Limiting Monoamines Degradation Increases L-DOPA Pro-Locomotor Action in Newborn Rats.

L-DOPA, the precursor of catecholamines, exerts a pro-locomotor action in several vertebrate species, including newborn rats. Here, we tested the hypothesis that decreasing the degradation of monoamines can promote the pro-locomotor action of a low, subthreshold dose of L-DOPA in five-day-old rats. The activity of the degrading pathways involving monoamine oxidases or catechol-O-methyltransferase was impaired by injecting nialamide or tolcapone, respectively. At this early post-natal stage, the capacity of the drugs to trigger locomotion was investigated by monitoring the air-stepping activity expressed by the animals suspended in a harness above the ground. We show that nialamide (100 mg/kg) or tolcapone (100 mg/kg), without effect on their own promotes maximal expression of air-stepping sequences in the presence of a sub-effective dose of L-DOPA (25 mg/kg). Tissue measurements of monoamines (dopamine, noradrenaline, serotonin and some of their metabolites) in the cervical and lumbar spinal cord confirmed the regional efficacy of each inhibitor toward their respective enzyme. Our experiments support the idea that the raise of monoamines boost L-DOPA's locomotor action. Considering that both inhibitors differently altered the spinal monoamines levels in response to L-DOPA, our data also suggest that maximal locomotor response can be reached with different monoamines environment.

Fictive locomotion in the adult decerebrate and spinal mouse in vivo.

Recently, transgenic mice have been created with mutations affecting the components of the mammalian spinal central pattern generator (CPG) for locomotion; however, it has currently only been possible to evoke fictive locomotion in mice, using neonatal in vitro preparations. Here, we demonstrate that it is possible to evoke fictive locomotion in the adult decerebrate mouse in vivo using l-3,4-dihydroxyphenylalanine methyl ester hydrochloride (l-DOPA) and 5-hydroxytryptophan (5HTP) following injection of the monoaminoxiadase inhibitor Nialamide. We investigate the effects of afferent stimulation and spinalization as well as demonstrate the possibility of simultaneous intracellular recording of rhythmically active motoneurones. Our results demonstrate that several features of the mouse locomotor CPG are similar to those that have been observed in rat, cat, rabbit and monkey suggesting a fairly conserved organisation and allowing for future results in transgenic mice to be extrapolated to existing knowledge of CPG components and circuitry obtained in larger species.

Development and validation of a hydrophilic interaction liquid chromatography-tandem mass spectrometry method for determination of isoniazid in human plasma.

An LC-MS/MS method for the determination of isoniazid in human plasma was developed and validated. Human plasma aliquots of 100 microL were used for analysis. The assay used nialamide as the internal standard. The calibration curve concentration range was 50-10,000 ng/mL. Sample preparation utilized protein precipitation, and the supernatant was directly injected onto silica column without reconstitution. The recovery was over 90% and matrix effect was negligible. The method is simple and fast, which is advantageous in respect to instability of isoniazid in human plasma and loss on reconstitution due to its low molecular weight.

Preproenkephalin knockout mice show no depression-related phenotype.

Clinical, preclinical, and pharmacological studies have suggested that decreased enkephalin tone is associated with depression-like symptoms and increase in enkephalin signaling could have a therapeutic value in the treatment of depression. In this study we demonstrate that, surprisingly, animals lacking enkephalin (preproenkephalin, Penk1(-/-)) showed no depression-related phenotype in the Porsolt forced swimming or tail suspension tests. Moreover, Penk1(-/-) mice had a lower frequency of depression-related behavior in stress-induced hypoactivity and ultrasonic vocalization models of depression, similar to animals treated with antidepressant drugs, although this effect was specific to the genetic background. In addition, there was no significant difference in the efficacy of antidepressant reference compounds in wild-type and knockout animals. Nialamide and amitriptyline were even slightly more effective in animals with genetic deletion of Penk1, whereas the minimal effective dose of imipramine and fluoxetine was the same in the two genotypes. The dual peptidase inhibitor RB-101 was also effective in Penk1(-/-) as well as in Penk1(-/-)/Pdyn(-/-) animals, although its efficacy was somewhat reduced compared with wild-type animals. This result was also surprising because the antidepressant effects of RB-101 were thought to be due to the elevation of enkephalin levels.

