Modulation of in situ murine neuroblastoma tumor growth by the adrenergic nervous system: differential response of clonal cell lines to chemical sympathectomy

The in situ growth characteristics of C-1300, N1E-115 and NS20Y murine neuroblastoma (MNB) tumor cell lines were compared in normal and sympathectomized A/J mice. Adrenergic nerve ablation was produced in neonatal mice by administration of 6-hydroxydopamine (6-OHDA) at a dose of 100 micrograms/gm body weight on post-natal days 4, 6, 8 and 10; controls received equivalent volumes of the vehicle solution (0.9% NaCl/0.1% Ascorbic Acid). All mice were inoculated subcutaneously with 10(6) viable MNB cells four to six weeks after treatment with 6-OHDA or vehicle. The growth rates of tumors produced by the adrenergic MNB cell lines, C-1300 and N1E-115, were significantly lower in sympathectomized mice when compared to control animals. In contrast, tumors induced by the cholinergic MNB cell line, NS20Y, grew at similar rates in both sympathectomized and control mice. All tumors obtained from control and sympathectomized mice regardless of whether they derived from cell lines characterized as cholinergic (NS20Y) or adrenergic (C-1300, N1E-115), contained both norepinephrine and dopamine. Depletion of adrenergic neurotransmitter in A/J mice was induced by administration of reserpine (5-10 micrograms/kg/day) beginning 30 days prior to implantation of the C-1300 MNB cell line and continuing until sacrifice of the animal. The effect of this treatment on organ and tumor catecholamine concentrations was confirmed by high-pressure liquid chromatography. Splenic catecholamine levels in reserpine-treated animals were reduced to 20% of controls as compared to 9% in the neonatally-sympathectomized group. However, there was no discernible effect on C-1300 MNB tumor growth in the reserpine-treated animals. C-1300 MNB tumor concentrations of nor-epinephrine and dopamine were significantly lower in the reserpine-treated animals than in controls. The suppression of tumor growth by adrenergic nerve ablation is selective for specific MNB tumor cell lines. An anatomically intact sympathetic nervous system appears to exert a greater influence than competency of adrenergic neuro-humoral transmission on MNB tumor growth. These data support the hypothesis that modulation of MNB tumor growth by the adrenergic nervous system is not mediated via catecholamines but may be modulated by endogenous growth factor(s).     

Immunohistochemical evidence for a plasma membrane localization of dopamine-beta-hydroxylase in the mouse neuroblastoma

Dopamine-beta-hydroxylase (D beta H), a glycoprotein enzyme which converts dopamine into noradrenaline, was purified from C1300 mouse neuroblastoma and used to raise antibodies in rabbits. Using an indirect immunofluorescence technique the cellular localization of D beta H in C1300 mouse neuroblastoma was compared with that of the superior cervical ganglion. C1300 neuroblastoma D beta H was found to be predominantly localized in the plasma membrane, in contrast to its intracellular localization in the superior cervical ganglion of A/J mice. At least part of the enzyme was found to be associated with the external side of the plasma membrane.     

Reserpine sensitivity of catecholamine metabolism in murine neuroblastoma clone N1E-115.

—Neuroblastoma cells of clone NIE-115, originally obtained from the murine tumor C1300, resemble normal noradrenergic neurons in that they have high levels of tyrosine 3-monooxygenase (EC 1.14.16.2; l -tyrosine, tetrahydropteridine: oxygen oxidoreductase (3-hydroxylating)) and dopamine β-monooxygenase (EC 1.14.17.1; 3,4-dihydroxyphenylethylamine, ascorbate: oxygen oxidoreductase (β-hydroxylating)) activities, dense core versicles (100–300 nm in dia), long neurites and excitable membranes. These studies show that reserpine, a blocker of vesicular uptake in noradrenergic neurons, inhibits the accumulation and storage of catecholamines, as well as the conversion of dopamine to NE in neuroblastoma cells. Differentiated monolayer cultures took up [³H]dopamine [10⁻⁴] at a rate of 37 pmol/min per mg protein. Reserpine [5 × 10⁻⁵m ] did not affect the initial rate of uptake, but reduced the extent of uptake at saturation by 60%. Chromatographic examination of cell extracts showed that dopamine was converted to NE in control cultures, but not in reserpine treated cultures. Cells labelled with [³H]dopamine for 60 min and then exposed to release buffer without dopamine for an additional 60 min, retained approximately 40% of the label, 10% as dopamine and 30% as NE. Thirty-five per cent of the radioactivity retained was found, after homogenization and high speed centrifugation, to be associated with a particulate, subcellular fraction. Reserpine, present during release incubations, also reduced the ability of cells to store catecholamines. These results show that N1E-115 cells synthesize and store NE by reactions similar to those in normal noradrenergic neurons.     

