Corticotropin-Releasing Factor Stimulates Catecholamine Release in Hypothalamus and Prefrontal Cortex in Freely Moving Rats as Assessed by Microdialysis

Abstract: In vivo microdialysis was used to measure changes in extracellular concentrations of catecholamines and indoleamines in freely moving rats in response to administration of corticotropin-releasing factor (CRF). Dialysis probes were placed stereotaxically in either the medial hypothalamus or the medial prefrontal cortex. We used a repeated-measures design in which each rat received artificial CSF or one dose of CRF 3–4 h apart, and each subject was retested with the same treatments in the reverse order 5–7 days later. With the dialysis probe in the hypothalamus, intracerebroventricular administration of CRF (17 or 330 pmol) dose-dependently increased dialysate concentrations of norepinephrine (NE), dopamine (DA), and all their measurable catabolites except normetanephrine. The effects on NE were substantially greater than those on DA. Dialysate concentrations of serotonin could not be measured reliably, but those of its catabolite, 5-hydroxyindoleacetic acid, were also elevated. Concentrations of NE and DA were elevated within the first one or two (20 min) collection periods, with a peak response at ∼ 1–2 h. Dialysate concentrations of catecholamines and metabolites normally returned to baseline within 3 h. Similar data were obtained with dialysis probes in the medial prefrontal cortex after intracerebroventricular administration of 17 or 167 pmol of CRF, except that the increases in DA exceeded those of NE in this region. Intraperitoneal administration of CRF (1 nmol) similarly elevated dialysate concentrations of NE, DA, 5-hydroxyindoleacetic acid, and all catecholamine catabolites except normetanephrine in both medial hypothalamus and medial prefrontal cortex. These results support earlier neurochemical data suggesting that CRF administered both centrally and peripherally stimulates the release of both DA and NE in the brain.     

Effects of Colchicine Application to Preganglionic Axons on Choline Acetyltransferase Activity and Acetylcholine Content and Release in the Superior Cervical Ganglion

Abstract: These experiments investigate the effect of block, by colchicine, of fast axonal transport in the cat's cervical sympathetic trunk (CST) on the superior cervical ganglion's choline acetyltransferase (ChAT) enzyme activity, acetylcholine (ACh) content, and ACh release. Electron microscopy on the segment of the CST exposed to colchicine 1 or 4 days earlier showed disappearance of microtubules and accumulation of vesicles and smooth membrane tubules but no disruption of the axonal cytomatrix. At 4 days following colchicine treatment, the number and size of synaptic boutons per grid square in the ganglion ipsilateral to the colchicine-treated CST were similar to those in the control ganglion. At 2 and 4 days following exposure of the CST to colchicine, ChAT activity in the ipsilateral ganglion was reduced to 76 ± 8 and 54 ± 6% of control values, respectively. ACh stores in the ganglia were also reduced (to 81 ± 6% of control values at 2 days and to 51 ± 5% of control values at 4 days). Ganglionic transmission and its sensitivity to blockade by hexamethonium during 2-Hz CST stimulation were not impaired at day 4 postcolchicine. ACh release evoked by 2-Hz stimulation of colchicine-treated axons was similar to release from untreated axons, despite the decrease in the ganglionic ACh content. In contrast, ACh release evoked by 20-Hz stimulation was depressed. The amount of ACh released during 5-Hz stimulation in the presence of vesamicol by the terminals of colchicine-treated axons was similar to that released by the terminals of untreated axons. These results suggest the following conclusions: (a) Colchicine-sensitive fast axonal transport contributes significantly to maintaining ChAT stores in preganglionic axon terminals. (b) The half-life of ChAT in sympathetic preganglionic terminals is ～4 days. (c) One consequence of colchicine-induced block of axonal transport is a reduced ACh content of preganglionic nerve terminals. (d) This decrease in ACh content appears to be the result of a loss in a reserve transmitter pool, whereas the size of the readily releasable compartment is maintained.     