On the mechanism by which extracellular sodium depletion causes 5-hydroxytryptamine release from rat brain synaptosomes.

The release of 3H-labelled 5-hydroxytryptamine (5-HT) from preloaded and superfused rat forebrain synaptosomes in response to extracellular Na+ depletion was studied. In the absence of monoamine oxidase inhibitors, the release of [3H]-5-HT caused by Na+ depletion was not affected by immobilizers of the plasma membrane 5-HT carrier. The release of [3H]-5-HT in response to Na+ depletion was also either independent of, or inversely related to the concentration of extracellular Ca2+ depending on the degree to which extracellular Na+ was reduced. The efflux of 45Ca2+ from prelabelled synaptosomes was decreased by Na+ reduction but the amplitude of the changes in 45Ca2+ efflux did not totally correlate with the changes in [3H]-5-HT efflux under the same experimental conditions. These results suggest that the release of [3H]-5-HT caused by Na+ depletion in drug-free synaptosomes is not mediated by 5-HT efflux through the plasma membrane carrier, nor to changes in cytosolic Ca2+ consequent to changes in Ca2+ fluxes across the plasma membrane. The results have been tentatively explained as an elevation of spontaneous 5-HT efflux caused by an increase in membrane fluidity mediated by the ionic manipulations used to produce the Na+-depleted media.

Catecholaminergic control of alpha-melanocyte-stimulating hormone (alpha MSH) release by frog neurointermediate lobe in vitro: evidence for direct stimulation of alpha MSH release by thyrotropin-releasing hormone.

The role of dopaminergic and adrenergic innervation of the intermediate lobe of amphibian pituitary in the release of alpha MSH has been studied in vitro. Neurointermediate lobes of frog (Rana ridibunda Pallas) have been perifused in amphibian culture medium (ACM) for 5-7 h. alpha MSH released in the effluent perifusate was measured by means of a sensitive and specific RIA. No significant morphological alteration of neurointermediate lobe cells was observed during the perifusion experiment, even at the electron microscopic level. The existence of dopaminergic receptors, responsible for an inhibition of frog melanotrophs, was shown using the dopaminergic agonists apomorphine (10(-6) M) and bromo-2-ergocryptine (10(-8) and 10(-7) M), which initiated a marked reduction of alpha MSH secretion. The effect of apomorphine was obliterated by the dopaminergic antagonist haloperidol. Haloperidol itself induced a dose-related stimulation, and the monoamine oxidase inhibitor nialamide (4 x 10(-3) M) inhibited alpha MSH secretion. In addition, haloperidol led to a complete reversal of the inhibitory effect of nialamide on alpha MSH secretion. These results demonstrate the existence, in the parenchyme of the intermediate lobe, of dopaminergic nerve fibers that are functionally active. The beta-adrenergic agonist isoproterenol was responsible for a dose-related stimulation of alpha MSH secretion; the stimulatory effect was reversed by the beta-adrenergic antagonist propranolol. TRH is a potent stimulator of alpha MSH secretion in amphibians. Since haloperidol and propranolol did not abolish the stimulation of alpha MSH release induced by TRH, it appeared that TRH action was not mediated via an inhibition of dopamine release or via a stimulation of adrenergic nerve fibers.

Dual action of antidepressant drugs (MAO inhibitors) on insulin release.

Experiments have been carried out to investigate the role of monoamine oxidase (MAO) in the mechanism of insulin release. Isolated islets and pieces of rat pancreas were incubated in media of high glucose content in the presence of various concentrations of MAO inhibitors. At the end of the incubation, the islet MAO activity and the concentration of insulin released into the medium was measured. The results have shown that: 1. Glucose-mediated insulin release was potentiated by low concentrations of MAO inhibitors, the highest effect being observed at 10 muM. 2. Islet MAO activity was completely abolished in the presence of 10 muM of MAO inhibitors, while at lower concentrations, partial inhibition of enzyme could be achieved. A rise in the concentration of MAO inhibitors was accompanied by a decline in their potentiating effect on insulin secretion. No potentiation was observed at 1 mM concentration, and total inhibition of glucose-mediated insulin release was obtained in the presence of 5-10 mM of MA9 inhibitors. The potentiation of insulin release caused by two of the MAO inhibitors studied was abolished by addition of 1 muM epinephrine into the incubation medium. The basal rate of insulin release was insensitive to low concentrations of MAO inhibitors, but an inhibitory effect was obtained when concentrations were raised to 10 mM. In a comparative study, it was found that MAO activity was greater in liver than in islet tissue, while islets contained three times the activity of exocrine pancreas, consistent with previous findings in islet-cell tumors. The data presented here clearly show that MAO inhibitors are capable of both potentiating and inhibiting insulin release in vitro, depending on their concentrations. It is concluded that the stimulation of glucose-mediated insulin secretion may be related to the MAO inhibitory effects of the drugs, while the inhibition observed at concentrations greater than 1 times 10-3M is due to some other unidentified mechanism.

