3 mo (MA-LT) were treated with E2. On Day 4, the rate constant of NE activity in microdissected hypothalamic nuclei was determined at 0900 h and 1500 h using the alpha-methyl-para-tyrosine method. Rate constants were compared by t-test to determine diurnal rhythmicity. Y-ST rats exhibited a diurnal rhythm in NE activity in the median eminence, which was absent in MA-ST rats. Long-term OVX spared animals this "age-related" loss in rhythmicity since MA-LT rats demonstrated a significant increase in NE activity from morning to afternoon.(ABSTRACT TRUNCATED AT 250 WORDS)     

Insights into a role of GH secretagogues in reversing the age-related decline in the GH/IGF-I axis

Growth hormone (GH) secretion and serum insulin-like growth factor-I (IGF-I) decline with aging. This study addresses the role played by the hypothalamic regulators in the aging GH decline and investigates the mechanisms through which growth hormone secretagogues (GHS) activate GH secretion in the aging rats. Two groups of male Wistar rats were studied: young-adult (3 mo) and old (24 mo). Hypothalamic growth hormone-releasing hormone (GHRH) mRNA and immunoreactive (IR) GHRH dramatically decreased (P < 0.01 and P < 0.001) in the old rats, as did median eminence IR-GHRH. Decreases of hypothalamic IR-somatostatin (SS; P < 0.001) and SS mRNA (P < 0.01), and median eminence IR-SS were found in old rats as were GHS receptor and IGF-I mRNA (P < 0.01 and P < 0.05). Hypothalamic IGF-I receptor mRNA and protein were unmodified. Both young and old pituitary cells, cultured alone or cocultured with fetal hypothalamic cells, responded to ghrelin. Only in the presence of fetal hypothalamic cells did ghrelin elevate the age-related decrease of GH secretion to within normal adult range. In old rats, growth hormone-releasing peptide-6 returned the levels of GH and IGF-I secretion and liver IGF-I mRNA, and partially restored the lower pituitary IR-GH and GH mRNA levels to those of young untreated rats. These results suggest that the aging GH decline may result from decreased GHRH function rather than from increased SS action. The reduction of hypothalamic GHS-R gene expression might impair the action of ghrelin on GH release. The role of IGF-I is not altered. The aging GH/IGF-I axis decline could be rejuvenated by GHS treatment.     

Is Hypothalamic GABA Involved in Immune Function in Relation to Dietary Protein During Aging?

Hypothalamic GABAergic activity and immune response in spleen were not significantly changed with the increase of age from 3 to 6 months in adult male albino rats. Further increase of age from 6 to 9 months increased the GABAergic activity and decreased the cell viability in spleen without any change in its T-lymphocyte cytotoxicity. Consumption of low protein diet (LPD) for a short-term period (STP; 7 consecutive days) increased the hypothalamie GABAergic activity without changing the immune response in 3 months old rats. When supplemented for a long-term period (LTP; 30 consecutive days) to 3 months old rats, a reduction of hypothalamie GABAergic activity and the immune response was observed. Intake of high protein diet (HPD) for both STP and LTP increased the GABAergic activity and immune response, but the increase of GABAergic activity in hypothalamus under STP was greater than that observed under LTP. In 6 months old rats consumption of LPD for STP reduced the GABAergic activity without any alteration of its immune response. Long-term supplementation of this LPD to the same age group increased GABAergic activity and the mitotic activity of spleen cells without any alteration of the functional activity of the T-cells in spleen. Consumption of HPD for STP failed to produce any change in hypothalamic GABAergic activity and the immune response of 6 months old rats. Supplementation of HPD for LTP reduced the hypothalamic GABAergic activity and the immune response of the same age group. The reduction in hypothalamic GABAergic activity without any change in the immune response was observed following the supplementation of low protein diet to 9 months old rat for STP. Intake of the LPD for LTP also reduced the hypothalamie GABAergic activity and the mitotic activity of the spleen cells without any alteration of the functional activity of the T-cells in spleen of 9 months old rats. Supplementation of HPD for STP to this aged rat, on the other hand, failed to produced any change in hypothalamic GABAergic activity and the immune response. Intake of HPD for LTP by this aged rats increased the hypothalamie GABAergic activity along with the immune response. The results of this study, thus, suggest that hypothalamic GABAergic activity during aging is an index of immune response and it is modulated following the short- and long-term consumption of protein poor and protein rich diet.     

