Effects of an organophosphate pesticide, quinalphos, on the hypothalamo-pituitary-gonadal axis in adult male rats

The effects of chronic sub-lethal doses (7-14 mg kg-1 a day for 15 days) of quinalphos were evaluated in adult male rats for changes in testicular morphology, circulatory concentrations of hormones (LH, FSH, prolactin and testosterone), activities of acetylcholinesterase (AChE) and angiotensin converting enzyme (ACE) as well as metabolism of biogenic amines (dopamine, noradrenaline and 5-hydroxytryptamine (5-HT)) in the hypothalamus and pituitary. Hormones were assayed by radioimmunoassay or chemiluminescent immunoassay (testosterone). The enzymes were estimated after spectrophotometry and the biogenic amines by HPLC-electrochemistry. Sub-lethal chronic administration of quinalphos resulted in: decreased testicular mass and AChE activity in central as well as peripheral organs; increased serum LH, FSH, prolactin and testosterone concentrations; decreased pituitary or increased testicular ACE activity; severe disruption of spermatogenesis with increasing doses of pesticide; and no significant effects on dopamine, noradrenaline or 5-HT concentrations in the hypothalamus or pituitary. Administration of oestradiol (50 micrograms per rat a day) during pesticide treatment resulted in: a significant decrease in the mass of the testis and accessory sex organs; decreases in serum LH, FSH, testosterone concentrations; an increase in prolactin concentration; and a decrease in dopamine or an increase in noradrenaline and 5-HT in the hypothalamus or pituitary. Oestradiol had a marked effect: in pesticide-treated animals, the pesticide effects were significantly reversed. This indicates that in pesticide toxicity, the hypothalamo-pituitary-gonadal axis is operational. Since many of the observed pesticide effects could be inhibited by oestradiol, it is suggested that the pesticide acts directly on the gonadotrophins. In conclusion, quinalphos decreases fertility in adult male rats by affecting the pituitary gonadotrophins.     

Sex differences in adrenocortical structure and function. V. The effects of postpubertal gonadectomy and gonadal hormone replacement on nuclear-cytoplasmic ratio, morphology and histochemistry of rat adrenal cortex.

Studies were carried out on adult rats of Wistar strain. Six weeks after postpubertal gonadectomy some of the orchiectomized rats were injected with a single dose of testosterone cypionate and ovari-ctomized rats with estradiol cypionate (17 beta-cyclopentyloproprionate esters of testosterone or estradiol). The control rats were sham operated. Investigations were carried out 8 weeks after surgery. Absolute and relative adrenal weight is lower in the male than in the female rat. Orchiectomy increases these weights while testosterone restores them to their normal level. Ovariectomy has no effect on the weight of adrenal, estradiol, however, increases the relative weight of the gland. The adrenal cortex of the adult female is wider than in the adult male rat and in female gland sudanophobic zone is lacking. Orchiectomy leads to the broadening of the adrenal cortex and testosterone replacement has an opposite effect. Ovariectomy has no effect on the structure of adrenal cortex and estradiol replacement resulted in the narrowing of zona glomerulosa. There were no differences in the nuclear-cytoplasmic ratio of zona glomerulosa cells in male and Female rats and neither gonadectomy nor gonadal hormone replacement has no effect on this parameter. The nuclear-cytoplasmic ratio of zona fasciculata cells in the male is markedly higher than in female. Orchiectomy lowers this ratio and testosterone restores it to the normal level. Neither ovariectomy nor estradiol replacement has effect on the nuclear-cytoplasmic ratio of zona fasciculata cells. Similar changes as those in the zona fasciculata were observed in zona reticularis cells. Among the oxidoreductases studied, the most marked sex differences were found in alpha-glycerophosphate dehydrogenase activity. In control female rats an intense reaction for this enzyme is observed in broad band of cells of the zona fasciculata interna, while in male rats only individual cells showed this reaction. In orchiectomized rats the reaction for this enzyme is similar as in control female rats and testosterone restores it to normal. Ovariectomy has no effect on localisation of reaction while after estradiol replacement reaction was more intense. Regarding the remaining oxidoreductases studied, in the adrenal cortex of rats of both sexes no marked differences were observed in localization, however, intensity of reaction depended upon applied experimental conditions. More distinct sex differences were observed in topochemistry of some hydrolases and intensity of reaction depended upon the applied experimental conditions.     