Nialamide, an MAO inhibitor, increases urinary excretion of endogenously produced bufotenin in man.

Nialamide, an MAO inhibitor, was given per os (PO) to a normal man who volunteered in two separate trials (total intake 300 mg and 1000 mg, respectively), and his bufotenin excretion was followed by consecutive urine samples. In both experiments the excretion rose well above the values measured from the same test subject when not taking nialamide (median 0.089 nmol/mmol creatinine, range 0.002-1.78). At its highest, the excretion was 16.5 nmol/mmol creatinine, and the maximum urinary output was 495 nmoles (56 micrograms) in 24 hr. The levels of bufotenin in plasma required for the excretion of the latter amounts are not far from those that produce psychic symptoms in man.

Characterization of the monoaminergic innervation of immature rat neocortex: a histofluorescence analysis.

In the neocortex of 6-day-old rat, abundant axon terminals which exhibit specific catecholamine fluorescence are found in all regions and throughout all cortical layers. The overall density of axons in 6-day-old cortex is similar to the density in the adult cortex. In immature cortex, there are two distinct fluorescent plexuses, both presumably noradrenergic, one in the molecular layer and another in the lower half of the cortex. The superficial plexus is composed primarily of horizontal fibers, and the deep plexus of a dense feltwork of obliquely oriented fibers suggestive of a terminal field. The cortical plate itself is traversed by a few vertical processes. Following lesions of the midbrain tegmentum no fluorescent axons are seen in cortex, providing evidence that the fluorescent axons in cortex arise from brain stem neurons. The deep and superficial plexuses can be differentially visualized depending on the histochemical techniques employed and on pharmacological treatment, such as loading with a monoamine congener. Both deep and superficial axons are shown to contain endogenous catecholamines but those fibers in the deep plexus are filled to far less than their maximum capacity. The pharmaco-histochemical differences between axons in the two plexuses suggest that there may exist two distinct catecholaminergic projections to lateral neocortex. The demonstration of an extensive cortical monoamine innervation early in ontogeny supports the possibility that monoamine neurons play an important role in information processing and/or developmental interactions in the immature brain.

Mode of distribution of aminergic fibers in the cerebellar cortex of the chicken.

The cerebellar cortex of adult hens contains a dense plexus of thin varicose nerve fibers which display a formaldehyde-induced green fluorescence. This plexus is not distributed at random in the cortical layers. Within the granular layer the plexus forms a netlike pattern. The fiber branches, which have numerous varicosities, are predominantly oriented in the traverse plane of the folium. In the molecular layer the fluorescent plexus shows some variations in the convex, flat and concave portions of the folia. Many of the fluorescent branches are oriented parallel to the course of the folium. They arise from a T-division of radially oriented axons resembling parallel fibers in Golgi sections. The meshes of the fluorescent plexus in the granular layer measure 10-60 mu. In the molecular layer (top of the folia) there are about 30 fluorescent fibers per 100 mu2. The fluorescent fibers originate from the locus coeruleus and form a rostral and a caudal bundle in the cerebellar peduncle. The mode of distribution of the fluorescent fibers in the cortical layers seems to depend on the organization of the innervated tissue. Light microscopy suggests that the aminergic fibers innervate more than one class of cerebellar neurons.

Ketamine inhibits serotonin uptake in vivo.