Hypothalamic luteinizing hormone-releasing hormone (LHRH) and LH secretion in aging female rats

Age-related changes in hypothalamic luteinizing hormone-releasing hormone (LHRH) and luteinizing hormone (LH) secretion were studied in young (6 months), middle-aged (12 months) and old (18 months) female rats. The LHRH levels in the mid-hypothalamic area were higher in intact middle-aged and old females than in young ones. Additionally, there was no age difference in the hypothalamic LHRH levels in male rats. In order to clarify the significance of this age-related increase in female rats, we examined the effects of progesterone treatment in estrogen-primed ovariectomized young and old rats on the LHRH levels in the median eminence (ME) and on plasma LH levels. We found phasic changes in ME-LHRH and plasma LH levels in estrogen-primed rats following progesterone treatment in rats of both ages, but the progesterone-induced change in ME-LHRH levels tended to be delayed in old rats compared with young females. This delay may correspond to the delayed onset, slow and low magnitude of plasma LH increase in old females. The ME-LHRH levels were generally higher in old rats than in young rats. Nevertheless, we found that the increase in plasma LH in response to progesterone treatment in estrogen-primed ovariectomized females was smaller in old rats than young rats. These results suggest that the LHRH secretory mechanism changes with age in female rats. Such alterations may result in the accumulation of LHRH in the mid-hypothalamic area and an increase in ME-LHRH.     

大鼠老化过程大脑皮层细胞核、染色质转录研究

 本实验对不同鼠龄(4—,16—17—,33—34—和99—103周)大鼠老化动物模型进行脑细胞核、染色质体外转录研究,结果表明:(1)大脑皮层细胞核、染色质转录活性在老化过程中呈下降趋势,其中RNA聚合酶Ⅰ、Ⅱ活性与染色质模板效率变化一致,说明染色质模板活性降低是导致细胞核转录功能减退的原因之一。(2)幼年鼠染色质RNA和NHCP含量高于老年鼠,提示染色质结合蛋白及RNA可能参与不同生理时期脑神经元染色质结构和功能的调节。(3)老年鼠脑染色质DNA抗DN-aseⅠ酶解能力增强,提示衰老导致转录活性染色质区域减少。     

Molecular origin of the aging effects in glyceraldehyde-3-phosphate dehydrogenase

Rat muscle glyceraldehyde-3-phosphate dehydrogenase was reacted with two reagents aimed at the highly reactive cysteine-149 residue in the active site of the enzyme. The enzyme was rapidly inactivated by iodine monochloride. Complete inactivation occured when approx. 6 mol ICl were added per mol enzyme, indicating that reactions which compete with the reagent's interaction with cysteine-149 take place. Iodine was also found to inactivate the enzyme rapidly and effectively, and, when not in excess, this reagent interacted specifically with cysteine-149. The fraction of original enzymatic activity which could be restored by 2-mercaptoethanol in enzyme samples inhibited by 4.2 mol I₂/mol enzyme, decreased with time to a limiting value of 0.6 reached after approx. 15 min. The enzyme thus treated showed a remarkable similarity to enzyme samples purified from old rats, both in its activity and in NAD⁺ binding patterns under various conditions. It is concluded that the structural modifications induced in the modified enzyme resemble the age-related modifications in native ‘old’ enzyme. These results demonstrate that the origin of the age-related effects in glyceraldehyde-3-phosphate dehydrogenase is in subtle, post-synthetic structural changes. The inactivation reactions described above require a non-reducing environment for the enzyme. Whether such conditions do exist in cells of old animals is the subject of future studies.     

Age Peculiarities of the Calpain/Calpastatin Cerebral System in Rats: Relation to the Hypothesis of Brain Aging

We studied age-related changes in the activity of calpain, those in the activity of its endogenous inhibitor calpastatin, and the ratio of these indices in the brain of rats of four age groups (2-3 weeks, 2-3, 5-6, and 24 months). The activity of calpain was estimated using FITC casein as the substrate. In a soluble fraction of the brain homogenate, the enzyme activity in general increased with age. In mature rats (5 to 6 months old), this index exceeded 3.65 times the corresponding index in juvenile (2 to 3 weeks old) animals, while in old animals this index somewhat decreased. In the fraction obtained after separation of calpain from other components using DEAE-cellulose chromatography, the age-related trend toward an increase in the activity of calpain was preserved, but it was much more moderate. The activity of calpastatin demonstrated an opposite direction of age changes: it was the maximum in 2-3-week-old animals and gradually decreased (by 27%) in old rats. We also found that the efficacy of inhibitory action of calpastatin in the cerebral tissue with respect to the activity of calpain was, as a rule, redundant. In this case, the ratio of inhibitor/enzyme activities decreased with age; this index was 1.65, 1.33, 1.1, and 1.0 or less in 2-3-week-old, 2-month-old, mature, and old animals. Therefore, we found that the intensity of calpain-mediated proteolysis in the rat brain increases from the juvenile period to the mature age and somewhat decreases in old individuals. Such alterations are developed at the expense of both an increase in the activity of the enzyme and weakening of the action of its inhibitor (calpastatin).     

Rat-liver superoxide dismutase. Purification and age-related modifications.

Cytoplasmic superoxide dismutase has been purified from livers of young (6 months) and old (27 months) rats. The enzyme purified from old animals shows an age-related reduction in the specific activity, accumulation of antigenically cross-reacting material and increased sensitivity to temperature. No differences were found in the molecular weight, electrophoretic mobility, antigenicity and Ki between enzymes purified from young and old rats. This is the first demonstration of age-related alterations in a purified form of a non-metabolic enzyme, which can be related to reduced activity. The possible role of this reduced activity in age-dependent deterioration of cellular functions is discussed.     