Effect of neurotransplantation on the content of catecholamines in the rat brain structures after heavy craniocerebral trauma

It was found that heavy craniocerebral trauma (CCT) in rats results in a drop in the dopamine and noradrenaline content in the damaged cerebral hemisphere, striatum, and hypothalamus. Transplantation of embryonic neural tissue after CCT in rats favors restoration of the level of catecholamines. Thirty days after the transplantation, the level of noradrenaline and dopamine in damaged structures mentioned above becomes similar to that in intact animals, or exhibits a tendency to be restored.     

Effect of a fall of blood pressure on the release of catecholamines in the hypothalamus

The posterior hypothalamus of anaesthetized cats was superfused through a push-pull cannula and the release of endogenous catecholamines was determined in the superfusate which was continuously collected in 15 min periods. Fall in blood pressure elicited by nitroprusside or bleeding led to an increased rate of release of noradrenaline, adrenaline and dopamine in the hypothalamus. Transection of the brain causal to hypothalamus greatly reduced the rate of resting release of the catecholamines and abolished the enhancing effects of bleeding and nitroprusside. Determination of the catecholamines in samples which were collected in 90 s periods suggested a different pattern of release of the three catecholamines. Further shortening of the collection period (10 s) showed that the fall in blood pressure immediately increased the release of dopamine, while the rates of release of noradrenaline and adrenaline were increased gradually. Hypotension did not influence the rates of release of the catecholamines in the anterior hypothalamus. It is concluded that dopamine, adrenaline and noradrenaline systems of the hypothalamus are involved in the regulation of the arterial blood pressure. The different patterns of release might indicate that dopamine exerts a different function from those of noradrenaline and adrenaline in the normalization of the blood pressure after acute hypotension.     

Effects of drugs on brain neurotransmitter and pituitary-testicular function in male rats.

The effects of different drugs influencing brain neurotransmitter contents have been tested on the pituitary-testicular function in male rats. L-dopa (200 mg/kg body weight, i.p.) increased the dopamine and noradrenaline contents of the hypothalamus, amygdala, striatum and mesencephalon, but it was ineffective as regards the 5-hydroxytryptamine contents of the same brain areas, and increased the plasma testosterone level. alpha-Methyl-p-tyrosine (250 mg/kg b.w., i.p.) decreased the dopamine and noradrenaline contents of these brain areas, but it was ineffective to 5-hydroxytryptamine, and decreased the plasma testosterone level. Diethyldithiocarbamate (400 mg/kg b.w., i.p. twice a day) increased the dopamine levels in the hypothalamus, amygdala, striatum and mesencephalon, decreased the noradrenaline contents in the same brain regions but had no effect on the 5-hydroxytryptamine contents of these brain areas or on the testosterone level in the peripheral blood. p-Chlorophenylalanine (300 mg/kg b.w., i.p.) decreased the 5-hydroxytryptamine contents of the different brain areas, while it had no effect on the dopamine and noradrenaline levels or on the plasma testosterone level. 5-Hydroxytryptophan (200 mg/kg b.w., i.p.) increased the 5-hydroxytryptamine contents of all brain areas studied, but was without effect on the dopamine and noradrenaline contents or the plasma testosterone level. The data suggest that both dopamine and noradrenaline may be involved in the regulation of the pituitary-testicular function, and the ratio of the two transmitters might be more important that their actual levels in definite brain areas.     

Tyrosine hydroxylase-synthesizing cells in the hypothalamus of prairie voles (Microtus ochrogaster): sex differences in the anteroventral periventricular preoptic area and effects of adult gonadectomy or neonatal gonadal hormones