Exocytotic release of dopamine in ventral tegmental area slices from C57BL/6 and dopamine transporter knockout mice

The present study used voltammetry to ascertain whether electrically stimulated somatodendritic dopamine release in ventral tegmental area slices from C57BL/6 and dopamine transporter knockout mice was due to exocytosis or dopamine transporter reversal, as has been debated. The maximal concentration of electrically evoked dopamine release was similar between ventral tegmental area slices from dopamine transporter knockout and C57BL/6 mice. Dopamine transporter blockade (10 μM nomifensine) in slices from C57BL/6 mice inhibited dopamine uptake but did not alter peak evoked dopamine release. In addition, dopamine release and uptake kinetics in ventral tegmental area slices from dopamine transporter knockout mice were unaltered by the norepinephrine transporter inhibitor, desipramine (10 μM), or the serotonin transporter inhibitor, fluoxetine (10 μM). Furthermore, maximal dopamine release in ventral tegmental area slices from both C57BL/6 and dopamine transporter knockout mice was significantly decreased in response to Na⁺ channel blockade by 1 μM tetrototoxin, removal of Ca²⁺ from the perfusion media and neuronal vesicular monoamine transporter inhibition by RO-04-1284 (10 μM) or tetrabenazine (10 and 100 μM). Finally, the glutamate receptor antagonists AP-5 (50 and 100 μM) and CNQX (20 and 50 μM) had no effect on peak somatodendritic dopamine release in C57BL/6 mice. Overall, these data suggest that similar mechanisms, consistent with exocytosis, govern electrically evoked dopamine release in ventral tegmental area slices from C57BL/6 and dopamine transporter knockout mice.     

Exocytotic release of dopamine in ventral tegmental area slices from C57BL/6 and dopamine transporter knockout mice

The present study used voltammetry to ascertain whether electrically stimulated somatodendritic dopamine release in ventral tegmental area slices from C57BL/6 and dopamine transporter knockout mice was due to exocytosis or dopamine transporter reversal, as has been debated. The maximal concentration of electrically evoked dopamine release was similar between ventral tegmental area slices from dopamine transporter knockout and C57BL/6 mice. Dopamine transporter blockade (10 μM nomifensine) in slices from C57BL/6 mice inhibited dopamine uptake but did not alter peak evoked dopamine release. In addition, dopamine release and uptake kinetics in ventral tegmental area slices from dopamine transporter knockout mice were unaltered by the norepinephrine transporter inhibitor, desipramine (10 μM), or the serotonin transporter inhibitor, fluoxetine (10 μM). Furthermore, maximal dopamine release in ventral tegmental area slices from both C57BL/6 and dopamine transporter knockout mice was significantly decreased in response to Na⁺ channel blockade by 1 μM tetrototoxin, removal of Ca²⁺ from the perfusion media and neuronal vesicular monoamine transporter inhibition by RO-04-1284 (10 μM) or tetrabenazine (10 and 100 μM). Finally, the glutamate receptor antagonists AP-5 (50 and 100 μM) and CNQX (20 and 50 μM) had no effect on peak somatodendritic dopamine release in C57BL/6 mice. Overall, these data suggest that similar mechanisms, consistent with exocytosis, govern electrically evoked dopamine release in ventral tegmental area slices from C57BL/6 and dopamine transporter knockout mice.     

Changes in plasma free and sulfoconjugated catecholamines before and after acute physical exercise: experimental and clinical studies.

