Effects of Handling or Immobilization on Plasma Levels of 3,4-Dihydroxyphenylalanine, Catecholamines, and Metabolites in Rats

In conscious animals, handling and immobilization increase plasma levels of the catecholamines norepinephrine (NE) and epinephrine (EPI). This study examined plasma concentrations of endogenous compounds related to catecholamine synthesis and metabolism during and after exposure to these stressors in conscious rats. Plasma levels of 3,4-dihydroxyphenylalanine (DOPA), NE, EPI, and dopamine (DA), the deaminated catechol metabolites 3,4-dihydroxyphenylglycol (DHPG), and 3,4-dihydroxyphenylacetic acid (DOPAC), and their O-methylated derivatives methoxyhydroxyphenylglycol (MHPG) and homovanillic acid (HVA) were measured using liquid chromatography with electrochemical detection at 1, 3, 5, 20, 60, and 120 min of immobilization. By 1 min of immobilization, plasma NE and EPI levels had already reached peak values, and plasma levels of DOPA, DHPG, DOPAC, and MHPG were increased significantly from baseline, whereas plasma DA and HVA levels were unchanged. During the remainder of the immobilization period, the increased levels of DOPA, NE, and EPI were maintained, whereas levels of the metabolites progressively increased. In animals immobilized briefly (5 min), elevated concentrations of the metabolites persisted after release from the restraint, whereas DOPA and catecholamine levels returned to baseline. Gentle handling for 1 min also significantly increased plasma levels of DOPA, NE, EPI, and the NE metabolites DHPG and MHPG, without increasing levels of DA or HVA. The results show that in conscious rats, immobilization or even gentle handling rapidly increases plasma levels of catecholamines, the catecholamine precursor DOPA, and metabolites of NE and DA, indicating rapid increases in the synthesis, release, reuptake, and metabolism of catecholamines.     

Effect of transient ischemia on monoamine levels in the cerebral cortex of gerbils

Abstract— Cortical monoamine changes during ischemic episodes of varied duration and their sequence of changes following cerebral reperfusion were studied in the gerbil. Forty-one percent of 280 animals exhibited signs of cerebral hemispheric ischemia (stroke) after unilateral common carotid artery occlusion. Norepinephrine (NE) levels decreased after 60 min in the occluded hemisphere of stroked animals but dopamine (DA) levels were unaltered. S-Hydroxytryptamine (5-HT) levels became bilaterally reduced in both stroked and non-stroked animals as soon as S min after occlusion. Upon reperfusion after periods of 30 or 60 min of occlusion there was a bilateral rebound increase of cortical NE and DA levels to well above control values in stroked and non-stroked animals. 5-HT levels remained reduced in both groups. Results suggest disorder of monoamine metabolism in ischemic brain which persists during the early reperfusion period, perhaps contributing to deficits in neurological function. Monoamine changes in contralateral non-ischemic hemispheres both during the occlusion and reperfusion periods are thought further evidence of diaschisis.     

Effect of restraint stress on rat brain serotonin

Restraint-induced stress in rats was found to enhance steady state concentrations of whole brain and hypothalamic serotonin, at 1,2 and 4 h after immobilization. The increase was maximal at 1 h and tended to decline thereafter. The rate of accumulation of rat brain serotonin, in pargyline pretreated animals, was significantly enhanced after restraint stress. Bilateral adrenalectomy and metyrapone, an endogenous corticoid synthesis inhibitor, failed to affect restraint stress (1h)-induced increase in rat brain serotonin levels. Thus restraint stress-induced autoanalgesia and potentiation of the pharmacological actions of several centrally acting drugs, in rats, are serotonin-mediated responses. The results also indicate that restraint stress-induced effects on rat brain serotonin are not dependent on endogenous corticoid activity.     