Anesthetic (120 and 160 mg/kg. i.p.) and subanesthetic (80 mg/kg) doses of ketamine HCl were found to prevent completely the depletion of whole brain serotonin (5-HT) by p-chloramphetamine (PCA). Furthermore, ketamine HCl (160 mg/kg) completely blocked the depletion of 5-HT by PCA in every individual brain region studied (Midbrain-thalamus, hypothalamus, striatum, hippocampus and cortex). Administration of ketamine alone had no effect on brain 5-HT levels. Nialamide (a monoamine oxidase (MAO) inhibitor) and fluoxetine (a selective 5-HT uptake inhibitor) also prevented the depletion of 5-HT by PCA. However, of these three agents, only nialamide prevented the depletion of 5-HT by reserpine. These results suggest that ketamine blocks PCA-induced 5-HT depletion by inhibiting 5-HT uptake and not by inhibiting MAO. Ketamine only weakly affected either [3H]5-HT or [3H]spiroperidol binding to 5-HT1 and 5-HT2 receptors respectively even at concentrations as high as 1 mM. These data support the contention that the primary direct effect of ketamine on serotonergic systems is the blockade of 5-HT uptake and that blockade of 5-HT uptake may mediate some of the behavioral effects of ketamine, such as analgesia.

Injections of deuterated tryptamine into the nucleus accumbens of the rat: effects on locomotor activity and monoamine metabolism.

Previous studies have shown that the systemic injection of tryptamine stimulates locomotion in rats and that the nucleus accumbens, a region involved in locomotion, contains the largest concentrations of binding sites for tryptamine in the brain of the rat. The present study examined the behavioral and neurochemical effects of bilateral injections into the accumbens of a deuterated analog of tryptamine, a,a-[2H]tryptamine. Injections of 25 micrograms a,a-[2H]tryptamine increased movements in rats at 25-70 min after injection and increased vertical (rearing) activity at 25-40 min. Injections of 50 micrograms of a,a-[2H]tryptamine produced a transient suppression of movement and vertical activity at 5-15 min, followed by increases in these activities at 40-65 min after injection that were comparable to the increases elicited by 10 micrograms of d-amphetamine. At 30 min after the injection of 50 micrograms a,a-[2H]tryptamine the concentration of dopamine in the nucleus accumbens was increased by 87%, and was preceded by a transient decrease in the level of the metabolite of dopamine homovanillic acid. The levels of 5-hydroxytryptamine and its major metabolite, 5-hydroxyindoleacetic acid in the nucleus accumbens were not changed. Thus, a,a-[2H]tryptamine may interact with tryptamine receptors in the nucleus accumbens to modulate locomotor behavior through mesolimbic dopamine neurons.

Effects of antidepressant drugs on a quickly-learned conditioned-suppression response in mice.

Mice experienced to electric shock, exhibited a marked suppression of motor activity when placed in the same cage 24 hr after administration of shocks. Acute administration of imipramine-HCl (10 mg/kg, i.p.), desipramine-HCl (5 and 10 mg/kg, i.p.) and amitriptyline-HCl (5 and 10 mg/kg, i.p.) caused marked reduction of the conditioned suppression of shocked mice, but reduced the motor activity of the non-shocked mice. Maprotiline, mianserin and dimetacrine did not cause reduction of the conditioned suppression. Nialamide (100 mg/kg, i.p.) and pargyline-HCl (100 and 200 mg/kg, i.p.) caused marked reduction of the conditioned suppression but did not increase the motor activity of the non-shocked mice, and tranylcypromine-HCl (10 and 20 mg/kg, i.p.) did not cause reduction of the conditioned suppression. Diphenhydramine-HCl (10 and 20 mg/kg, i.p.) reduced the conditioned suppression of shocked mice in a dose-related manner. Chronic administration of imipramine-HCl (1 and 5 mg/kg, i.p.) for 14 days significantly reduced the conditioned suppression but did not influence the motility rate of the non-shocked mice. Also, chronic administration of amitriptyline (1 mg/kg, i.p.), desipramine (5 mg/kg, i.p.) and dimetacrine (10 mg/kg, i.p.), for 10 days, significantly reduced the conditioned suppression, but did not influence the motility rate of the non-shocked mice. Chronic administration of maprotiline reduced the conditioned suppression. On the other hand, chronic administration of mianserin (5 mg/kg, i.p.) and diphenhydramine (10 mg/kg, i.p.) did not cause a reduction of the conditioned suppression.