The vertebrate hypothalamus and surrounding region contain a large population of cells expressing tyrosine hydroxylase (TH), the rate limiting enzyme for synthesis of dopamine and other catecholamines. Some of these populations are sexually dimorphic in rats. We here examined sex differences in TH-immunoreactive populations in the forebrain of gonadally intact and gonadectomized prairie voles (Microtus ochrogaster), a species that sometimes shows unusual sexual differentiation of brain and behavior. A sex difference was found in the anteroventral periventricular preoptic area (AVPV; likely analogous to the rat rostral A14) only in gonadectomized subjects, which was due to a 50% reduction in the number of TH-immunoreactive cells after castration in males. There was no significant sex difference or effects of gonadectomy on the number of TH-immunoreactive cells in the anteroventral preoptic area (AVP), periventricular anterior hypothalamus (caudal A14), arcuate nucleus (A12), zona incerta (A13), or posterodorsal hypothalamus (A11). In a second experiment, testosterone propionate (TP; 500 microg), diethylstilbestrol (DES; 1 microg), or estradiol benzoate (EB; 30 microg) injected daily during the first week after birth each significantly reduced later TH expression in the AVPV of females by approximately 40-65% compared to oil-treated controls. Unlike rats, therefore, a sex difference in TH expression in the prairie vole AVPV is found only after removal of circulating gonadal hormones in males. Furthermore, unlike our previous findings on the generation of sex differences in extra-hypothalamic arginine-vasopressin expression in prairie voles, TH expression in the AVPV of female prairie voles can be highly masculinized by neonatal exposure to either aromatizable androgens or estrogens.     

Genetic-neurochemical analysis of the role of brain catecholamines in the manifestation of dominance in male mice in micropopulations

The ability of male mice to dominate in micropopulations, in connection with brain catecholamines was studied. It was shown that the F1 obtained from breeding PT and CBA/Lac which have respectively large and small numbers of high rank males were of high level of dominance in micropopulations. Moreover, they had high levels of noradrenaline and dopamine in hippocampus, striatum and brain stem, and low--in hypothalamus.     

Sexual hormones terminate in the rat: the significantly enhanced catecholaminergic/serotoninergic tone in the brain characteristic to the post-weaning period

The amount of dopamine released from the striatum, substantia nigra and tuberculum olfactorium, noradrenaline from locus coeruleus and serotonin from the raphe, was significantly higher in four and five weeks old rats than in three month old ones, proving that the catecholaminergic/serotoninergic activity enhancer (CAE/SAE) regulation works unrestrained during developmental longevity and is restricted thereafter. As the dampening of the CAE/SAE regulation (end to the second month of age) coincided temporally with the appearance of sexual hormones, we castrated three weeks old male and female rats and measured at the end of the third month of their life the release of catecholamines and serotonin from selected discrete brain regions. The amount of catecholamines and serotonin released from the neurons was significantly higher in castrated than in untreated or sham operated rats, signalting that sexual hormones inhibit the CAE/SAE regulation in the brain. We therefore treated male and female rats s.c. with oil (0.1 ml/rat), testosterone, (0.1 mg/rat), estrone (0.01 mg/rat) and progesterone (0.5 mg/rat), respectively, and measured their effect on the CAE/SAE regulation. Twenty-four hours after a single injection with the hormones, the release of noradrenaline, dopamine and serotonin was significantly inhibited in the testosterone or estrone treated rats, but remained unchanged after progesteron treatment. In rats treated with a single hormone injection, testosterone in the male and estrone in the female was the significantly more effective inhibitor. Remarkably, the reverse order of potency was found in rats treated with daily hormone injections for 7 or 14 days. After two-week treatment with the hormones estrone was in the male and testosterone in the female the significantly more potent inhibitor of the CAE/SAE regulation. The data indicate that sexual hormones terminate the hyperactive phase of adolescence by dampening the impulse propagation mediated release of catecholamines and serotonin in the brain.     

Sex differences in adrenocortical structure and function. III. The effects of postpubertal gonadectomy and gonadal hormone replacement on adrenal cholesterol sidechain cleavage activity and on steroids biosynthesis by rat adrenal homogenates.

In rats postpubertal orchiectomy results in an increase in the adrenal weight, testosterone replacement restores the adrenal weight to the normal level. Neither ovariectomy (8 weeks of duration) nor estradiol replacement has an effect on adrenal weight in female rats. Pregnenolone synthesis as well as corticosterone and blue tetrazolium-positive steroids secretion is significantly higher in homogenates of adrenals from female rats than from males. Orchiectomy results in a marked increase in pregnenolone biosynthesis, testosterone replacement restores the value to the normal levels. Neither ovariectomy nor estradiol replacement has an effect on pregnenolone synthesis in v i t r o. In both sexes gonadectomy causes a marked decrease in corticosterone output by adrenal homogenates, concomitantly the increase in the adrenal 5alpha-reductase activity is observed. The ratio of secreted corticosterone to pregnenolone is significantly lower in gonadectomized rats of both sexes than in control animals. Estradiol or testosterone replacement inhibits the adrenal 5alpha-reductase activity and restores the corticosterone output as well as corticosterone/pregnenolone ratio to the normal values. The above described findings show that the sex differences in steroids secretion by the rat adrenal are partially conditioned by a cholesterol sidechain cleavage activity. Testosterone inhibits this activity while estradiol under applied experimental conditions has no effect on the cholesterol sidechain cleavage activity.     