To elucidate whether sulfoconjugated catecholamines in plasma, especially dopamine, serve as a source of free catecholamines, we examined the change in afterload on the deconjugating activity of catecholamines in isolated Langendorff perfused rat hearts. Dopamine-sulfate was administered under ordinary or high-work-load conditions. Free dopamine in the effluent was increased by the high-work-load of the hearts, whereas conjugated dopamine showed an apparent decrease. These results indicate the possibility that deconjugation of sulfoconjugated catecholamines is accelerated by a high-work-load. To obtain further evidence in humans, we also examined the changes in the plasma levels of free and sulfoconjugated catecholamines in healthy volunteers before and after marathon running. Free dopamine increased 1.99-fold from the baseline value after exercise, whereas conjugated dopamine decreased by 12%. Similarly, the plasma levels of free noradrenaline and adrenaline increased after exercise to 2.45- and 1.51-fold their respective baseline values, while conjugated noradrenaline and adrenaline both decreased. These clinical results, as well as those of the experimental studies, suggest that the increase in plasma free catecholamines after exercise is due not only to increased release from the sympathoadrenal system but also to accelerated conversion from sulfoconjugated catecholamines in the plasma.     

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.     

Effects of gonadectomy and sex hormones on the levels of noradrenaline and dopamine in rat hypothalamus

The levels of noradrenaline and dopamine were determined in the homogenates of the hypothalamus of rats of either sex. The determinations were done in intact rats, after sham gonadectomy, 6 and 9 weeks after gonadectomy, and in gonadectomized rats receiving 6 weeks after gonadectomy one dose of oestradiol cypionate (females) or testosterone cypionate (males). Catecholamines were determined fluorimetrically. The changes of the determined catecholamines differed in the hypothalamic homogenates in males and females after gonadectomy. Following orchidectomy the noradrenaline level rose, while after ovariectomy the level of this catecholamine decreased. Contrary to this, in ovariectomized rats the dopamine level was significantly raised after the operation. This change was reversible as observed after administration of oestradiol cypionate. Orchidectomy and testosterone cypionate injection had no effect on the dopamine level in the hypothalamus. The role of these catecholamines in the processes connected with the regulation of the hypothalamus-hypophysis-gonad axis is discussed.     

Plasma gonadotropin,prolactin levels and hypothalamic tyrosine hydroxylase activity in rats during estrous cycle,after overiectomy and after blockade of catecholamine biosynthesis

Plasma gonadotropin, prolactin levels and hypothalamic tyrosine hydroxylase activity were evaluated at 0900, 1200 and 1700 h during diestrus, proestrus and estrus, ovariectomized and after systemic administration of reserpine or α-methyl p-tyrosine, which interfere with catecholamine biosynthesis, in rats. Gonadotropin and prolactin levels showed peak values during the afternoon of proestrus, while hypothalamic tyrosine hydroxylase activity was markedly lowered at 1200 on proestrus. Gonadotropin levels were slightly lowered whereas prolactin concentrations and hypothalamic tyrosine hydroxylase activity were significantly increased by reserpine. Depletion of hypothalamic dopamine by reserpine apparently resulted in significant elevation of prolactin levels which inturn induce tyrosine hydroxylase. Gonadotropin levels and hypothalamic tyrosine hydroxylase activity were significantly suppressed after the administration of α-methyl p-tyrosine. Prolactin levels, however, were elevated significantly. These results indicate that catecholamines are involved in the control of gonadotropin and prolactin release during estrous cycle and inhibition of catecholamines biosynthesis by α-methyl p-tyrosine could result in suppression of gonadotropin levels, whereas removal of tonic inhibition of hypothalamic dopamine by α-methyl-p-tyrosine elevate prolactin levels.     

Action of dopamine on the human iris

Dopamine eye drops produce marked dilatation of the pupil in man. This mydriatic effect is inhibited by pretreatment with guanethidine. It is therefore concluded that dopamine acts indirectly via adrenergic nerve endings, rather than exerting a direct effect on adrenergic receptors in the dilator pupillae muscle. In this respect dopamine resembles the phenyl-alkylamines, such as tyramine, rather than the catecholamines, adrenaline and noradrenaline.If dopamine acts by releasing noradrenaline from adrenergic nerve endings, high concentrations of dopamine could lead to depletion of noradrenaline stores, since synthesis might be unable to keep pace with release. This could be the explantion for the orthostatic hypotension found in patients taking L-dopa for Parkinsonism.     