Effects of monoamine oxidase inhibitors on levels of catechols and homovanillic acid in striatum and plasma.

Levels of homovanillic acid (HVA), dihydroxyphenylacetic acid (DOPAC) and dihydroxyphenylglycol (DHPG) in plasma and the striatium were measured after inhibition of monoamine oxidase type A (MAO-A) by clorgyline (4 mg/kg i.p.), MAO-B by (-)deprenyl (1 mg/kg i.p.), both MAO-A and MAO-B by nialamide (75 mg/kg i.p.) or peripheral neuronal MAO by debrisoquin (40 mg/kg i.p.). Levels of HVA in plasma decreased by about 60% after single doses of nialamide or clorgyline, by about 80% after repeated doses of nialamide, by about 40% after a single dose of debrisoquin and by about 50% after repeated doses of debrisoquin. The administration of clorgyline, nialamide or debrisoquin significantly decreased concentrations of DOPAC and DHPG in plasma, whereas (-)deprenyl did not affect levels of DHPG or HVA. None of the MAO inhibitors produced more than about 80% depression of levels of any of the deaminated metabolites. The results suggest that most of the HVA in plasma is derived from deamination of DA by MAO-A in peripheral neurons; that DOPAC in plasma is derived from cells outside the central nervous system; that DHPG in plasma is derived virtually exclusively from the metabolism of norepinephrine in sympathetic nerve endings and that residual levels of HVA after treatment with debrisoquin provide an improved but limited indication of central dopaminergic activity.

Bromocriptine enhances the behavioural effects of apomorphine and dopamine after systemic or intracerebral injection in rats.

The ergot alkaloid bromocriptine, given intraperitoneally produced dose-dependent, long-lasting stereotyped behaviour in rats which was partly antagonised by the injection of trifluoperazine into the caudate nucleus. The stereotyped behaviour produced by apomorphine (s.c.) in both naïve and catecholamine-depleted rats was significantly enhanced by prior treatment with bromocriptine (i.p.). The bilateral application of bromocriptine (2.5-40 micrograms/side in either 0.5% tartaric acid or 50% propylene glycol aqueous vehicles) to the nucleus accumbens (NAC) of rats had no effect on locomotion over a 12 hr period after injection. In contrast, another ergot alkaloid, ergometrine, dissolved in the propylene glycol vehicle, and dopamine (DA) dissolved in either of the vehicles or in saline, produced marked stimulation of locomotion. As well as being inactive after direct application to the nucleus accumbens, bromocriptine (10-160 micrograms/side) did not induce stereotyped behaviour after bilateral injection into the caudate nucleus. However, the local application of bromocriptine (10 micrograms/side) to the nucleus accumbens, while itself inactive, significantly enhanced the locomotor stimulant effect of DA (5 micrograms/side) applied to the same nucleus. The data suggest that bromocriptine is able to enhance the effects of agonists such as DA and apomorphine at DA receptors, even under conditions where bromocriptine itself is inactive.

Immunocytochemical localization and circadian variations of serotonin and N-acetylserotonin in photoreceptor cells. Light and electron microscopic study in the teleost pineal organ.

Using two immunocytochemical procedures (i.e., immunofluorescence and the unlabeled peroxidase-antiperoxidase method), the localization of a serotonin(HT)-like and of a N-acetylserotonin (aHT)-like immunoreactivity in the pineal organ of the pike was studied during winter. It was shown that immunostaining was exclusively restricted to the cells of the receptor line (CRL = typical and modified photoreceptors). The intensity of the reactions varied through the light-dark cycle, HT-like immunoreactivity being high during the photophase and low during the scotophase. In contrast, aHT-like immunoreactivity was highest at the beginning of the scotophase. HT and aHT-like immunoreactivities were detected in all cell types of the pineal epithelium after administration of a monoamine oxidase inhibitor. Up to now, only HT immunoreactivity could be localized at the ultrastructural level. In a number of typical and modified photoreceptors, a HT-positive staining seemed to be confined within the hyaloplasm of the inner segment, particularly with that of the perikaryon and basal pedicle. Our previous and present results strongly suggest that indole compounds, which are involved in the regulation of various neuroendocrine processes in fish, are synthetized within the CRL. Taking into account that the CRL of the pike are also photosensitive, it appears more and more likely that they are photoneuroendocrine cells involved in mediating the effects of the photoperiod on various physiological and behavioral processes.

