Cocaine-induced stimulation of the rat hypothalamic-pituitary-adrenal axis is progressively attenuated following hourly-interval regimens of the drug.

The role of multiple (iv) injections of cocaine on the rat hypothalamic-pituitary-adrenal (HPA) axis was examined using four different temporal regimens of drug exposure. In intact rats, cocaine (5 mg/kg) consistently stimulated the secretion of adrenocorticotropin hormone (ACTH) and corticosterone over a 6 hr interval regimen. In all experimental groups, administration of the vehicle alone failed to measurably alter the secretion of the aforementioned hormones. When rats where exposed to the drug over a 4 hr interval regimen, a modest attenuation of ACTH, but not corticosterone, secretion was observed following the third and last cocaine injection. To test whether the attenuation of ACTH secretion to cocaine administration was caused by corticosterone-mediated negative feedback, the response of intact and adrenalectomized (ADX) rats over 2 hr and 1 hr interval regimens was compared. In intact rats, both drug interval regimens resulted in a significant attenuation of ACTH secretion following, the second and third injections of the drug. ADX rats, on the other hand, exhibited significant increases in ACTH levels following either interval regimens, though we observed a modest blunting of pituitary responsiveness to the 1 hr regimen. From these results we conclude that in intact rats the activity of the HPA axis is significantly attenuated in response to multiple, acute cocaine injections, and that this decreased response may be at least in part caused by a negative corticoid feedback mechanism.     

Influence of Melatonin on the Rate of Rana pipiens Tadpole Metamorphosis In Vivo and Regression of Thyroxine-Treated Tail Tips In Vitro

Metamorphosis of Rana pipiens tadpoles may be retarded when the light phase of the light/dark (LD) cycle is shortened or when thyroxine (T₄) is given in the dark because melatonin peaks during the dark. Injection of premetamorphic tadpoles in spontaneous metamorphosis with melatonin (15 μg) retarded tail growth and hindlimb development on 18L:6D but had no significant effect on 6L:18D. During induced metamorphosis (30 μg/liter T₄), melatonin injections retarded tail resorption on 18L:6D and accelerated it on 6L:18D, but did not affect the hindlimb. When melatonin was injected during T₄ immersion at different times in the photophase on 18L:6D (L onset 0800 hr), tail regression was retarded by melatonin at 1430 or 2030 hr. At 0830 hr, shrinkage of tail length was accelerated whereas tail height was not affected. Tail tips in vitro induced to resorb by 0.2 μg/ml T₄ in Niu-Twitty solution regressed more slowly in the presence of melatonin (10 or 15 μg/ml) than with T₄ alone on both 6L:18D and 18L:6D. The findings implicate melatonin in LD cycle effects on tadpole metamorphic rate in vivo , show the importance of the time of melatonin injections, and indicate that melatonin antagonizes the metamorphic action of T₄ at the tissue level.     

Thyroxine elicits divergent changes in mRNA levels for two urea cycle enzymes and one gluconeogenic enzyme in tadpole liver

Thyroxine-induced metamorphosis of the tadpole to the frog (Rana catesbeiana) is marked by increased activities of the urea cycle enzymes in liver. Cloned cDNAs for two mammalian urea cycle enzymes--carbamyl-phosphate synthetase I and argininosuccinate synthetase--were shown to cross-hybridize with the corresponding mRNAs in tadpole liver. Thyroxine treatment produced nearly 10-fold, coordinate increases in hybridizable mRNA levels for these two enzymes in tadpole liver. This increase is sufficient to account for reported increases in enzyme levels and synthesis rates, demonstrating that thyroxine largely regulates concentrations of these enzymes at a pretranslational step(s). In contrast, levels of phosphoenolpyruvate carboxykinase mRNA in tadpole liver decreased by more than 90% following thyroxine treatment. This differs from the thyroxine-induced increases in synthesis rates of enzyme and mRNA reported for phosphoenolpyruvate carboxykinase in rat liver. However, the decreased levels of this mRNA in tadpole liver may represent a secondary response due to thyroxine-stimulated release of insulin.     