Hypothalamic Catecholamine Metabolism in Diabetic Rats: The Effect of Insulin Deficiency and Meal Ingestion

The effect of moderate insulin deficiency of 2 weeks in duration on hypothalamic catecholamine metabolism in food-deprived and meal-fed rats was evaluated. Hypothalamic tyrosine content in food-deprived (from 0700 to 1600 h), diabetic rats was normal. Also normal were the rates of 3,4-dihydroxyphenylalanine accumulation following aromatic amino acid decarboxylase inhibition, norepinephrine and 3,4-dihydroxyphenylethylamine (dopamine) clearance after tyrosine hydroxylase inhibition, and intraneuronal amine accumulation following monoamine oxidase inhibition. Differences in hypothalamic amine metabolism were apparent, however, when diabetic and normal rats were fed 2-g meals. The 3-methoxy-4-hydroxyphenylethyleneglycol sulfate accumulation rate was depressed in diabetic rats by the carbohydrate meal but was stimulated by the tyrosine-supplemented protein meal. In contrast, the tyrosine-supplemented diet had no effect on 3,4-dihydroxyphenylacetic acid accumulation in diabetic animals, whereas the production rate in normal rats was increased. We conclude that normal responses occurring in hypothalamic catecholamine metabolism after the consumption of a meal are modified by the presence of diabetes.     

Transsynaptic Regulation of Olfactory Bulb Catecholamines in Mice and Rats

Norepinephrine (NE), dopamine (DA), 3,4-dihydroxyphenylalanine (DOPA), and 3,4-dihydroxyphenylacetic acid (DOPAC) were measured simultaneously by high performance liquid chromatography with electrochemical detection in extracts of olfactory bulbs at various intervals after chemical or surgical deafferentation. Chemical deafferentation of mice by intranasal irrigation with Triton X-100 or of rats by olfactory axotomy resulted in a rapid progressive decline of DA and DOPAC and an associated rise in NE in the olfactory bulb. However, after several weeks, these values returned to prelesion levels concomitant with reinnervation of the bulb by the afferent neurons. In contrast, deafferentation by procedures known to prevent reinnervation of the bulb by the afferent chemoreceptor neurons (i.e., a ZnSo4 solution in mice or a surgical procedure in rats) completely blocked the return to pre-lesion values of DA, DOPAC, and NE. The specificity of these effects was demonstrated by the inability of intranasal administration of the neurotoxin 6-hydroxydopamine to alter DA levels, resulting instead in a significant decline in olfactory bulb NE content. These data demonstrate that the DA content of the olfactory bulb can be influenced by either chemical or surgical modulation of the afferent pathway in two different species. This offers additional support for our hypothesis of transsynaptic regulation of intrinsic DA neurons of the bulb by the afferent olfactory chemoreceptor neurons.     

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.     

Differential Effects of Chloral Hydrate and Pentobarbital Sodium on a Cocaine Level and Its Catecholamine Response in the Medial Prefrontal Cortex: A Comparison with Conscious Rats

Abstract: Adult male Sprague-Dawley rats anesthetized with chloral hydrate and pentobarbital sodium were used as two different treatment groups. Conscious rats were used as a control group. By using baseline (precocaine) concentration as 100%, after cocaine administration (3.0 mg/kg i.v.), the maximal dopamine (DA) increase occurring at the first microdialysis collection period (20 min) in the medial prefrontal cortex was 299 ± 46% for the chloral hydrate group, 168 ± 12% for the pentobarbital sodium group, and 325 ± 23% for the conscious group. At the same time, norepinephrine (NA) increases reached a maximum and were 162 ± 20%, 100 ± 5%, and 141 ± 17%, respectively. The maximal changes of DA and NA in the chloral hydrate group and in the control group were both significantly higher than that in the pentobarbital sodium group. Meanwhile, the cocaine concentration was higher over a 100-min period of time in the chloral hydrate group when compared with the pentobarbital group and the control group. The peak cocaine concentration in dialysate occurred in the same time slot of maximal DA and NA responses, which were 0.65 ± 0.08, 0.30 ± 0.02, and 0.41 ± 0.05 µ M , respectively. Anesthetics suppress the pharmacologic response of neurons, which may explain the difference in catecholamine response between the pentobarbital sodium and the conscious groups. Conversely, because there was no significant difference in DA and NA response between the chloral hydrate group and the conscious group, it may possibly be due to the balancing effect between the higher existing cocaine concentration and the anesthetic suppression on pharmacological response of neurons in the chloral hydrate group. The effect of guide cannula implantation on the cocaine-induced catecholamine response was also evaluated.     

