Monoamine Oxidase Activity and Monoamine Metabolism in Brains of Parkinsonian Patients Treated with l-Deprenyl

Monoamine oxidase (MAO) type A and type B were measured using kynuramine, 3,4-dihydroxyphenylethylamine (dopamine, DA), and 5-hydroxytryptamine (5-HT, serotonin) in 20 brain areas. The highest activities were found in the striatum (caudate nucleus, putamen, globus pallidus, and substantia nigra), hypothalamus, and c-mammilare. The ratio of DA to 5-HT deamination varied in the different regions, being in favor of DA in the striatum. With kynuramine as the substrate IC50 values of a number of inhibitors indicated that l-deprenyl was far more potent an inhibitor of human brain MAO than clorgyline or harmaline. N-Desmethylpropargylindane hydrochloride (AGN 1135) was also shown to have MAO-B inhibitory selectivity similar to that of l-deprenyl. Brains obtained at autopsy from l-deprenyl-treated Parkinsonian patients showed that, whereas MAO-B was fully inhibited by the therapeutic doses of l-deprenyl, substantial MAO-A activity was still evident. These results are matched by the significant increases of DA noted in caudate nucleus, globus pallidus, putamen, and substantia nigra and the unaltered 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) in the same regions. These data indicate that the therapeutic actions of l-deprenyl may lie in its selective inhibition of MAO-B resulting in increased brain levels of DA formed from L-dihydroxyphenylacetic acid (L-DOPA).     

A and B Forms of Monoamine Oxidase Within the Monoaminergic Neurons of the Rat Brain

The inhibition of the A and B forms of monoamine oxidase (MAO) inside and outside serotonergic, noradrenergic, and dopaminergic synaptosomes in homogenates of rat hypothalamus or striatum by clorgyline, a selective and irreversible MAO-A inhibitor, and selegiline, a selective and irreversible MAO-B inhibitor, was examined. Intrasynaptosomal deamination at low concentrations of the substrates [14C]5-hydroxytryptamine ([14C]5-HT; 0.1 microM), [14C]noradrenaline (0.25 microM), [14C]3,4-dihydroxyphenylethylamine ([14C]dopamine; 0.25 microM), and [14C]tyramine (0.25 microM) was hindered by selective uptake inhibitors (citalopram, maprotiline, and amfonelic acid) in the incubation media. Thus, the difference between the deamination of 14C-amine in the absence and presence of the appropriate selective uptake inhibitor provided a measure of deamination in the specific aminergic synaptosomes. This was verified by determining the loss of MAO activity within noradrenergic and serotonergic systems after degeneration of the nerve terminals by the neurotoxins N-chloroethyl-N-ethyl-2-bromobenzylamine and p-chloroamphetamine. Results with the two inhibitors revealed that the A and B forms were responsible for 80 and 20%, respectively, of the deamination of [14C]5-HT within serotonergic synaptosomes from the hypothalamus. The deamination of [14C]noradrenaline within the noradrenergic synaptosomes from the hypothalamus and that of [14C]dopamine and [14C]tyramine within the striatal dopaminergic synaptosomes were due to MAO-A. About 10% of the deamination of [14C]noradrenaline, [14C]dopamine, and [14C]tyramine outside the noradrenergic or dopaminergic synaptosomes was brought about by the B form, with the remainder being deaminated by MAO-A.     

3,4-Dihydroxyphenylethylamine and 5-Hydroxytryptamine Metabolism in the Rat: Acidic Metabolites in Cisternal Cerebrospinal Fluid Before and After Giving Probenecid