Role of alpha-1-adrenergic receptors in the regulation of corticotropin-releasing hormone mRNA in the paraventricular nucleus of the hypothalamus during stress

SUMMARY 1. The role of 1-adrenergic receptors on CRH mRNA levels in the PVN was studied in control and stressed rats receiving i.c.v. injections of the 1-adrenergic agonist, methoxamine, or the 1- antagonist, prazosin.2. Plasma ACTH increased significantly 60 min and 4 hr after a single injection of methoxamine (100 g, i.c.v.). No desensitization of this response was observed after repeated injections every 6 hr for 24 hr. Concomitantly, POMC mRNA in the anterior pituitary increased by 25% at 4 hr after a single injection and by 96% after repeated injections.3. CRH mRNA levels in the PVN increased by 131% after repeated injections for 24 hr, but were unchanged 4 hr after a single injection. Central 1-adrenergic blockade with prazosin did not prevent the increases in CRH mRNA following 4 hr of acute stress, but significantly reduced the increases observed 24 hr after an i.c.v. injection of 75 g of colchicine or after repeated i.p. hypertonic saline injections every 8 hr.4. These studies demonstrate that while 1-adrenergic receptors contribute to long-term increases of CRH mRNA levels in the PVN during prolonged stress, other factors are likely to be involved in the stimulation of CRH mRNA following acute stimulation.     

Influence of androgen on the development of sexual behavior in rats : I. Time of administration and masculine copulatory responses, penile reflexes, and androgen receptors in females

The objective of the present study was to investigate the effect of the time of administration of androgen, during the neonatal period, on the development of masculine copulatory behavior in female rats. In addition, the influence of androgen, administered neonatally, on the development of penile reflexes and cytoplasmic androgen receptor levels in the hypothalamic-preoptic area (HPOA) was examined. Female rats were injected with 0.5 mg testosterone propionate (TP) at either 1, 8, or 24 hr after birth and again 24 hr after the first injection. Fifty percent of the females treated with TP at 1 and 8 hr after birth displayed the ejaculatory response when tested in adulthood. In contrast, 93 and 87.5% of oil-treated males and females, respectively, which were androgenized at 24 hr after birth exhibited this response. The results indicate that a considerable amount of masculinization occurs postnatally in the rat. However, none of the androgenized females displayed any penile reflexes even when tested following the display of an ejaculatory response. HPOA androgen receptor levels were somewhat higher in the oil-treated females than in males but were not correlated with the ability to exhibit ejaculation patterns.     

Endogenous hormone response and fertility in dairy heifers after treatment with Lutalyse and GnRH

Fifty-five dairy heifers were given two injections of Lutalyse 11 days apart. Twenty-one of the heifers were also given an injection of GnRH 48 hr after the second Lutalyse injection (Group G). Of the remaining 34 animals, 19 were randomly allotted to be inseminated 12 hr after observed estrus following Lutalyse (Group E), while 15 were inseminated 80 hr after the second Lutalyse injection (Group P). The intervals from second Lutalyse injection to occurrence of both estrus and peak gonadotropin concentrations were variable among animals receiving only Lutalyse. GnRH injections reduced variation (P<.01) in the interval from second Lutalyse injection to occurrence of peak gonadotropin concentrations, but did not improve fertility. Pregnancy rates did not differ (P>.05) among treatment groups. The failure of GnRH administration following Lutalyse to improve pregnancy rates indicates that GnRH administration followed by insemination 12 hr later is not effective in increasing pregnancy rates above those attained in animals inseminated at either 12 hr post estrus or 80 hr after second Lutalyse injection.     

Effect of some antiestrogens and aromatase inhibitors on androgen induced sexual behavior in castrated male rats

The effect of antiestrogens (MER-25, ICI-46474, and cis-clomiphene) and aromatase inhibitors (5-α-androstanedione, metopirone, and aminoglutethimide) on androgen induced copulatory behavior was tested in sexually inexperienced castrated male tats. Daily injections of 1 mg testosterone (T) for 21 days induced sexual activity in most subjects (61% mounting). Daily pretreatment with MER-25 or cis-clomiphene at three dose levels did not block the behavioral response to T. ICI-46474 at the high dose level (1 mg/kg) elicited a significant depressory effect on the sexual behavior of the T treated castrated rats. A single injection of 6 mg testosterone propionate (TP) induced mounting behavior in 56% of the tested rats within 120 hr. Treatment with metopirone or 5 α-androstanedione (injections every 12 hr for 96 hr) did not inhibit the response to TP. By contrast, aminoglutethimide (5 or 15 mg every 12 hr for 96 hr) abolished the behavioral response to androgen.     

Prostaglandin F2α-induced stimulation of estrone and estradiol-17β secretion in cattle

Four of 5 Holstein heifers given intra-ovarian injections of 300 μg of prostaglandin F_2α (PGF_2α) showed transient, but statistically significant, depressions in plasma progesterone levels which returned to near normal levels within 24 hr. The same 4 animals also exhibited significant elevations in plasma estrone and estradiol-17β levels during the initial 24 hr. period following treatment, although no animals were observed in estrus during this time. Plasma levels of progesterone, estrone, estradiol-17β and PGF showed little change in control heifers receiving intra-ovarian injections of the buffer solution used as a vehicle for PGF_2α. It is concluded that PGF_2α stimulates estrogen secretion, presumably by follicular elements of the ovary.     