Biochemistry of Somatodendritic Dopamine Release in Substantia Nigra: An In Vivo Comparison with Striatal Dopamine Release

Abstract: The somatodendritic release of dopamine in substantia nigra previously has been suggested to be nonvesicular in nature and thus to differ from the classical, exocytotic release of dopamine described for the dopaminergic nerve terminal in striatum. We have compared the effects of reserpine, a compound that disrupts vesicular sequestration of monoamines, on the storage and release of dopamine in substantia nigra and striatum of rats. Reserpine administration (5 mg/kg, i.p.) significantly decreased the tissue level of dopamine in substantia nigra pars reticulata, substantia nigra pars compacta, and striatum. In these brain areas, reserpine-induced reductions in tissue dopamine level occurred within 2 h and persisted at 24 h postdrug. In vivo measurements using microdialysis revealed that reserpine administration rapidly decreased the extracellular dopamine concentration to nondetectable levels in substantia nigra as well as in striatum. In both structures, it was observed that reserpine treatment significantly attenuated the release of dopamine evoked by a high dose of amphetamine (10 mg/kg, i.p.) given 2 h later. In contrast, dopamine efflux in response to a low dose of amphetamine (2 mg/kg, i.p.) was not altered by reserpine pretreatment either in substantia nigra or in striatum. The present data suggest the existence, both at the somatodendritic and at the nerve terminal level, of a vesicular pool of dopamine that is the primary site of transmitter storage and that can be displaced by high but not low doses of amphetamine. The physiological release of dopamine in substantia nigra and in striatum is dependent on the integrity of this vesicular store.     

Evidence against an essential role of endogenous brain dopamine in methamphetamine-induced dopaminergic neurotoxicity

The present studies examined the role of endogenous dopamine (DA) in methamphetamine (METH)-induced dopaminergic neurotoxicity while controlling for temperature-related neuroprotective effects of the test compounds, reserpine and alpha-methyl-p-tyrosine (AMPT). To determine if the vesicular pool of DA was essential for the expression of METH-induced DA neurotoxicity, reserpine (3 mg/kg, given iintraperitoneally 24-26 h prior to METH) was given prior to a toxic dose regimen of METH. Despite severe striatal DA deficits during the period of METH exposure, mice treated with reserpine prior to METH developed long-term reductions in striatal DA axonal markers, suggesting that vesicular DA stores were not crucial for the development of METH neurotoxicity, but leaving open the possibility that cytoplasmic DA might be involved. To evaluate this possibility, cytoplasmic DA stores were depleted with AMPT prior to METH administration. When this study was carried out at 28 degrees C, complete neuroprotection was observed, likely due to lingering effects on core temperature because when the same study was repeated at 33 degrees C (to eliminate AMPT's hypothermic effect in METH-treated animals), the previously observed neuroprotection was no longer evident. In the third and final set of experiments, mice were pretreated with a combination of reserpine and AMPT, to deplete both vesicular and cytoplasmic DA pools, and to reduce striatal DA levels to negligible values during the period of METH administration (< 0.05%). When core temperature differences were eliminated by raising ambient temperature, METH-induced DA neurotoxic changes were evident in mice pretreated with reserpine and AMPT. Collectively, these findings bring into question the view that endogenous DA plays an essential role in METH-induced DA neurotoxicity.     

Amperozide, a putative anti-psychotic drug: uptake inhibition and release of dopamine in vitro in the rat brain

The effects of amperozide (a diphenylbutylpiperazinecarboxamide derivative) on the uptake and release of 3H-dopamine in vitro were investigated. Amperozide inhibited the amphetamine-stimulated release of dopamine from perfused rat striatal tissue in a dose-dependent manner. With 1 and 10 microM amperozide there was significant inhibition of the amphetamine-stimulated release of dopamine, to 44 and 36% of control. In contrast, 10 microM amperozide significantly strengthened the electrically stimulated release of dopamine from perfused striatal slices. Amperozide 1-10 microM had no significant effect on the potassium-stimulated release of dopamine. 10 microM amperozide also slightly increased the basal release of 3H-dopamine from perfused striatal tissue. These effects on various types of release are similar to those reported for uptake inhibitors (Bowyer et al, 1984). The uptake of dopamine in striatal tissue was inhibited by amperozide with IC50 values of 18 microM for uptake in chopped tissue and 1.0 microM for uptake in synaptosomes. Amperozide also inhibited the uptake of serotonin in synaptosomes from frontal cortex, IC50 = 0.32 microM and the uptake of noradrenaline in cortical synaptosomes, IC50 = 0.78 microM. In conclusion, amperozide shows uptake-inhibiting properties in both release and uptake studies done in vitro on the rat. In the in vivo studies, however, amperozide differs from dopamine uptake inhibitors.     