Tyrosine hydroxylase‐synthesizing cells in the hypothalamus of prairie voles (Microtus ochrogaster): Sex differences in the anteroventral periventricular preoptic area and effects of adult gonadectomy or neonatal gonadal hormones

The vertebrate hypothalamus and surrounding region contain a large population of cells expressing tyrosine hydroxylase (TH), the rate limiting enzyme for synthesis of dopamine and other catecholamines. Some of these populations are sexually dimorphic in rats. We here examined sex differences in TH‐immunoreactive populations in the forebrain of gonadally intact and gonadectomized prairie voles (Microtus ochrogaster), a species that sometimes shows unusual sexual differentiation of brain and behavior. A sex difference was found in the anteroventral periventricular preoptic area (AVPV; likely analogous to the rat rostral A14) only in gonadectomized subjects, which was due to a 50% reduction in the number of TH‐immunoreactive cells after castration in males. There was no significant sex difference or effects of gonadectomy on the number of TH‐immunoreactive cells in the anteroventral preoptic area (AVP), periventricular anterior hypothalamus (caudal A14), arcuate nucleus (A12), zona incerta (A13), or posterodorsal hypothalamus (A11). In a second experiment, testosterone propionate (TP; 500 μg), diethylstilbestrol (DES; 1 μg), or estradiol benzoate (EB; 30 μg) injected daily during the first week after birth each significantly reduced later TH expression in the AVPV of females by approximately 40–65% compared to oil‐treated controls. Unlike rats, therefore, a sex difference in TH expression in the prairie vole AVPV is found only after removal of circulating gonadal hormones in males. Furthermore, unlike our previous findings on the generation of sex differences in extra‐hypothalamic arginine‐vasopressin expression in prairie voles, TH expression in the AVPV of female prairie voles can be highly masculinized by neonatal exposure to either aromatizable androgens or estrogens. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2006     

Effects of gonadectomy and steroid treatment on renal prostaglandin 9-ketoreductase activity in the rat

The effect of gonadectomy and treatment with sex-steroids on renal prostaglandin 9-ketoreductase activity in 10-11 week old male and female rats was determined. Rats were gonadectomized or subjected to sham operation at 3 weeks of age. During week 7, rats were injected s.c. twice over a 6-day interval with vehicle (peanut oil, 0.5 ml X kg-1) or with depot forms of testosterone (5 mg X kg-1), estradiol (0.02 mg X kg-1), progesterone (5 mg X kg-1), or estradiol and progesterone combined. Renal prostaglandin 9-ketoreductase activity was about 50% higher in female rats than in males. Gonadectomy decreased 9-ketoreductase activity in females, but not in males, and eliminated the gender difference in enzyme activity. Treatment with estradiol elevated 9-ketoreductase activity in males and females, while treatment with testosterone or progesterone was without effect. Progesterone did, however, antagonize the elevation in 9-ketoreductase activity produced by estradiol.     

The brain catecholamines during domestication of the silver fox Vulpes fulvus

Studies have been made on the content of catecholamines (noradrenaline and dopamine) as well as metabolites of dopamine (3,4-dihydroxyphenylacetic and homovanillic acids) in the brain structures of silver foxes which differ in their handling reactions. The level of noradrenaline was found to be significantly higher in the anterior hypothalamus of domesticated animals; no differences in noradrenaline content were found in the frontal cortex, hippocamp, posterior hypothalamus and midbrain in animals from aggressive and domesticated groups. Dopamine content was higher in the tuberculum olfactorium of domesticated animals, being lower in the striatum and n. accumbens. Metabolite level remained unaffected which is presumably due to changes in dopamine synthesis in the investigated structures. It was concluded that domestication of animals favours the specimens with an altered state of catecholaminergic system of the brain.     