Roles for inhibition: studies on networks controlling swimming in young frog tadpoles

The hatchling frog tadpole provides a simple preparation where the fundamental roles for inhibition in the central nervous networks controlling behaviour can be examined. Antibody staining reveals the distribution of at least ten different populations of glycinergic and GABAergic neurons in the CNS. Single neuron recording and marker injections have been used to study the roles and anatomy of three types of inhibitory neuron in the swimming behaviour of the tadpole. Spinal commissural interneurons control alternation of the two sides by producing glycinergic reciprocal inhibition. By interacting with the special membrane properties of excitatory interneurons they also contribute to rhythm generation through post-inhibitory rebound. Spinal ascending interneurons produce recurrent glycinergic inhibition of sensory pathways that gates reflex responses during swimming. In addition their inhibition also limits firing in CPG neurons during swimming. Midhindbrain reticulospinal neurons are excited by pressure to the head and produce powerful GABAergic inhibition that stops swimming when the tadpole swims into solid objects. They may also produce tonic inhibition while the tadpole is at rest that reduces spontaneous swimming and responsiveness of the tadpole, keeping it still so it is not noticed by predators.     

Pharmacological aspects of the influence of melanocortins on the formation of regenerative peripheral nerve sprouts

Adrenocorticotropin (ACTH) and alpha-melanocyte-stimulating hormone (alpha-MSH) stimulate the initial sprouting response in the crushed rat sciatic nerve. In this report a detailed analysis of the neurotrophic action of Org.2766 [a degradation resistant ACTH(4-9) analog] and alpha-MSH is described. Org.2766 treatment results in enhanced numbers of outgrowing sprouts in the damaged nerve. The growth velocity of the sprouts is not affected. The peptide effect is dose-dependent. A single peptide injection administered immediately following the crush stimulates the formation of sprouts significantly. Continued high blood levels of Org.2766 are probably not critical for the neurotrophic effect of these peptides, since a more moderate dosing protocol (injections given every 48 hr) was more effective than more frequent injections (injections given every 12 hr). The present results further the understanding of the mode of action of ACTH/alpha-MSH-like peptides and underscore the necessity to test a wide range of doses and injection protocols to avoid false negative results in clinical work being planned to start in the near future.     

Frequency of cocaine administration affects behavioral and endocrine responses in male and female Fischer rats.

Accumulating evidence has shown disparate behavioral responses to cocaine in male and female rats. To date, there is a lack of understanding of how cocaine administration frequency affects sexually dimorphic behavioral responses. In the present study we investigated the behavioral and endocrine responses to single (1 x 15 mg/kg) and "binge" (3 x 15 mg/kg) cocaine administration in male and female Fischer rats. Overall, females showed a more prolonged and robust behavioral response to both acute and "binge" pattern cocaine administration. Furthermore, sex-dependent behavioral topographies emerged during binge-pattern cocaine administration; female rearing activity increased across "binge" injections while ambulatory activity decreased. In contrast, male ambulatory and rearing behaviors remained constant across injections of "binge" cocaine. At the hormonal level, both single and "binge" pattern cocaine administration decreased testosterone levels in male rats. However, cocaine's modulation of testosterone levels was transient since testosterone levels were decreased by cocaine 30 min but not 3 hr following a single injection. In both male and female rats, "binge" cocaine increased plasma progesterone levels. However, acute cocaine administration increased progesterone levels transiently in only female rats. Our results show that pattern of administration affects both cocaine-stimulated behavioral and endocrine responses in male and female rats.     

Iron-induced accumulation of hepatic metallothionein: the lack of glucocorticoid involvement

The process(es) by which parenteral iron effects the accumulation of hepatic metallothionein (MT) is not known. The present study examined glucocorticoids as potential mediators of this process. Chicks were given either one injection (ip) of iron (+1FE) at 10 mg Fe/kg, two injections of iron (+2FE) given 24 hr apart, or a single injection of saline. Plasma corticosterone was evaluated at various times following the last injection. Plasma corticosterone increased approximately 50% following +1FE but more than 200% at 2 and 4 hr following a second injection of iron (+2FE). Plasma zinc showed a transient increase followed by a considerable depression. Coincidentally, the accumulation (determined at 24 hr) of zinc MT in liver of +2FE chicks was three times higher than that of +1FE chicks. In another experiment, markedly greater changes, at similar time intervals, in plasma corticosterone were effected by multiple subcutaneous injections of adrenocorticotropic hormone (ACTH) (either 5 IU ACTH or 20 IU ACTH/kg). Subsequent analysis of hepatic zinc MT showed only minor changes as a result of ACTH injections. These results indicate that a change in the plasma glucocorticoid corticosterone is not a primary component in the process(es) by which parenteral iron effects an increase in hepatic zinc MT.     