Emotional manifestations in the electrical stimulation of the hypothalamic nuclei in adult and old rats

In experiments on adult (9-10 months) and old (24-26 months) white Wistar rats behavioural manifestations under electrical stimulation of the ventromedial hypothalamus nucleus and self-stimulation (SS) of the lateral hypothalamic region were studied. It has been found that with age electrical thresholds of negative emotional manifestations decrease with invariable SS thresholds. In old rats, in comparison with the adult ones, SS frequency is lower, maximum SS proceeds at lower currents, the range of currents capable to evoke an intensive SS is narrower, SS motivational component is less expressed. The obtained data testify that in old rats there exist neurophysiological preconditions for prevailing of negative emotional manifestations.     

Features, causes and consequences of splanchnic sequestration of amino acid in old rats

Rationale

In elderly subjects, splanchnic extraction of amino acids (AA) increases during meals in a process known as splanchnic sequestration of amino acids (SSAA). This process potentially contributes to the age-related progressive decline in muscle mass via reduced peripheral availability of dietary AA. SSAA mechanisms are unknown but may involve an increased net utilization of ingested AA in the splanchnic area.

Objectives

Using stable isotope methodology in fed adult and old rats to provide insight into age-related SSAA using three hypotheses: 1) an increase in protein synthesis in the gut and/or the liver, 2) an increase in AA oxidation related to an increased ureagenesis, and 3) Kupffer cell (KC) activation consequently to age-related low-grade inflammation.

Findings

Splanchnic extraction of Leu (SPELeu) was doubled in old rats compared to adult rats and was not changed after KC inactivation. No age-related effects on gut and liver protein synthesis were observed, but urea synthesis was lower in old rats and negatively correlated to liver Arg utilization. Net whole-body protein synthesis and arterial AA levels were lower in old rats and correlated negatively with SPELeu.

Conclusion

SSAA is not the consequence of age-related alterations in ureagenesis, gut or liver protein synthesis or of KC activity. However, SSAA may be related to reduced net whole-body protein synthesis and consequently to the reduced lean body mass that occurs during aging.     

Effect of hypophysectomy on liver nuclear ribonucleic acid synthesis in aging rats.

Changes in RNA synthesis in liver nuclei were observed at different ages and after hypophysectomy and hormone replacement in female Sprague-Dawley rats. As determined by the incorporation of [3H]UMP into an acid-insoluble product, RNA synthesis decreased by about 75% in intact rats from 6 months to 24 months of age. This decline with age was not observed in liver nuclei from 24-month-old rats that had been hypophysectomized at 12 months and maintained on a minimal hormone-replacement therapy. Thyroid hormones and somatotropin (growth hormone) had an additive effect on RNA synthesis in liver nuclei from these hypophysectomized rats. The same hormones had no significant effect on intact, age-matched rats. With advancing age, nuclei of intact rats had an increase in the pool of free RNA polymerase and an apparent decrease in the enzyme activity bound to nuclear chromatin. There was no change in total enzyme with age. In hypophysectomized, hormone-treated rats, free RNA polymerase activity decreased and chromatin-bound activity increased. There was no difference in total nuclear RNA polymerase activity between operated or intact rats. However, the ratio of the bound to the free activity was different. These results suggest that the ability of RNA polymerase to bind to chromatin may be involved in the age-related decrease in liver nuclear RNA synthesis of intact rats.     

Age-associated oxidative modifications of mitochondrial α-subunit of F1 ATP synthase from mouse skeletal muscles

Abstract

The objective of this study was to investigate the pattern of age-associated oxidative post-translational modifications in the skeletal muscles of a mammalian species and to address whether the modifications result in the loss of function of the oxidatively modified protein(s). Accordingly, proteins in the mitochondrial matrix of the hind limb of C57BL/6Nnia mice were examined for modifications by carbonylation—an established marker of oxidative post-translational modifications—by Western blotting using anti-2,4-dinitrophenyl antibodies and tritiated sodium borohydride methods. An age-associated increase in carbonylation of mitochondrial matrix proteins was observed, but not all proteins were equally susceptible. A 55 kDa protein, identified as the α-subunit of the F1 complex of ATP synthase (ATP phosphohydrolase [H⁺-transporting]), had approximately 17% and 27% higher levels of protein carbonyls in adult and old animals, respectively, in comparison to the young controls as estimated using tritiated sodium borohydride. In addition, an age-associated decline in its activity was observed, with approximately 9% and 28% decrease in the activity in the adult and old animals, respectively, in comparison to young controls. It may be concluded that such oxidative post-translational modifications and the resultant attenuation of the protein activity may contribute to the age-related energy loss and muscular degeneracy.