3,4-Dihydroxyphenylethylamine (DA, dopamine) and 5-hydroxytryptamine (5-HT) turnover values were determined in freely moving male rats by measuring the rates of accumulation of the acidic metabolites of the above transmitters, i.e., 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA) in cisternal cerebrospinal fluid (CSF) samples after probenecid (200 mg/kg i.p.) administration. Determinations on samples before and after acid hydrolysis showed that the latter procedure was necessary for DA turnover determination. Thus whereas total (DOPAC + HVA) increased linearly with time after probenecid, free (DOPAC + HVA) did not. This was because the percentage of DOPAC + HVA in conjugated form increased with time. Determinations on a group of 28 rats during the dark (red light) period showed that cisternal amine metabolite concentrations before probenecid injection did not parallel turnover values. This was probably because individual differences in metabolite egress strongly affect the pre-probenecid values. The poor correlations between CSF tryptophan and 5-HT turnover suggested that differences of brain tryptophan concentration were not major determinants of differences of brain 5-HT metabolism within this group of normal rats. Considering that the rats were of similar weight and that the turnover values were all determined at approximately the same time of day, the three- to fourfold ranges of the turnover values are remarkable. The positive correlation between the DA and 5-HT turnovers of individual rats suggests the existence of common effects on DA and 5-HT turnover in normal rats.     

Effect of Intrastriatal Injection of Diisopropylfluorophosphate on Acetylcholine, Dopamine, and Serotonin Metabolism

Diisopropylfluorophosphate (81.5 nmol) was injected directly into the striata of rats to study changes in striatal metabolism of acetylcholine (ACh), 3,4-dihydroxyphenylethylamine (dopamine), and 5-hydroxytryptamine (serotonin) at early time points following acute irreversible inhibition of cholinesterase. Twenty minutes following the intrastriatal injection of diisopropylfluorophosphate, levels of striatal acetylcholine were elevated by 50%, but a decrease in KACh compensated for this change. At 1 h, levels of ACh were still elevated, but not significantly different from control values. However, KACh and, hence, ACh turnover were greatly enhanced at this time. Finally, at 24 h, striatal ACh content was only slightly elevated and KACh and the turnover rate of ACh had returned to control values. Striatal cholinesterase activity remained significantly inhibited at all three times. At none of these times was ACh content or turnover affected in the parietal cortex, hippocampus, hypothalamus, or medulla/pons. Neither dopamine and its metabolites 3,4-dihydroxyphenylacetic acid and homovanillic acid nor serotonin and its metabolite 5-hydroxyindoleacetic acid were significantly affected at any of the three times by intrastriatal diisopropylfluorophosphate treatment. Possible mechanisms of the changes in cholinergic parameters are discussed.     

Synaptosomal Calcium Metabolism During Hypoxia and 3,4-Diaminopyridine Treatment

A decline in the calcium-dependent release of neurotransmitters appears to underlie the decreased neuronal function that accompanies reduced oxygen tensions (hypoxia). To determine if alterations in calcium uptake are primary to these changes, synaptosomal calcium uptake was measured in the presence of 100%, 2.5%, or 0% oxygen. Calcium uptake declined 60.2 +/- 0.1 and 82.4 +/- 2.5% with 2.5% and 0% when compared with 100% oxygen, respectively. 3,4-Diaminopyridine stimulated calcium uptake by synaptosomes when they were incubated in low-potassium media. It also diminished the hypoxic-induced decline in calcium uptake to 30.6 +/- 3.1 and 33.5 +/- 3.1% with 2.5% and 0% oxygen, respectively. External binding to the synaptosomal plasma membrane declined to 29.2 +/- 0.3 or 11.8 +/- 0.9% when the oxygen tension was reduced to 2.5% or 0% oxygen. 3,4-Diaminopyridine increased this superficial binding from 111.7 +/- 0.3 to 86.5 +/- 0.9 or 23.4 +/- 0.9% with 100%, 2.5%, or 0% oxygen when compared with 100% oxygen without 3,4-diaminopyridine, respectively. Thus, the decline in neuronal processing that accompanies acute hypoxia may be due to altered calcium homeostasis, which diminishes neurotransmitter release.     