Sex differences in response to discrete estradiol injections

Developmental effects of perinatal androgens render adult male rats refractory to the activation of feminine sexual behavior by estradiol (E2) and progesterone (P). Recent evidence suggested that fluctuating levels of systemic E2, which are thought to approximate the ovarian secretion under physiological conditions, may reverse this insensitivity to E2 and, particularly, to the synergistic effects of P in male rats of the Wistar strain. We examined whether this hormonal regimen would reverse this insensitivity in Sprague-Dawley rats. Gonadectomized animals received two injections of E2 (1 microgram per injection) 12 hr apart at 0900 and 2100 hr followed by P (0.5 mg) or oil, at 35 hr, and a mating behavior test, at 38 hr, subsequent to the initial E2 administration. This treatment was repeated four times at 4-day intervals. The inability of Sprague-Dawley male rats to respond to E2 and P was unaffected by this pattern of exposure to exogenous E2. Receptivity scores, lordosis quotients, and proceptivity were negligible in males, and significantly less than that displayed by females. In addition, the levels of sexual receptivity and proceptivity were facilitated by the availability of P following E2 in females, but not in males. The present findings fail to support a general hypothesis that "discontinuous" E2 stimulation, achieved by two spaced injections of this hormone, reverses developmental determinants of sex differences in responsiveness to hormones mediating female sexual behaviors.     

Dexamethasone and estradiol benzoate induced parturition in dairy cattle

Fifteen 2-year-old Holstein cows and 21 mature Holstein cows were assigned to one of three groups. Cows in Group I calved spontaneously. Cows in Groups II and III received single intramuscular injections of 20 mg dexamethasone and 25 mg estradiol benzoate to induce parturition prematurely. In addition, cows in Group III received a single intramuscular injection of 12.5 mg estradiol benzoate 48 hr prior to dexamethasone and estradiol benzoate. The objective of the experiment was to determine the effectiveness of estradiol benzoate in combination with dexamethasone on traits at parturition and on productive and reproductive characteristics following parturition. Induction of parturition shortened gestation length and increased the incidence of retained placentas (both P < .01). All induced cows calved between 21 and 59 hr postinjection with less (P < .05) udder edema when compared to control cows. Mean plasma estrogen concentrations, using an assay system which does not measure estradiol benzoate, were not different among groups following injections of estradiol benzoate. Mean estradiol-17β concentrations in induced cows, however, using an assay system which does recognize estradiol benzoate (70.8% crossreactivity), were higher (P < .01) following estradiol benzoate injection, tended to be higher through parturition, and remained elevated (P < .01) at 12 and 24 hr following parturition when compared to cows calving spontaneously. Mean monthly milk production and the 2x, 305-ME records for milk, fat and FCM were not different among groups.     

Effect of aflatoxin B1 on phosphoinositide signal transduction pathway during regeneration of liver cells following partial hepatectomy.

Aflatoxin B1 (AFB1) when administered to partially hepatectomised rats 4 hr prior to sacrifice, activated the signalling pathway in regenerating rat liver. The activity of phosphatidylinositol (PI) kinase was found decreased at 30 min but increased at 24 hr and returned to normal at 48 hr. At 30 min, inositol-1,4,5-triphosphate (IP3) level increased significantly whereas diacylglycerol (DAG) level dropped. However, at 24 hr and 48 hr, DAG and IP3 showed the same trend i.e. an increase in their levels. Phosphatidylinositol-4-phosphate levels were found to increase at 24 hr. Protein kinase C (PKC), activity from the particulate fraction was significantly inhibited at 30 min, followed by increase in activity at 24 hr and return to normal at 48 hr. Cytosolic PKC showed a decrease at 24 hr and a significant increase at 48 hr. At the peak of DNA synthesis (24 hr) following partial hepatectomy, all these signalling steps had earlier been found to be inhibited, but the present study shows that aflatoxin B1 administration 4 hr prior to sacrifice reverses the action. Activation of PKC by aflatoxin B1, during regeneration of liver cells when PKC in normally inhibited, may possibly create conditions conducive to carcinogenesis.     

Characteristics of the isoprenaline stimulated proliferative response of rat submaxillary gland.