Stress mediated changes in hypothalamic corticotropin releasing factor-like immunoreactivity

Hypothalamic corticotropin releasing factor-like immunoreactivity (CRF-LI), plasma ACTH and corticosterone levels were measured by radioimmunoassay over a two hour period of restraint stress. The results of this study demonstrate a significant decrease in hypothalamic CRF-LI levels 15 and 30 minutes after the start of restraint stress which is followed by a significant increase at 60 minutes that is abolished by cycloheximide pretreatment. Plasma ACTH and corticosterone levels were significantly elevated after 15, 30, 60, 90, and 120 minutes of restraint stress. These results are consistent with a release of CRF from the hypothalamus during stress. The cycloheximide-sensitive increase in hypothalamic CRF-LI indicates that synthesis of CRF-41 occurs during prolonged stress. These results suggest that the response of an organism to exposure to a long-term, high intensity stress involves both the release and synthesis of CRF-41.     

Alterations in Norepinephrine and Dopamine Content in Selected Brain Areas of Guinea Pigs Adapted to Simulated High Altitude

Abstract: The alterations in brain content of norepinephrine (NE) and dopamine (DA) were studied in guinea pigs adapted to simulated high altitude (hypobaric hypoxia) equivalent to 5500 meters. The animals were adapted for 46 days over a period of 82 days to a pressure of 375 mm Hg. The animals were then killed and the following brain parts dissected: cerebellum, neocortex, caudate head/basal forebrain, diencephalons/rhinencephalon, and brain stem. NE and DA content were analyzed by high pressure liquid chromatography with electrochemical detection by a technique described. Results showed a significant increase of NE and DA in neocortex; a significant increase of DA but not NE in caudate/basal forebrain, and a significant decrease of NE and not DA in diencephalons/rhinencephalon.     

Serotonin in brain regions of rats differing in their inborn locomotor activity

Serotonin contents in the paraventricular hypothalamic nucleus (PVN) and dorsal hippocampus of rats with different levels of inborn motor activity were studied by microdialysis in basal and stimulated conditions. Rats were exposed to elevated platform and forced swimming stress. In basal conditions, differences in serotonin contents between rats with different levels of inborn motor activity were found neither in hippocampus nor in PVN. In both kinds of stress conditions, serotonin content in hippocampus increased only in rats with higher level of inborn motor activity. Serotonin content in PVN dramatically increased during forced swimming in both rat groups. This increase was significantly more pronounced in rats with low activity. The data suggest that serotonin release in stress depends on inborn motor activity, brain area dialyzed, and the stressor the animals were exposed to.     

Time-dependent changes in brain biogenic amine dynamics following castration in male rats

—Alterations in whole-brain and hypothalamic levels of serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), norepinephrine (NE), dopamine (DA) as well as the turnover rates of NE and DA of adult male rats were analysed fluorometrically at either 3 weeks or 6 weeks following castration. Significant increases were observed in whole-brain (minus hypothalamus) 5-HIAA levels and hypothalamic DA levels, fractional rate constants and utilization rates at the 3 but not the 6 week intervals. Elevated levels of 5-HT were observed at both time intervals while an increase in whole-brain DA was seen only at the 6 week interval. Whole brain NE turnover rates of castrated animals did not differ significantly from those of sham-castrate control animals at either test interval. However, a tendency toward increased hypothalamic NE turnover rates was seen in the castrated animals. These biochemical changes resulted in decreased NE/5-HT and DA/5-HT ratios for the castrate rats as compared to controls. The results are discussed in relation to emotional and aggressive behavior and are interpreted as being consistent with the hypothesis purporting an inhibitory role for 5-HT and excitatory role for NE and DA in sex-specific behavior patterns including aggression.     

Alterations in Regional Brain Catecholamine Concentrations After Experimental Brain Injury in the Rat