Phosphatidylinositol Metabolism During In Vitro Hypoxia

The effects of in vitro histotoxic hypoxia (0.5 mM KCN) on potassium-stimulated phosphatidylinositol turnover were determined. In rat cortical slices that were prelabeled with [2-3H]inositol, depolarization with 60 mM KCl increased [2-3H]inositol monophosphate and [2-3H]inositol bisphosphate accumulation in a Ca2+-dependent manner. At early times (10 s and 1 min), histotoxic hypoxia enhanced potassium-stimulated [2-3H]inositol monophosphate and inositol bisphosphate accumulation. Under basal conditions, hypoxia did not alter the accumulation of [2-3H]inositol phosphates. These results are consistent with the following hypothesis. The hypoxic-induced increase in cytosolic free calcium that we reported previously may lead to the early stimulation of inositol phosphates formation during hypoxia through activation of phospholipase C. The impairment of inositol phosphates formation during more prolonged hypoxia may be due to negative feedback regulation of the phosphatidylinositol cascade by protein kinase C or to a reduction in ATP levels.     

Decreases in Amino Acid and Acetylcholine Metabolism During Hypoxia

Abstract: Hypoxia impairs brain function by incompletely defined mechanisms. Mild hypoxia, which impairs memory and judgment, decreases acetylcholine (ACh) synthesis, but not the levels of ATP or the adenylate energy charge. However, the effects of mild hypoxia on the synthesis of the glucosederived amino acids [alanine, aspartate, γ-amino butyric acid (GABA), glutamate, glutamine, and serine] have not been characterized. Thus, we examined the incorporation of [U-¹⁴C]glucose into these amino acids and ACh during anemic hypoxia (injection of NaNO₂), hypoxic hypoxia (15 or 10% O₂), and hypoxic hypoxia plus hypercarbia (15 or 10% O₂ with 5% CO₂). In general, the synthesis of the amino acids and of ACh declined in parallel with each type of hypoxia we studied. For example, anemic hypoxia (75 mg/kg of NaNO₂) decreased the incorporation of [U-¹⁴C]glucose into the amino acids and into ACh similarly. [Percent inhibition: ACh (57.4), alanine (34.4), aspartate (49.2), GABA (61.9). glutamine (59.2), glutamate (51.0), and serine (36.7)]. A comparison of several levels (37.5, 75, 150, 225 mg/kg of NaNO₂) of anemic hypoxia showed a parallel decrease in the flux of glucose into ACh and into the amino acids whose synthesis depends on mitochondrial oxidation: GABA (r= 0.98), glutamate (r= 0.99), aspartate (r= 0.96), and glutamine (r= 0.97). The synthesis of the amino acids not dependent on mitochondrial oxidation did not correlate as well with changes in ACh metabolism: serine (r= 0.68) and alanine (r= 0.76). The decreases in glucose incorporation into ACh and into the amino acids with hypoxic hypoxia (15% or 10% O₂) or hypoxic hypoxia with 5% CO₂ were very similar to those with the two lowest levels of anemic hypoxia. Thus, any explanation of the brain's sensitivity to a decrease in oxygen availability must include the alterations in the metabolism of the amino acid neurotransmitters as well as ACh.     

Effect of Decreased Oxygen on In Vitro Release of Endogenous 3,4-Dihydroxyphenylethylamine from Mouse Striatum

The effects of hypoxia on release of endogenous 3,4-dihydroxyphenylethylamine (DA, dopamine) were investigated in mouse striatal slices. Following a 60-min preincubation, potassium increased DA release 12 times between zero and 1 min. By 10 min, uptake processes exceeded release and DA levels in the media decreased. Hypoxia (low oxygen) and anoxia (no oxygen) increased DA in the media by 120 and 205%, respectively, but did not alter dihydroxyphenylacetic acid concentrations. Under similar conditions, anoxia increased [3H]DA uptake eight-fold. For the uptake studies, the amount of DA added to the media was critical; in the presence of high concentrations of DA, anoxia reduced reuptake. Regardless of exogenous DA, hypoxia and anoxia increased extracellular DA, which may play a role in ischemic cell damage.     