Immobilization stress-induced changes in catecholamine levels in the individual cerebral nuclei in rats of different zoosocial ranks

Immobilization stress induced catecholamines level changes were studied in brain nuclei, taking part in blood pressure central regulation in rats with different zoosocial position. The significant differences in catecholamines level between the zoosocial groups of rats were shown in dopamine synthesized hypothalamic nuclei-n. arcuatus, n. periventricularis and noradrenaline synthesized-n. subcoeruleus++. The dominant rats were characterized by the highest catecholamines level.     

Estradiol is responsible for reduced renal prostaglandin dehydrogenase activity in female rats

The contribution of sex steroids to sex-related differences in renal prostaglandin dehydrogenase activity and urinary prostaglandin excretion was examined in 7-8-week-old male and female rats subjected to sham-operation or gonadectomy at 3 weeks of age. Rats were injected subcutaneously twice over a 6-day interval with vehicle (peanut oil, 0.5 mg/kg) or with depot forms of testosterone (10 mg/kg), estradiol (0.1 mg/kg), progesterone (5 mg/kg), or with estradiol and progesterone combined (0.1 and 5 mg/kg). After the second injection, 24-h urine samples were collected for prostaglandin measurement by radioimmunoassay; the rats were killed, and renal and pulmonary prostaglandin dehydrogenase activities were determined by radiochemical assay. Renal prostaglandin dehydrogenase activity was 10-times higher in intact male rats than in intact females. Gonadectomy increased renal prostaglandin dehydrogenase activity 4-fold in females, but had no effect in males; estradiol, alone or combined with progesterone, markedly suppressed renal prostaglandin dehydrogenase activity in both sexes, while testosterone or progesterone alone had no effect. Pulmonary prostaglandin dehydrogenase did not differ between the sexes and was unaffected by gonadectomy or sex-steroid treatment. Intact female sham-operated rats excreted 70-100% more prostaglandin E2, prostaglandin F2 alpha, and 6-keto-prostaglandin F1 alpha in urine than did males; gonadectomy abolished the difference in urinary prostaglandin E2 excretion. Estradiol decreased urinary prostaglandin E2 in females but not in males; treatment with other sex steroids did not alter urinary prostaglandin excretion.     

Effects of gonadal steroids on tuberoinfundibular and tuberohypophysial dopaminergic neuronal activity in male and female rats

The activities of tuberoinfundibular and tuberohypophysial dopamine (DA) neurons were estimated by measuring the turnover of DA in terminals of these neurons in the median eminence and in the neural and intermediate lobes of the pituitary, respectively. The rate of DA turnover (alpha-methyltyrosine-induced decline of DA) in the median eminence was two to three times faster in females than in males, but no sexual differences in DA turnover rates were noted in the neural and intermediate lobes. Two weeks following gonadectomy the rate of DA turnover in the median eminence was increased in the male but decreased in the female. These effects were reversed by testosterone and estrogen replacement in gonadectomized males and females, respectively. Neither gonadectomy nor steroid replacement altered DA turnover in the neural or intermediate lobes of either males or females. These results indicate that estrogen stimulates and testosterone inhibits tuberoinfundibular DA neuronal activity while neither steroid affects tuberohypophysial DA neuronal activity.     

Effect of age, gonadectomy and hypophysectomy on mitochondrial hydroxylation of vitamin D3 (cholecalciferol) and of 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol in female and male rat liver.

In a previous study we found that liver mitochondrial side-chain hydroxylation of vitamin D3 (cholecalciferol) and of 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol was higher in female than in male rats [Saarem & Pedersen (1987) Biochem. J. 247, 73-78]. The present paper describes the effects of age, gonadectomy and hypophysectomy on these activities. The sex difference became manifest above the age of 7 weeks. Ovariectomy and/or injection of oestradiol valerate had no effect on the hydroxylase activities in adult females. Castration increased, and subsequent testosterone treatment decreased, the hydroxylase activities in adult males. Hypophysectomy had no effect in females, but increased the hydroxylase activities in males. Testosterone treatment had no effect in hypophysectomized females or males. Injection of oestradiol valerate had no effect on the hydroxylase activities in hypophysectomized females. In hypophysectomized males this treatment had no effect on the vitamin D3 25-hydroxylase activity, but decreased the C27-steroid 27-hydroxylase activity in males. Microsomal 1 alpha-hydroxyvitamin D3 25-hydroxylase activity was lower in females than in males in all age groups. Castration or hypophysectomy decreased the activity in male rats. It is concluded that, in adult female rats, the mitochondrial side-chain hydroxylation of vitamin D3 and of 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol is independent of sex hormones. In males these activities are regulated by influence of sex hormones on the hypophysis, probably by the presence of androgens in the neonatal period. Different effects on the two hydroxylases indicate the presence of at least two different cytochromes P-450 in rat liver mitochondria.     