Abstract: Although activation of brain catecholaminergic systems has been implicated in the cerebrovascular and metabolic changes during subarachnoid hemorrhage, cerebral ischemia, cortical ablation, and cortical freeze lesions, little is known of the response of regional brain catecholamine systems to traumatic brain injury. The present study was designed to characterize the temporal changes in concentrations of norepinephrine (NE), dopamine (DA), and epinephrine (E) in discrete brain regions following experimental fluid-percussion traumatic brain injury in rats. Anesthetized rats were subjected to fluid-percussion brain injury of moderate severity (2.2–2.3 atm) and killed at 1 h, 6 h, 24 h, 1 week, and 2 weeks postinjury (n = 6 per timepoint). Control animals (surgery and anesthesia without injury) were killed at identical timepoints (n = 6 per timepoint). Tissue concentrations of NE, DA, and E were evaluated using HPLC. Following brain injury, an acute decrease was observed in DA concentrations in the injured cortex ( p < 0.05) at 1 h postinjury, which persisted up to 2 weeks postinjury. Striatal concentrations of DA were significantly increased ( p < 0.05) only at 6 h postinjury. Hypothalamic concentrations of DA and NE increased significantly beginning at 1 h postinjury ( p < 0.05 and p < 0.05, respectively) and persisted up to 24 h for DA ( p < 0.05) and 1 week ( p < 0.05) for NE. These data suggest that acute alterations occur in regional concentrations of brain catecholamines following brain trauma, which may persist for prolonged periods postinjury.     

Brain catecholaminergic and tryptophan responses to restraint are attenuated by nitric oxide synthase inhibition

Increases in the brain concentrations of tryptophan and in serotonin (5-HT) metabolism are commonly observed in animals under stress. Previous experiments indicated that the increase in brain tryptophan and 5-hydroxyindoleacetic acid (5-HIAA) observed in response to administration of endotoxin (lipopolysaccharide, LPS) and interleukin-1 (IL-1) were largely prevented by pretreatment with N-nitro-L-arginine methylester (L-NAME), an inhibitor of NO synthase (NOS). Therefore we tested whether the increases in tryptophan and 5-HT metabolism observed following restraint and footsthock were similarly affected. Mice were injected with L-NAME (30 mg/kg) or saline and restrained for 40 min. Restraint caused increases in concentrations of tryptophan and the catabolites of dopamine (DA), norepinephrine (NE) and 5-HT in the medial prefrontal cortex, hypothalamus, and brain stem. The L-NAME pretreatment significantly attenuated, but did not prevent, the changes in tryptophan and catecholamine metabolism, with a very small effect on the increase in plasma corticosterone. When mice pretreated with L-NAME were subjected to 30 min footshock, the NOS inhibitor had no statistically significant effects on the increases in DA, NE and 5-HT metabolism, but tended to attenuate the increases in tryptophan. We interpret these results to indicate that NOS plays a relatively small role in the cerebral neurochemical responses to restraint and footshock, but the role in the restraint-induced changes was greater than that in the footshock-induced ones. The attenuation of the restraint-related effects on the catecholamines most probably reflects a contribution to the CNS responses from peripheral vascular changes which are likely to be limited by the inhibition of NOS.     

Role of hypothalamic inputs in maintaining pituitary-adrenal responsiveness in repeated restraint

The role of hypothalamic structures in the regulation of chronic stress responses was studied by lesioning the mediobasal hypothalamus or the paraventricular nucleus of hypothalamus (PVH). Rats were acutely (60 min) and/or repeatedly (for 7 days) restrained. In controls, a single restraint elevated the plasma adrenocorticotropin (ACTH), corticosterone, and prolactin levels. Repeated restraint produced all signs of chronic stress, including decreased body and thymus weights, increased adrenal weight, basal corticosterone levels, and proopiomelanocortin (POMC) mRNA expression in the anterior pituitary. Some adaptation to repeated restraint of the ACTH response, but not of other hormonal responses, was seen. Lesioning of the mediobasal hypothalamus abolished the hormonal response and POMC mRNA activation to acute and/or repeated restraint, suggesting that the hypothalamo-pituitary-adrenal axis activation during repeated restraint is centrally driven. PVH lesion inhibited the ACTH and corticosterone rise to the first restraint by approximately 50%. In repeatedly restrained rats with PVH lesion, the ACTH response to the last restraint was reduced almost to basal control levels, and the elevation of POMC mRNA level was prevented. PVH seems to be important for the repeated restraint-induced ACTH and POMC mRNA stimulation, but it appears to partially mediate other restraint-induced hormonal changes.     