Subsynaptosomal Calcium Distribution During Hypoxia and 3,4-Diaminopyridine Treatment

Previous results demonstrate that hypoxia (low oxygen) diminishes calcium uptake by synaptosomes. The present studies examined the effects of low oxygen on calcium homeostasis in the digitonin-resistant (mitochondrial) and the digitonin-labile (nonmitochondrial) compartments of intact synaptosomes and their relation to altered membrane potentials. A 10-min hypoxic incubation in low-potassium media reduced total (-38.3%), mitochondrial (-43.3%), and nonmitochondrial (-27.8%) calcium uptake. In high-potassium media, low oxygen reduced mitochondrial (-41.2%) and total (-34.4%) uptake whereas nonmitochondrial (+ 6%) calcium uptake was essentially unaffected. A temporal analysis of nonmitochondrial calcium uptake revealed an initial depression (0-5 min) followed by a stimulation (5-10 min). Hypoxic-induced alterations in the subsynaptosomal distribution of calcium resembled those produced by uncouplers [FCCP (carbonylcyanide-p-trifluoromethoxyphenylhydrazone) or rotenone plus oligomycin]. 3,4-Diaminopyridine partially ameliorated the hypoxic- and FCCP-induced decreases in synaptosomal calcium uptake. Low oxygen reduced the total synaptosomal membrane potential (i.e., plasma plus mitochondrial membrane potential) as measured by an increased efflux of tetraphenylphosphonium ion. This hypoxic-induced efflux of tetraphenylphosphonium was slowed by pretreatment with 3,4-diaminopyridine. Thus, both drug and membrane potential studies suggest that hypoxic-induced alterations in the subcellular distribution of calcium may be due to an uncoupling mechanism and a collapse of the synaptosomal mitochondrial membrane potential.     

Monoamine Synthesis and Concentration in Neonatal Rat Brain During Hypercapnia and Recovery

One-day-old rats were exposed to a gas mixture of 15% CO2-21% O2-64% N2 for a 30-min period. Monoamine synthesis in whole brain was measured during, and at various intervals after, hypercapnia by estimating the accumulation of dihydroxyphenylalanine (DOPA) and 5-hydroxytryptophan (5-HTP) after inhibition of aromatic L-amino-acid decarboxylase with NSD 1015. Endogenous concentrations of tyrosine, dopamine (DA), noradrenaline (NA), tryptophan, 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) were measured at the same intervals. Exposure to CO2 induced an increased synthesis of catecholamines and 5-HT. Further, an increase in DA concentration was seen during hypercapnia, while NA and 5-HT were unchanged. After the CO2 exposure the increased in vivo synthesis rates of catecholamines and 5-HT were rapidly normalized, as was the endogenous DA concentration. A slight increase in 5-HT and 5-HIAA concentrations was seen immediately after CO2 exposure. These results indicate that in neonatal animals, hypercapnia induces changes in central monoamine neurons, primarily an increased synthesis. These alterations may be relevant to some physiological changes seen during CO2 exposure, such as the alteration in central respiratory performance.     

Transient Hypoxia Alters Striatal Catecholamine Metabolism in Immature Brain: An In Vivo Microdialysis Study

Microdialysis probes were inserted bilaterally into the striatum of 7-day-old rat pups (n = 30) to examine extracellular fluid levels of dopamine, its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), and the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA). The dialysis samples were assayed by HPLC with electrochemical detection. Baseline levels, measured after a 2-h stabilization period, were as follows: dopamine, not detected; DOPAC, 617 +/- 33 fmol/min; HVA, 974 +/- 42 fmol/min; and 5-HIAA, 276 +/- 15 fmol/min. After a 40-min baseline sampling period, 12 animals were exposed to 8% oxygen for 120 min. Hypoxia produced marked reductions in the striatal extracellular fluid levels of both dopamine metabolites (p less than 0.001 by analysis of variance) and a more gradual and less prominent reduction in 5-HIAA levels (p less than 0.02 by analysis of variance), compared with controls (n = 12) sampled in room air. In the first hour after hypoxia, DOPAC and HVA levels rose quickly, whereas 5-HIAA levels remained suppressed. The magnitude of depolarization-evoked release of dopamine (elicited by infusion of potassium or veratrine through the microdialysis probes for 20 min) was evaluated in control and hypoxic animals. Depolarization-evoked dopamine efflux was considerably higher in hypoxic pups than in controls: hypoxic (n = 7), 257 +/- 32 fmol/min; control (n = 12), 75 +/- 14 fmol/min (p less than 0.001 by analysis of variance). These data demonstrate that a brief exposure to moderate hypoxia markedly disrupts striatal catecholamine metabolism in the immature rodent brain.     