Serotonin-stimulated cyclic AMP synthesis in the rabbit corneal epithelium.

Serotonin increases the level of cyclic AMP in incubated rabbit corneas; the concentration of agonist producing half-maximal stimulation is approximately 1.5 microM. Nialamide, an inhibitor of monoamine oxidase, potentiates the response to serotonin but not to epinephrine. Amitriptyline, an inhibitor of neuronal uptake of serotonin, does not potentiate the stimulation of cyclic AMP synthesis. Lysergic acid diethylamide, but not timolol, antagonizes the response to serotonin; the half-maximal inhibitory concentration is approximately 6 nM lysergic acid diethylamide. A comparison of the time course of the increase in cyclic AMP synthesis after addition of serotonin or epinephrine to the incubation media indicates that serotonin, but not epinephrine, must penetrate a barrier to its free diffusion. We conclude that the corneal epithelium contains specific serotonergic receptors that, upon activation, cause the synthesis of cyclic AMP, which mediates the stimulation of chloride transport (c.f. companion article, Klyce et al.). The serotonergic receptors must be at a location posterior to the beta-adrenergic receptors, which are on the anterior-surface of the apical cells.

Neural serotonin stimulates chloride transport in the rabbit corneal epithelium.

Evidence is presented that serotonin acts as a neurotransmitter in the cornea of the adult rabbit. Serotonin was localized to granules in a sparse population of subepithelial corneal nerves by an electron microscopic histochemical procedure. Significant endogenous levels of serotonin and its principal metabolite, 5-hydroxyindoleacetic acid, were detected in the central cornea by a fluorometric assay. Exogenous serotonin stimulated ion transport by corneal epithelium. This effect was potentiated by monoamine oxidase inhibition and was unaffected by an alpha-adrenergic receptor antagonist. Serotonin-stimulated ion transport was inhibited by the specific antagonist, methysergide, and by the replacement of Cl- with an impermeable anion. In tracer experiments, the serotonin-stimulated ion transport was shown to be caused by increased epithelial Cl- secretion. The serotonin response was partially inhibited by the beta-adrenergic antagonist, timolol. In a companion article, assay of corneal cyclic AMP showed stimulation of cyclic AMP synthesis by serotonin, inhibition by the specific antagonist, lysergic acid diethylamide, and potentiation by monoamine oxidase inhibition. We postulate that specific serotonergic receptors are present in the corneal epithelium and that activation of these receptors by serotonin released from serotonergic neurons increases the level of cyclic AMP, which stimulates active Cl- secretion by the corneal epithelium.

Histochemical localization of adrenergic nerves in the guinea-pig trachea.

1. Specific catecholamine fluorescence was demonstrated in guinea-pig trachea in fine, varicose, nerve fibres running parallel to the tracheal smooth muscle fibres.2. The density of nerves in tracheal smooth muscle was greater at the laryngeal end than at the bronchial end of the trachea.3. The findings confirm pharmacological evidence for an adrenergic innervation of the guinea-pig isolated tracheal chain preparation.

Variation in activity of monoamine metabolizing enzymes in rat liver during pregnancy.

1. Catechol-0-methyltransferase (COMT) and monoamine oxidase (MAO) activities in rat liver were measured during pregnancy, parturition and postpartum. Compared with activity in non-pregnant controls, both enzymes showed a significant decrease in activity which was most pronounced at day 18. 2. The metabolism of intravenously infused [3H]-adrenaline to [3H]-metanephrine and to [3H]-acidic metabolites was also significantly depressed during pregnancy but had returned to control values by the 21st day. 3. The effects of reserpine and/or nialamide on hepatic COMT and MAO were studied in control and 20-day-pregnant rats. Their action on COMT activity differed in the two groups. MAO was inhibited to a similar extent in these groups whether the drugs were given separately or in combination. 4. It seems possible that the changes in endocrine function which occur during pregnancy are responsible for the observed alterations in enzyme activity.