A simple procedure for distinguishing dopamine from noradrenaline in peripheral nervous structures in the fluorescence microscope

The difference between dopamine and noradrenaline after ordinary histofluorescent procedures cannot be discerned. Reserpine treatment results in depletion of fluorescent material from dopaminergic and noradrenergic peripheral nervous structures. Administration of reserpine, 1 mg/kg subcutaneously for 3 hr, followed by intraperitoneal injection of 200 mg/kg levodopa methyl ester on 0.9% saline for 90 min, result in refluorescence of dopaminergic (glomus cells of the carotid body) but not noradrenergic (sympathetic ganglion cells, nerves of atrial heart muscle and blood vessels) structures. Hence, the sequential administration of these readily available drugs and the application of ordinary histofluorescent techniques result in a simple procedure for distinguishing dopamine from noradrenaline in the fluorescence microscope.     

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.     

Stimulation of the locus coeruleus elicits noradrenaline and dopamine release in the medial prefrontal and parietal cortex

Our previous studies have suggested that dopamine and noradrenaline may be coreleased from noradrenergic nerve terminals in the cerebral cortex. To further clarify this issue, the effect of electrical stimulation of the locus coeruleus on extracellular noradrenaline, dopamine and DOPAC in the medial prefrontal cortex, parietal cortex and caudate nucleus was analysed by microdialysis in freely moving rats. Stimulation of the locus coeruleus for 20 min with evenly spaced pulses at 1 Hz failed to modify cortical catecholamines and DOPAC levels. Stimulation with bursts of pulses at 12 and 24 Hz increased, in a frequency-related manner, not only noradrenaline but also dopamine and DOPAC in the two cortices. In both cortices noradrenaline returned to baseline within 20 min of stimulation, irrespective of the stimulation frequency, whereas dopamine returned to normal within 20 and 60 min in the medial prefrontal cortex and within 60 and 80 min in the parietal cortex after 12 and 24 Hz stimulation, respectively. DOPAC remained elevated throughout the experimental period. Phasic stimulation of the locus coeruleus at 12 Hz increased noradrenaline in the caudate nucleus as in the cerebral cortices but was totally ineffective on dopamine and DOPAC. Tetrodotoxin perfusion into the medial prefrontal cortex dramatically reduced noradrenaline and dopamine levels and suppressed the effect of electrical stimulation. These results indicate that electrical stimulation-induced increase of dopamine is a nerve impulse exocytotic process and suggest that cortical dopamine and noradrenaline may be coreleased from noradrenergic terminals.     

Reserpine induced intraneuronal dopamine oxidation: reversal by MPP+ action.

1-methyl-4-phenylpyridinium ion (MPP+) was tested for its effects upon dopamine level after incubating striatal synaptosomes in medium with and without reserpine. In the absence of reserpine, MPP+ enhanced the total incubation mixture dopamine level when tyrosine was present in the medium but that enhancing effect was considerably weaker when tyrosine was replaced by alpha methyl p-tyrosine. 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) also had effects upon dopamine but likely due to MPP+, which was formed from MPTP by free mitochondrial MAO present in the tissue preparation. The incubation mixture dopamine level was drastically reduced by the addition of only reserpine and its presence in the medium markedly raised the ability of MPP+ to increase dopamine; the effects of MPTP in this medium were weaker than those of MPP+. Pargyline also raised dopamine levels under these conditions but only at concentrations much higher than those of MPP+. The particulate uptake of MPP+, at several medium concentrations, and the corresponding value of dopamine increase above the basal level were determined; the dopamine increase in p-moles was much greater than the p-moles of MPP+ uptake. These results indicate that, in the presence of reserpine, MPP+ has a potent action and that may lead to a release of intraneuronal free dopamine; this action is also likely to be independent of the countertransport from MPP+ uptake. The possibility of MPP+ being a potent inhibitor of intraneuronal MAO may have to be considered.     