Cytosolic Free Calcium and Gene Expression During Chemical Hypoxia

Understanding the cellular response to hypoxia may help elucidate the role of altered oxidation in neuronal death or abnormal cell function. In PC12 cells, 30 min of chemical hypoxia (i.e., KCN) reduced ATP concentrations by 92%, but diminished viability by only 10%. Ten minutes of hypoxia increased cytosolic free calcium ([Ca2+]i) 2.5-fold above control, but after 30 min of hypoxia, [Ca2+]i was slightly below that of nonhypoxic cells. Short periods of hypoxia also exaggerated the K(+)-induced elevation of [Ca2+]i, but by 30 min these ATP-depleted cells reestablished a calcium gradient that was equal to nonhypoxic, K(+)-depolarized cells. Thus, 30 min of severe ATP depletion left [Ca2+]i and viability relatively unaffected. Nerve growth factor caused slight, but significant, improvements in ATP and viability of hypoxic cells, but had no effect on [Ca2+]i. Although [Ca2+]i was equivalent in control and hypoxic cells after 30 or 60 min, hypoxia abolished the K(+)-stimulated elevation of [Ca2+]i. The nerve growth factor induction of c-fos, an indicator of the genomic response, was diminished by approximately 80%. Thus, hypoxic PC12 cells with greatly reduced ATP stores maintained normal [Ca2+]i, but their ability to respond to external stimulation was impaired. Further, the reduced oxidation that occurs in the brain in a variety of pathological conditions may interfere with the cellular response to stimulation and growth factors.     

Circadian Rhythms of Locomotor Activity in the Nile Tilapia Oreochromis niloticus

Behavioral rhythms of the Nile tilapia were investigated to better characterize its circadian system. To do so, the locomotor activity patterns of both male and female tilapia reared under a 12:12 h light-dark (LD) cycle were studied, as well as in males the existence of endogenous rhythmicity under free-running conditions (DD and 45 min LD pulses). When exposed to an LD cycle, the daily pattern of activity differed between individuals: some fish were diurnal, some nocturnal, and a few displayed an arrhythmic pattern. This variability would be typical of the plastic circadian system of fish. Moreover, reproductive events clearly affected the behavioral rhythms of female tilapia, a mouth-brooder teleost species. Under DD, 50% (6 of 12) of male fish showed circadian rhythms with an average period (τ) of 24.1±0.2 h, whereas under the 45 min LD pulses, 58% (7 of 12) of the fish exhibited free-running activity rhythms with an average τ of 23.9±0.5 h. However, interestingly in this case, activity was always confined to the dark phase. Furthermore, when the LD cycle was reversed, a third of the fish showed gradual resynchronization to the new phase, taking 7–10 days to be completely re-entrained. Taken together, these results suggest the existence of an endogenous circadian oscillator that controls the expression of locomotor activity rhythms in the Nile tilapia, although its anatomical localization remains unknown.     

Social Interactions and the Circadian Rhythm in Locomotor Activity in the Cockroach Leucophaea maderae

The role of social interactions in entrainment has not been extensively studied in the invertebrates. Leucophaea maderae is a gregarious species of cockroach that exhibits extensive social interactions. Social interactions associated with copulation between the sexes have been shown to be regulated by the circadian system. We show here that social interactions between males are also under circadian control. We examined the question of whether or not these rhythmic social contacts could function as zeitgebers capable of regulating circadian phase and period. Animals initially in phase that were housed as groups or pairs of single sex or mixed sex in constant darkness for 2–7 weeks were found to drift out of phase. Their behavior was not significantly different from individual animals maintained in isolation. Further, animals that were initially out of phase by 12 h housed as groups or pairs were not significantly different in phase from animals that were isolated. The results show the circadian clocks of cockroaches are remarkably insensitive to the extensive social interactions that occur between individuals.     