Effect of steroid aromatase inhibitors on the catecholamine content of the hypothalamus of neonatally androgenized rats

Administration of 50 micrograms of testosterone propionate to newborn female rats on the 5th day of life provoked a reliable increase in noradrenaline and dopamine concentrations in the hypothalamus of 10-day-old rats. Neonatal administration of aromatase inhibitors on the 5th and 7th days of life prevented testosterone-induced increase in catecholamine concentrations. The data obtained prove the integration of the processes of testosterone aromatization and catecholamine accumulation in androgen-dependent brain differentiation.     

Differential effects of experimentally induced blood pressure changes on the release of catecholamines in hypothalamic and limbic areas of rats

The effects of longer lasting blood pressure changes on the release of endogenous catecholamines (CA) in limbic and hypothalamic areas were studied in anaesthetized rats. For this purpose the central nucleus of the amygdala (AC), ventral hippocampus (VH) and medial hypothalamus (MH) were simultaneously superfused through push-pull cannulae with artificial cerebrospinal fluid and the release of the endogenous catecholamines dopamine (DA), noradrenaline (NA) and adrenaline (A) was determined before and after blood pressure manipulations. A fall in blood pressure elicited by the ganglionic blocking agent chlorisondamine resulted in different changes of the various CA release patterns in AC. Short lasting increased CA release rates as compared to prehypotension levels could be observed in the hippocampus. The activity of catecholaminergic neurons in MH remained unchanged. A rise in arterial blood pressure induced by intravenous injection of tramazoline did not change the release rates of DA in all 3 brain areas studied. In hippocampus, NA levels in the superfusates decreased initially during hypertension but returned to normal values 40 min after drug injection. In the late phase of hypertension increased rates of release of NA in the amygdala and of A in the hypothalamus could be observed. The different patterns in the release of CA suggest that DA, NA and A are differentially implicated in the regulation of experimentally induced blood pressure changes.     

Endogenous Noradrenaline and Dopamine in Nerve Terminals of the Hippocampus: Differences in Levels and Release Kinetics

The presence and release of endogenous catecholamines in rat and guinea pig hippocampal nerve terminals was studied by fluorimetric HPLC analysis. In isolated nerve terminals (synaptosomes) the levels and breakdown of endogenous catecholamines were determined and the release process was characterized with respect to its kinetics and Ca2+ and ATP dependence. Endogenous noradrenaline and dopamine, but not adrenaline, were detected in isolated hippocampal nerve terminals. For dopamine both the levels and the amounts released were more than 100-fold lower than those for noradrenaline. In suspension, released endogenous catecholamines were rapidly broken down. This could effectively be blocked by monoamine oxidase inhibitors, Ca(2+)-free conditions, and glutathione. The release of both noradrenaline and dopamine was highly Ca2+ and ATP dependent. Marked differences were observed in the kinetics of release between the two catecholamines. Noradrenaline showed an initial burst of release within 10 s after K+ depolarization. The release of noradrenaline was terminated after approximately 3 min of K+ depolarization. In contrast, dopamine release was more gradual, without an initial burst and without clear termination of release within 5 min. It is concluded that both catecholamines are present in nerve terminals in the rat hippocampus and that their release from (isolated) nerve terminals is exocytotic. The characteristics of noradrenaline release show several similarities with those of other classical transmitters, whereas dopamine release characteristics resemble those of neuropeptide release in the hippocampus but not those of dopamine release in other brain areas. It is hypothesized that in the hippocampus dopamine is released from large, dense-cored vesicles, probably colocalized with neuropeptides.