Effect of 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) on monoamine neurotransmitters in mouse brain & heart

The effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was studied on dopamine (DA), norepinephrine (NE), serotonin (5HT) and γ-aminobutyric acid (GABA) neurons in mouse brain and on NE neurons of mouse heart. MPTP (45 mg/kg) was administered s.c. to mice twice daily for 2 consecutive days. This dosage regimen produced a decrease in the forebrain concentrations of DA and NE at 7 and 20 days after injection. In contrast, the forebrain concentrations of 5HT and GABA were not significantly decreased at either time. MPTP administration also produced a marked decrease in the uptake of ³H-DA into striatal slices and ³H-NE into cerebral cortical slices. In contrast, the uptake of ³H-NE into hypothalamic slices and the uptake of ³H-5HT into slices from several brain regions were not altered. MPTP initially reduced the concentration of NE in the heart, but unlike the persistent decreases in the forebrain concentrations of NE and DA, the NE concentration in the heart returned to control levels at approximately 20 days after MPTP administration. These results, showing that MPTP can produce a long lasting and selective decrease in the forebrain concentrations of NE and DA and in the uptake of radioactive DA and NE into brain slices, suggest that MPTP can cause the destruction of catecholamine neurons in mouse brain. In contrast, MPTP administration does not appear to produce long term changes in either 5HT or GABA neurons.     

Feeding increases 5-hydroxytryptamine and norepinephrine within the hypothalamus of chicks

It is thought that hypothalamic 5-hydroxytryptamine (5HT) and norepinephrine (NE) are involved in the regulation of feeding in chicks. The present study was conducted to elucidate changes in the levels of extracellular 5HT and NE in the hypothalamus during feeding of chicks. In order to measure 5HT, NE and 4-hydroxy-3-methoxyphenylglycol (MHPG), which is a major metabolite of NE, we used brain microdialysis and high-pressure liquid chromatography with an electrochemical detector. After collecting samples to determine the basal levels of 5HT, NE and MHPG, food-deprived birds were given access to food. 5HT levels in the medial hypothalamus (MH) and lateral hypothalamus (LH) increased during the first 30 min of feeding, and then returned to basal levels. NE and MHPG in the LH increased during feeding, and remained elevated throughout the experiment. This study supports an idea that hypothalamic monoamines in the chick brain are involved in the regulation of feeding.     

The agouti coat-color locus may influence brain catecholamines: Regional differences in norepinephrine and dopamine in the brains of two color-morphs of deermice (Peromyscus maniculatus)

Levels of norepinephrine (NE) and dopamine (DA) were measured by HPLC from the cortex, striatum and brain stem of agouti and nonagouti deermice (Peromyscus maniculatus gracilis). In the brain stem, agouti animals exhibited significantly higher NE whereas nonagouti animals had significantly higher DA. In contrast, agouti deermice had nearly twice the DA levels of nonagouti animals in both the cortex and striatum. Levels of cortical and striatal NE were similar in both color morphs, although NE in nonagouti animals was 8% higher and less variable. The variability of both NE and DA was much lower in the brain stem than in either the striatum or the cortex. This is the first evidence of a pleiotropic effect of the agouti coat-color locus on brain catecholamines.     

Stress-Related Changes in Cerebral Catecholamine and Indoleamine Metabolism: Lack of Effect of Adrenalectomy and Corticosterone