Differential Effects of Chemical Sympathectomy on Expression and Activity of Tyrosine Hydroxylase and Levels of Catecholamines and DOPA in Peripheral Tissues of Rats

Tyrosine hydroxylase (TH) mRNA and activity and concentrations of 3,4-dihydroxyphenylalanine (DOPA) and catecholamines were examined as markers of sympathetic innervation and catecholamine synthesis in peripheral tissues of sympathectomized and intact rats. Chemical sympathectomy with 6-hydroxydopamine (6-OHDA) markedly decreased norepinephrine and to a generally lesser extent TH activities and dopamine in most peripheral tissues (stomach, lung, testis, duodenum, pancreas, salivary gland, spleen, heart, kidney, thymus). Superior cervical ganglia, adrenals and descending aorta were unaffected and vas deferens showed a large 92% decrease in norepinephrine, but only a small 38% decrease in TH activity after 6-OHDA. Presence of chromaffin cells or neuronal cell bodies in these latter tissues, indicated by consistent expression of TH mRNA, explained the relative resistance of these tissues to 6-OHDA. Stomach also showed consistent expression of TH mRNA before, but not after 6-OHDA, suggesting that catecholamine synthesizing cells in gastric tissue are sensitive to the toxic effects of 6-OHDA. Tissue concentrations of DOPA were mainly unaffected by 6-OHDA, indicating that much of the DOPA in peripheral tissues is synthesized independently of local TH or sympathetic innervation. The differential effects of chemical sympathectomy on tissue catecholamines, DOPA, TH mRNA and TH activity demonstrate that these variables are not simple markers of sympathetic innervation or catecholamine synthesis. Other factors, including presence of neuronal cell bodies, parenchymal chromaffin cells, non-neuronal sites of catecholamine synthesis and alternative sources of tissue DOPA, must also be considered when tissue catecholamines, DOPA and TH are examined as markers of sympathetic innervation and local catecholamine synthesis.     

Effect of Lesion of A5 and A7 Brainstem Noradrenergic Areas or Transection of Brainstem Pathways on Sympathoadrenal Activity in Rats During Immobilization Stress

Both A5 and A7 brainstem noradrenergic cell groups innervate dorsal horns of the spinal cord. Moreover, A5 cell group directly innervates sympathetic preganglionic neurons. Thus, A5 and A7 noradrenergic neurons could modulate the sympathoadrenal system (SAS) activity. We investigated the role of A5 and A7 noradrenergic cell groups in regulation of the SAS activity under control and stressful conditions. We evaluated the effect of electrolytical lesions of A5 or A7 cell groups and also the effect of bilateral brainstem cuts interrupting brainstem pathways on tyrosine hydroxylase gene expression in A5 and A7 areas and on the SAS activity measured by plasma epinephrine and norepinephrine levels. We have found that immobilization stress increases activity of the A5 and A7 brainstem areas and also levels of the gene expression of tyrosine hydroxylase, the rate-limiting catecholamine biosynthetic enzyme. Immobilization of sham-operated and brainstem pathways transected or A5 or A7 lesioned animals induced a similar, highly significant increase in plasma epinephrine and norepinephrine levels in both sham-operated and A5 or A7 destroyed or transected groups. Our data suggest that both A5 and A7 noradrenergic cell groups are activated during immobilization stress. However, transection of brainstem pathways innervating A5 and A7 neurons or lesion of A5 or A7 cell groups is not sufficient enough for changes in immobilization stress-induced activation of the SAS. We suggest that neither A5 and A7 noradrenergic neurons nor the transected brainstem pathways represent structures crucial for an activation of the SAS during immobilization stress. We hypothesize that during regulation of the stress response, various areas and pathways are involved and the elimination just one of them might be compensated by the remained intact areas and pathways.Special Issue Dedicated to Miklós Palkovits.     

Brain Catecholamine Alterations and Pathological Features with Aging in Parkinson Disease Model Rat Induced by Japanese Encephalitis Virus

We analyzed two disease model groups with rats infected by Japanese encephalitis virus (JEV), a 90-day group and a 180-day group after JEV infection. The time measured by the modified pole test showed that motor activities in these two groups were slower than those of age-matched control groups. Striatal dopamine (DA) levels were significantly decreased in all JEV-infected rats. Norepinephrine concentration in brain regions in the 180-day group was significantly decreased in the medulla oblongata and hypothalamus as compared with the control and 90-day group. Tyrosine hydroxylase-positive neurons were significantly decreased in both JEV-infected rat groups. These results suggest that DA decrease and pathological changes in JEV-infected model rats persist for a long time, at least up to 180 days, and this model will be useful for the evaluation of new anti-parkinsonian agents.     