Simultaneous radioenzymatic determination of plasma and tissue adrenaline,noradrenaline and dopamine within the femtomole range

A radiometric-enzymatic assay for measuring simultaneously femtomole quantities of adrenaline, noradrenaline and dopamine has been developed. The three catecholamines are first converted to their O-methylated analogues by catechol-O-methyltransferase in the presence of S-adenosyl-methionine-³H and thereafter extracted following addition of sodium tetraphenylborate. This extraction, together with an improved quick chromatographic separation and the oxidation of the adrenaline and noradrenaline derivatives to vanillin, yields an extremely high sensitivity and specificity of the method.The present assay allows the determination of adrenaline, noradrenaline and dopamine in tissue samples with a protein content of 100 μg or less and in plasma volumes of 20 – 100 μl. The amine content of 40 – 50 samples can be determined in two days by one person.Due to the high sensitivity achieved, this method promises to be a valid alternative to the gas chromatography-mass spectrometry technique.     

Lack of effect of corticotropin releasing factor on hypothalamic dopamine and serotonin synthesis turnover rates in rats

Catecholamine and serotonin neurons in the hypothalamus regulate the secretion of corticotropin releasing factor (CRF). We considered the possibility that CRF might in turn affect the activity of these aminergic neurons. We examined the effect of intracisternal administration of synthetic CRF on the synthesis turnover rates of dopamine and serotonin in the hypothalamus of adult male rats using two different methods to assess turnover. In one study, we measured the accumulation of L-dihydroxyphenylalanine (L-DOPA) or 5-hydroxytryptophan (5-HTP) in mediobasal hypothalamus after L-aromatic amino acid decarboxylase inhibition with m-hydroxybenzylhydrazine 20 min before sacrifice, and in the second study we measured the accumulation of dopamine, norepinephrine, epinephrine and serotonin after monoamine oxidase inhibition with pargyline 20 min before sacrifice. The commercial CRF which we administered intraarterially increased plasma ACTH and corticosterone concentrations. Intracerebral CRF 5 to 20 micrograms 20 min before sacrifice or 20 micrograms 110 min before sacrifice did not alter the m-hydroxybenzylhydrazine-induced accumulation of L-DOPA or 5-HTP when compared with saline vehicle-injected controls. CRF 20 micrograms did not alter basal concentration or pargyline-induced accumulation of the catecholamines or serotonin in whole hypothalamus when compared with saline vehicle-injected controls. Thus, intracisternal administration of CRF did not alter hypothalamic dopamine or serotonin synthesis rates as assessed by two nonsteady state turnover methods. The data suggest that the release of CRF from neurons in hypothalamus does not alter the activity of catecholamine or serotonin neurons in the hypothalamus of normal adult male rats.     

Dissociate destruction of noradrenaline and dopamine descending projections in the thoracic spinal cord of the rat

Intracisternal administration of 6-hydroxydopamine to male Wistar rats produced a near complete depletion of noradrenaline levels, as measured by a radioenzymatic assay in micropunches sampled from the dorsal, lateral and ventral horns of the thoracic spinal cord. This drastic effect was reversed by pretreatment with desipramine, a pharmacological inhibitor of noradrenergic neuron uptake. Surprisingly, dopamine content was not significantly reduced. The question as to whether such a lack of concomitant dopamine decrease might be inherent to the dopamine assay itself could be answered by the results obtained with both pharmacological (reserpine) treatments and interference determinations in the dopamine assay. The relative potency of 6-hydroxydopamine to destroy noradrenergic and dopaminergic neurons might account for their differential behavior. Conversely, a large midbrain section performed by knife cut could decrease both dopamine and DOPAC (one of its major acid metabolites) in the thoracic lateral horn and partly in the ventral one. The noradrenaline content was not reduced. Results are discussed in light of recently reported data on dopaminergic descending projections to the spinal cord. The lesion procedures presented here seem to provide valuable tools to dissociate noradrenergic from dopaminergic spinal projections, which is necessary for further anatomical and functional studies on these systems.