Distribution of the Two Forms of Monoamine Oxidase Within Monoaminergic Neurons of the Guinea Pig Brain

The relative distribution of type A and type B monoamine oxidase (MAO) inside and outside the monoaminergic synaptosomes in preparations from hypothalamus and striatum of the guinea pig was determined by incubation of synaptosomal preparations of these regions with low concentrations of [14C]5-hydroxytryptamine (5-HT), noradrenaline, and dopamine. The deamination within the monoaminergic synaptosomes was hindered by selective amine uptake inhibitors. In the absence of these inhibitors, both intra- and extraneuronal deamination was measured. The two forms of the enzyme were differentiated with the irreversible and selective MAO-A and MAO-B inhibitors clorgyline and selegiline (l-deprenyl), respectively. [14C]5-HT was deaminated greater than 90% by MAO-A both inside and outside the 5-hydroxytryptaminergic synaptosomes prepared from the guinea pig hypothalamus. The deamination of [14C]noradrenaline within the noradrenergic synaptosomes of the hypothalamic preparation was in the ratio 75:25% for MAO-A:MAO-B; the corresponding ratio outside these synaptosomes was 45:55%. The deamination of [14C]dopamine within dopaminergic synaptosomes in the striatal preparation was 65% type A:35% type B, whereas outside these synaptosomes the ratio was 35:65%. Because the relative amounts and the distribution of the two forms of MAO in the guinea pig brain seem to be similar to those previously detected for the human brain, the MAO in the guinea pig brain may be a good model for the MAO in the human brain.     

Monoamine Metabolism in the Locus Coeruleus Measured Concurrently with Behavior During Opiate Withdrawal: An In Vivo Microdialysis Study in Freely Moving Rats

Abstract: Using microdialysis, changes in monoamine metabolism were monitored in the locus coeruleus of freely moving rats during opiate withdrawal concomitantly with behavioral symptoms. Rats were infused with morphine (2 mg/kg/h, s.c.) or saline for 5 days and challenged with naltrexone (100 mg/kg, s.c.) on day 6. Following naltrexone challenge, the classic behavioral symptoms of morphine withdrawal were observed in rats treated with morphine but not in saline-infused rats. In morphine-dependent rats, naltrexone induced a marked increase (280%) in dialysate concentrations of 3,4-dihydroxyphenylacetic acid, an index of the functional activity of the noradrenergic neurons in the locus coeruleus. The local concentrations of the serotonin metabolite 5-hydroxyindoleacetic acid were also increased (70%) during morphine withdrawal. Taken together, these results (a) confirm in unanesthetized rats the hypothesis of an activation by opiate withdrawal of noradrenergic neurons in the locus coeruleus and (b) suggest an increase in serotonergic transmission in the same nucleus during morphine withdrawal.     

Determination of 3-Methoxytyramine in Rat Brain by HPLC with Electrochemical Detection and Its Correlation with Dopamine Function After Administration of a Monoamine Oxidase Inhibitor and L-3,4-Dihydroxyphenylalanine