Extracellular Dopamine, Norepinephrine, and Serotonin in the Nucleus Accumbens of Freely Moving Rats During Intracerebral Dialysis with Cocaine and Other Monoamine Uptake Blockers

Abstract: Monoamine-uptake blockers were applied focally (0.1–1,000 µ M ) through a dialysis probe in the nucleus accumbens of freely moving rats, and the extracellular concentrations of dopamine, norepinephrine, and serotonin were measured. The selective dopamine-uptake blocker GBR 12935 increased dopamine preferentially with only a small effect on norepinephrine, whereas the selective serotonin-uptake blocker fluoxetine increased serotonin output preferentially. In contrast, the selective norepinephrine-uptake blockers desipramine and nisoxetine enhanced not only norepinephrine, but also serotonin and dopamine appreciably. Cocaine increased all three amines with the greatest effects on dopamine and serotonin. As in our previous study on the ventral tegmental area, there was a positive association between dopamine and norepinephrine output when all blocker data were taken together. The present results suggest a contribution of the increase in norepinephrine, but not serotonin, to the enhancement of dopamine after cocaine applied focally in the nucleus accumbens.     

Leptin Levels Are Associated with Fat Oxidation and Dietary‐Induced Weight Loss in Obesity

Objective: To examine the relationship between fasting plasma leptin and 24‐hour energy expenditure (EE), substrate oxidation, and spontaneous physical activity (SPA) in obese subjects before and after a major weight reduction compared with normal weight controls. To test fasting plasma leptin, substrate oxidations, and SPA as predictive markers of success during a standardized weight loss intervention. Research Methods and Procedures: Twenty‐one nondiabetic obese (body mass index: 33.9 to 43.8 kg/m²) and 13 lean (body mass index: 20.4 to 24.7 kg/m²) men matched for age and height were included in the study. All obese subjects were reexamined after a mean weight loss of 19.2 kg (95% confidence interval: 15.1–23.4 kg) achieved by 16 weeks of dietary intervention followed by 8 weeks of weight stability. Twenty‐four‐hour EE and substrate oxidations were measured by whole‐body indirect calorimetry. SPA was assessed by microwave radar. Results: In lean subjects, leptin adjusted for fat mass (FM) was correlated to 24‐hour EE before (r = ?0.56, p < 0.05) but not after adjustment for fat free mass. In obese subjects, leptin correlated inversely with 24‐hour and resting nonprotein respiratory quotient (r = ?0.47, p < 0.05 and r = ?0.50, p < 0.05) both before and after adjustments for energy balance. Baseline plasma leptin concentration, adjusted for differences in FM, was inversely related to the size of weight loss after 8 weeks (r = ?0.41, p = 0.07), 16 weeks (r = ?0.51, p < 0.05), and 24 weeks (r = ?0.50, p < 0.05). Discussion: The present study suggests that leptin may have a stimulating effect on fat oxidation in obese subjects. A low leptin level for a given FM was associated with a greater weight loss, suggesting that obese subjects with greater leptin sensitivities are more successful in reducing weight.     

A Low Sympathoadrenal Activity is Associated with Body Weight Gain and Development of Central Adiposity in Pima Indian Men

TATARANNI P ANTONIO JAMES B YOUNG, CLIFTON BOGARDUS, ERIC RAVUSSIN. A low sympathoadrenal activity is associated with body weight gain and development of central adiposity in Pima Indian men. To investigate the possible role of impaired sympathetic nervous system and/or adrenal medullary function in the etiology of human obesity, we studied 64 Pima Indian men (28 ± 6 years, 101 ± 25 kg, 34 ± 9% body fat, mean ± SD) in whom sympathoadrenal function was estimated at baseline by measurements of 24-hour urinary norepinephrine (NE) and epinephrine (Epi) excretion rates under weight-maintenance conditions. Body weight, body composition (hydrodensitometry), and body fat distribution (waist-to-thigh circumference ratio, W/T) were measured at baseline and follow-up. Follow-up data were available on 44 subjects who gained on average 8.4 ± 9.5 kg over 3.3 ± 2.1 years. In these subjects, baseline NE excretion rate, adjusted for its determinants (i.e., fat free mass, fat mass, and W/T), correlated negatively with bodyweight gain (r=?0.38; p=0.009). Baseline Epi excretion rate correlated negatively with changes in W/T (r=?0.44; p=0.003). In conclusion, our data show for the first time that a low sympathetic nervous system activity is associated with body weight gain in humans. Also, a low activity of the adrenal medulla is associated with the development of central adiposity.