A simple stochastic model has been developed to determine the cell cycle kinetics of the isoprenaline stimulated proliferative response in rat acinar cells. The response was measured experimentally, using 3H-TdR labelling of interphase cells and cumulative collections of mitotic cells with vincristine. The rise and fall of the fraction of labelled interphase cells and of metaphase cells is expressed by the product of the proliferative fraction and a difference of probability distributions. The probability statements of the model were formulated and then compared by an iterative fitting procedure to experimental data to obtain estimates of the model parameters. The model when fitted to the combined fraction labelled interphase (FLIW) and fraction metaphase (FMWa) waves gave a mean Gis transit time of 21-2 hr, mean Gis +S transit time of 27-0 hr, and mean Gis + S + G2 transit time of 35-8 hr for a single injection of isoprenaline, where Gis is the initiation to S phase time. When successive injections of isoprenaline were given at intervals of 24 and 28 hr the corresponding values after the third injection were 12-4 hr, 20-8 hr and 25-7 hr respectively. The variance of the Gis phase dropped from 18-1 to 1-3 while the other variances remained unchanged. The estimated proliferative fraction was 0-24 after a single injection of isoprenaline, and 0.31 after three injections of the drug. Independently determined values of the proliferative fraction, obtained from repeated 3H-TdR injections, were 0-21 and 0-36 respectively.     

Feeding and drinking responses in the golden hamster following treatment with cholecystokinin and angiotensin II

Drinking and feeding behaviours of female golden hamsters were examined following intracerebroventricular injections of angiotensin II or systemic and intracerebroventricular injections of cholecystokinin octapeptide. Injections of angiotensin II into the brain produced a dose-dependent drinking response in water repleted animals. Systemically, a low dose (0.5 microgram/kg body wt) of cholecystokinin was ineffective at reducing food intake of fasted animals during a 1 hr test. Larger peripheral doses (1.0 to 4.0 microgram/kg body wt), however, were effective at decreasing food intake. Injected in the lateral cerebral ventricle, nanogram doses of cholecystokinin decreased food consumption in a dose dependent manner. These results are discussed in relation to how these peptides regulate feeding and drinking behaviours in other species.     

Effects of superovulatory doses of pregnant mare serum gonadotropin on oocyte quality and ovulatory and steroid hormone responses in rats

Immature rats (aged 28 days) were injected with 4, 20, or 40 IU pregnant mare serum gonadotropin (PMSG) and sacrificed every 6 or 12 hr. Control rats (4 IU) ovulated between 60 and 72 hr, whereas rats given superovulatory doses of PMSG (20 and 40 IU) ovulated between 24 and 72 hr. The oocyte count from the superovulated rats increased slightly between 24 and 36 hr and markedly between 48 and 72 hr. Degenerated oocytes were recovered 48 and 36 hr after administration of 20 and 40 IU PMSG, respectively. Thereafter, the proportion of degenerated oocytes was dose dependent and reached a maximum at 72 (30.9%, 20 IU) and 60 hr (61.0%, 40 IU). 17β-estradiol content of the superovulated ovaries increased significantly (P < 0.01) from 36 hr and was maximal at 60 (20 IU) or 54 hr (40 IU), when compared to the control regimen. Administration of 40 IU PMSG resulted in a biphasic increase of progesterone content with the peaks at 36 and 60 hr. Androgen content of the superovulated ovaries was lower than control levels during the first 36 hr but was significantly (P < 0.01) higher thereafter. The results suggest that these alterations in the steroid response (particularly androgens) from 36 hr onward following superovulation may be responsible for the coincidental occurrence of abnormal oocytes, possibly by disturbing the specific intrafollicular steroid environment essential for complete maturation. In addition, oocyte aging that is due to earlier activation by the exogenous luteinizing hormone activity may be a contributing factor.     

Heat shock protects cultured neurons from glutamate toxicity.

Expression of heat shock proteins (HSPs) occurs in brain after ischemia and status epilepticus. We report that induction of the heat shock response in cortical cultures protects neurons from glutamate-induced excitotoxicity. Cultures heated to 42.2 degrees C for 20 min showed an overall decrease in protein synthesis but an increase in the synthesis of approximately 72 and approximately 85 kd proteins and in the levels of HSP70 mRNA. Heat shock inhibited excitotoxicity in cells exposed to glutamate at 3 or 24 hr following heat exposure, but not when the interval between heat and glutamate exposure was shortened to 15 min or lengthened to 48 hr. Protection due to heat shock required new protein synthesis, since it did not occur when protein or RNA synthesis inhibitors were added. By ameliorating excitotoxic processes, HSPs may attenuate brain injury in certain pathologic conditions.