3-Methoxytyramine (3-MT), normally a minor metabolite of 3,4-dihydroxyphenylethylamine (dopamine) in brain, becomes the sole product of metabolism following the administration of a monoamine oxidase (MAO) inhibitor. A simplified reverse-phase HPLC method for 3-MT employing electrochemical detection is fully described. This method has a detection limit of 0.1 microgram/g brain wet weight and is sensitive enough to detect 3-MT in individual brain regions after rats have been pretreated with an MAO inhibitor. Administration of tranylcypromine (TCP, 10 mg/kg) and L-3,4-dihydroxyphenylalanine (L-DOPA) (10-50 mg/kg) produced a dose-dependent linear increase in 3-MT concentrations in the dopaminergic brain regions n. caudatus (r = 0.95; p less than 0.01) and n. accumbens (r = 0.96; p less than 0.01). This treatment also produced a dose-dependent increase in behavioural activity in rats (r = 0.88; p less than 0.01). Furthermore, a good correlation was found between the activity responses of individual rats and the accumulation of 3-MT after TCP/L-DOPA in both n. caudatus (r = 0.76; p less than 0.01) and n. accumbens (r = 0.84; p less than 0.01). These data describe a simple and sensitive HPLC analysis technique for 3-MT and demonstrate that following administration of an MAO inhibitor this metabolite may provide a useful monitor of central dopamine function.     

Circadian Locomotor Activity Rhythm During Ontogeny in Crayfish Procambarus Clarkii

The characteristics of the circadian rhythm of locomotor activity in the crayfish Procambarus clarkii during ontogeny under constant darkness and light-dark (LD 12:12) conditions were studied in 132 juvenile crayfish, aged 10-140 days, divided in four groups. All animals were individually monitored with a motor activity recording system. Activity was quantitatively and qualitatively analyzed. All ages showed a circadian rhythm, although the probability of its appearance increased with age. Period values oscillated between 25.0 h in group I (2-4-week-old animals) and 24.3 h in group IV (16-20-week-old animals with more than 6 molts), but always with a high standard deviation. Groups II (5-10-week-old animals) and IV showed a statistically significant bimodal nonrandom synchrony of phases. The activity/ rest relationship diminishes as development progresses and is most uniform in group IV. We discuss the possibility that the pacemaker system responsible for this rhythm might be present from the moment of eclosion, but the coupling strength of this system with the effectors might change along development. The results presented in this work seem to indicate that the central pacemakers responsible for the activity and the ERG rhythm are not the same.     

Stocking Density Affects Circadian Rhythms of Locomotor Activity in African Catfish,Clarias gariepinus

The effect of stocking density on the locomotor activity of African catfish C. gariepinus under different light regimes was investigated. C. gariepinus were stocked under different densities (1, 5, or 10 fish/tank), and their locomotor activity recorded under light-dark (LD), constant light (LL), constant darkness (DD), and LD-reversed (DL) regimens. Under the LD cycle, catfish showed a crepuscular activity pattern, irrespective of stocking density, with most of the daily activity concentrated around the light-onset and light-offset times. When fish were subjected to DD, all 4 tanks with medium (5 fish) and high (10 fish) stocking densities showed circadian rhythmicity, with an average period (τ) of 23.3?±?0.5 and 24.6?±?0.5?h, respectively. In contrast, only 2 low (1 fish) density tanks showed free-running rhythms. Under LL, activity levels decreased significantly in comparison with levels observed under LD and DD. Moreover, fish of 1, 2, and 3 out of the 4 tanks with low, medium, and high densities, respectively, showed free-running rhythms under these conditions. When the photocycle was reversed (DL), fish of 3, 2, and 4 out of the 4 tanks with low, medium, and high stocking densities, respectively, showed gradual resynchronization to the new phase, and transient cycles of activity were observed. These results suggest that stocking density of fish affected the display of circadian rhythmicity and the intensity of activity levels. Thus, fish kept in higher densities showed more robust rhythmicity and higher levels of daily activity, indicating that social interactions may have an influence on behavioral patterns in the African catfish. (Author correspondence: lmvera@um.es)     

Cerebrovascular Endothelial Cell Culture: Metabolism and Synthesis of 5-Hydroxytryptamine

The presence of 5-hydroxytryptamine was investigated in cultured and propagated cerebrovascular endothelium using immunohistochemistry and high pressure liquid chromatography. These studies demonstrate that the endothelium has the ability to take up and metabolize 5-hydroxytryptamine as well as to synthesize this amine from its precursor L-tryptophan, thus providing evidence for extraneural synthesis of 5-hydroxytryptamine in the central nervous system.