The concentrations of catecholamine and indoleamine metabolites were measured in intact and adrenalectomized mice to determine whether adrenal hormones mediate or modulate the stress-induced responses. Thirty minutes of footshock resulted in significant increases of the ratios of the dopamine (DA) catabolite, dihydroxyphenylacetic acid (DOPAC), to DA in prefrontal cortex, nucleus accumbens, striatum, hypothalamus, and brainstem, and of homovanillic (HVA)/DA ratios in nucleus accumbens, striatum, amygdala, and hypothalamus. Ratios of 3-methoxy-4-hydroxyphenylethyleneglycol to norepinephrine (NE) were also increased in prefrontal cortex, nucleus accumbens, septum, amygdala, hypothalamus, hippocampus, and brainstem. The concentration of NE was decreased in amygdala. 5-Hydroxyindoleacetic acid (5-HIAA)/5-hydroxytryptamine (5-HT, serotonin) ratios and free tryptophan were also increased in every brain region. Very similar data were obtained from mice restrained for 30 min. Adrenalectomy resulted in increased HVA/DA ratios in prefrontal cortex and striatum, and 5-HIAA/5-HT in septum. The stress-related changes were largely similar in adrenalectomized mice. Significant interactions between adrenalectomy and footshock treatment occurred in prefrontal cortical DOPAC/DA and hypothalamic NE which was depleted only in adrenalectomized mice, suggesting tendencies for these measures to be more responsive in adrenalectomized mice. Corticosterone administration (0.5-2.0 mg/kg s.c.) which resulted in plasma concentrations in the physiological range did not alter the concentrations of the cerebral metabolites measured in any region. We conclude that adrenal hormones do not mediate cerebral catecholamine or indoleamine metabolism in stress, although adrenalectomy may affect HVA and 5-HIAA metabolism, and there was a tendency for catecholamines to be more sensitive to stress in adrenalectomized animals.     

Catecholamine level in cerebrospinal fluid of epileptics

We estimated catecholamine levels in CSF of 15 epileptics and 75 non-neurological patients utilizing a high performance liquid chromatograph with a highly sensitive fluorometer and found the following results: The dopamine (DA) levels in males were significantly higher than those in females, while norepinephrine (NE) levels in males were the same as in females. The DA levels were significantly lower and NE levels significantly higher in epileptics than in non-neurological patients. DA and NE in petit mal patients were on the average lower than in grand mal patients, but untreated grand mal patients had higher NE levels. These results suggest that epilepsy may be associated with a disturbance of DA and/or NE metabolism or release in the brain.     

Effects of estradiol on circulating levels of prolactin in female rats bearing ectopic pituitaries

The existence of local mechanisms controlling the prolactin (PRL) release from anterior pituitaries (AP) grafted to an ectopic location has been recently described. To study if these mechanisms are affected by estrogens, pituitary-grafted (GRAFT) and sham-operated (SHAM) rats were injected with a single dose of estradiol benzoate (EB), their plasma PRL levels as well as their hypothalamic and AP contents of norepinephrine (NE) and dopamine (DA) being analyzed. Administration of EB to GRAFT animals produced a small increase in their previously high plasma PRL levels, with both an increased NE and a decreased DA content in the ectopic AP. Since NE enhances the PRL release from ectopic AP and DA partially inhibits this secretion these changes may explain such a small increase in PRL levels. However, an additional increase in the decreased PRL release from the in situ AP of these animals cannot be discarded since EB produced also a decrease of the DA content in this tissue with an unaltered hypothalamic content. Finally, administration of this steroid to SHAM animals produced an important increase in plasma PRL levels. Since this increase was correlative to a decrease in DA and NE hypothalamic contents and unaltered AP contents. EB may be supposed to be able to reduce the DA synthesis in the tuberoinfundibular neurons, while the changes in noradrenergic inputs could be more related to the feedback effects of estrogens on the gonadotrophin release.     

Chronic Blockade of Nitric Oxide Synthesis Elevates Plasma Levels of Catecholamines and Their Metabolites at Rest and During Stress in Rats

Formation of nitric oxide, an endothelium-derived relaxing factor, can be inhibited by administration of N-nitro-L-arginine methylesther (L-NAME). In the present study, the activity of the sympathoadrenal system in rats with blood pressure (BP) elevation induced by L-NAME was investigated. L-NAME was administered in a dose of 50 mg/kg, i.p. every 12 h for 4 days. Blood samples were collected via chronically inserted arterial catheters in conscious, freely moving rats at rest and during immobilization stress. Plasma epinephrine (EPI), norepinephrine (NE), and dopamine (DA), as well as catecholamine metabolites dihydroxyphenylglycol (DHPG) and dihydroxyphenylacetic acid (DOPAC) were measured by HPLC method. In L-NAME treated animals, which showed a significant increase in BP, plasma EPI levels were markedly elevated both before and during stress. Plasma NE levels were not significantly increased, however, DHPG levels, which indicate NE turnover and reuptake, were highly elevated. Plasma DA levels were not changed after L-NAME administration but DA metabolite DOPAC showed a significant elevation both under basal conditions and during stress. Thus, the present results indicate that the prolonged blockade of nitric oxide synthesis that causes arterial hypertension is associated with an activation of the sympathoadrenal system.     

The influence of alpha-fluoromethylhistidine on the regional brain histamine and plasma corticosterone levels in aging

alpha-Fluoromethylhistidine, a histidine decarboxylase inhibitor, induced a significant depletion in the hypothalamic, midbrain, and cortical brain histamine amounts in 12- and 3-month-old rats. In all three brain regions the most evident depletion occurred 2 h after treatment. In both groups of rats midbrain histamine levels returned to control values 6 h after treatment; however, hypothalamic histamine depletion was still significant and more evident in the old than in the young animals. Cortical brain histamine also remained significantly depleted in old rats, but returned to control values in young animals 6 h after alpha-fluoromethylhistidine treatment. These results suggest that old rats show a slower rate of new histamine synthesis in the cortex and hypothalamus. Regional brain histamine depletion was associated with a very significant decrease in plasma corticosterone levels, which indicates that brain histamine-corticosterone interactions do occur.     

Influence of restraint and ketamine anesthesia on adrenal steroids, progesterone, and gonadotropins in rhesus monkeys

Changes in gonadotropins, progesterone, cortisol, DHA, and DHAS were monitored in 10 female rhesus monkeys (Days 20-23 of the menstrual cycle) subjected to cage restraint with or without ketamine anesthesia for successive venipunctures. All animals were bled without sedation for 2 hr at 30-min intervals. Then 4 of the animals were anesthetized with ketamine-HCl and bleedings in all animals were continued for an additional 2.5 hr. FSH and progesterone were not appreciably affected by either restraint technique. LH declined steadily for the duration of the bleedings (P less than 0.05). Serum levels of cortisol and the adrenal androgens increased twofold (P less than 0.05). Anesthesia with ketamine had no effect on any of the six variables when compared with saline controls. Cortisol and dehydroepiandrosterone (DHA) levels tended to plateau (P less than 0.01) after 2 hr in both treated and control groups. In contrast, dehydroepiandrosterone sulfate (DHAS) levels increased continuously throughout the entire study period. These data indicate that ketamine anesthesia does not alter endocrine responses to venipuncture when administered following cage restraint of conscious animals. These findings further confirm the difficulties in obtaining estimates of basal levels of hormones which are responsive to stress and suggest that the first sample may provide the best estimate.     

Characterization of pituitary–adrenocortical activity in the Malayan flying fox (Pteropus vampyrus)

Pituitary–adrenocortical and gonadal endocrine activity was investigated in a captive colony of Pteropus vampyrus, a highly social Old World fruit bat. Both cortisol and corticosterone were present in plasma, at a ratio of approximately 5:1, respectively. Glucocorticoid but not testosterone levels significantly increased prior to and concomitant with the evening active period. Restraint stress for 15–60 min resulted in a significant and rapid increase in plasma levels of adrenocorticotropic hormone (ACTH) and glucocorticoids. ACTH levels quickly returned to baseline following restraint whereas glucocorticoid levels remained elevated for at least 30 min after restraint ended. Plasma ACTH levels after stress were similar to levels reported after stress in other mammals. Stress-induced glucocorticoid levels were several-fold greater than those reported for most mammals. Restraint for 15 min significantly inhibited testosterone levels. Restraint stress did not affect hormone levels on the morning following restraint. Brief capture, handling, and release of the animals did not elicit increases in these hormones. The physiological responsiveness of the pituitary and adrenal glands, along with P. vampyrus’s documented seasonality and range of social behaviors, makes these bats an excellent model for exploring the general physiology of the hypothalamic–pituitary–adrenal and hypothalamic–pituitary–gonadal axes, as well as social influences on these axes.