The 5,7-DHT-induced anticonflict effect is dependent on intact adrenocortical function.

We have recently reported that the anxiolytic-like effect observed in rats severely depleted of brain serotonin (5-HT) by means of 5,7-DHT is indirect and probably involves the GABA(A)/benzodiazepine chloride ionophore receptor complex (GABAA/BDZ-RC). One tentative explanation for this effect considered the involvement of corticosteroids. In the present series of experiments we have therefore investigated the effect of adrenalectomy (ADX) on the 5,7-DHT-induced anxiolytic-like effect displayed by rats in Vogel's conflict test. ADX totally abolished the anticonflict effect of the 5,7-DHT lesion. Replacement treatment with corticosterone, but not with dexamethasone, reinstated the anticonflict effect. These results indicate that an intact adrenocortical function, possibly via brain steroid type I receptors, is required for the expression of the 5,7-DHT-induced anxiolytic-like effect. It is postulated that ADX lowers the concentration of endogenous positive modulators at the GABAA/BDZ-RC to a level no longer sufficient to produce anxiolytic-like effects in 5,7-DHT-lesioned animals. The finding that 5,7-DHT-lesioned animals were more sensitive than sham-lesioned controls to the anticonflict effect of the barbiturate-like corticosteroid THDOC provides further support for the contention that an increased endogenous activity at the GABAA/BDZ-RCes is involved in the anxiolytic-like effect observed in rats with a severe depletion of brain 5-HT.     

Involvement of the GABAA/benzodiazepine chloride ionophore receptor complex in the 5,7-DHT Induced anticonflict effect.

The effects of drugs interacting with the GABAA/benzodiazepine chloride ionophore receptor complex (GABAA/BDZ-RC) on the anticonflict and biochemical effects observed after intracerebroventricular (i.c.v.) administration of 5,7-dihydroxytryptamine (5,7-DHT; 450 micrograms -14 days) were investigated in the rat using a modified Vogel's drinking conflict test. The GABAergic antagonistic drugs bicuculline, picrotoxin and Ro 15-4513 all counteracted the 5,7-DHT induced anxiolytic-like action in doses that did not alter the behavior per se, whereas flumazenil was ineffective in this respect. Also i.c.v. administration of 5-HT antagonized the 5,7-DHT induced anticonflict effect. Furthermore, 5,7-DHT-lesioned animals appeared more sensitive to the anticonflict effects of diazepam than sham-lesioned controls. The 5,7-DHT treatment produced marked depletions of 5-HT in the limbic system (80-90%) and hippocampus (90-95%), and an increase in the 5-HIAA/5-HT quotient in hippocampus. The effects on the levels of noradrenaline were comparatively small. The doses of bicuculline and picrotoxin antagonizing the 5,7-DHT induced anticonflict effect did not uniformly influence 5-HT levels or 5-HIAA/5-HT quotients. It is suggested that the anxiolytic-like effect observed in 5,7-DHT-lesioned rats in Vogel's drinking conflict test involves enhanced transmission at the GABAA/BDZ-RC.     

Effect of the local administration of 5,7-DHT and 6-OHDA into the neocortex on the learning and exploratory behavior of rats in an open field

On Wistar rats characteristics were studied of investigating behaviour in the open field, of learning of conditioned food-reinforced reaction and also of BA and their metabolites content in various brain structures under local intracerebral injections of specific neurotoxins; 6-hydroxydopamine (6-OHDA) and 5,7-dihydroxytryptamine (5,7-DHT), abolishing correspondingly catecholaminergic and serotoninergic terminals. Bilateral injection of 6-OHDA in the neocortex led to a weakening of rats investigating activity in the open field and to an increase of the time of fulfillment of the forming of conditioned food-reinforced reaction. Administration of 5,7-DHT was accompanied by an increase of the investigating behaviour in the open field and a reduction of the duration of the forming of conditioned reaction. Administration of 6-OHDA to the neocortex caused a lowering of catecholamines level in the frontal area of the neocortex and the hippocampus. Analogous administration of 5,7-DHT elicited simultaneously with a deep level lowering of 5-HT and its metabolite in these structures, a change of catecholamines content which testifies to a lesser specificity of the neurotoxin 5,7-DHT in comparison with 6-OHDA. Structures lesion of serotoninergic and catecholaminergic systems of the frontal cortex and the hippocampus brought about by a local administration of 6-OHDA and 5,7-DHT in the neocortex was accompanied by differently directed changes in animals behaviour.     

Intraventricular 5,7-Dihydroxytryptamine Increases Thyrotropin-Releasing Hormone Content in Regions of Rat Brain

Rats received intraventricular (i.v.t.) injections of 5,7-dihydroxytryptamine (5,7-DHT) (100-600 micrograms). Some animals also received intraperitoneal injections of the 5-hydroxytryptamine uptake blocker fluoxetine (FX) (20 mg/kg) or the norepinephrine uptake blocker desmethylimipramine (DMI) (48 mg/kg) 30-90 min prior to i.v.t. 5,7-DHT. Rats were killed between 2 and 35 days following i.v.t. 5,7-DHT, brains were dissected, and regions were assayed for thyrotropin-releasing hormone (TRH) by radioimmunoassay. Dose-dependent increases in TRH content following i.v.t. 5,7-DHT were noted in the brainstem and hippocampus. DMI pretreatment blocked the increase in hippocampal TRH, but not in brainstem TRH. FX pretreatment was ineffective in blocking any increases in TRH content. These results suggest differential regulation of regional TRH content by interactions with specific neurotransmitter systems.     

Effects of the intraventricular administration of 5,7-dihydroxytryptamine on FSH, LH and prolactin secretion in neonatally estrogenized male rats

The role of the serotoninergic system in the control of LH, FSH and prolactin secretion was analyzed in control and neonatally estrogenized male rats. Animals injected s.c. with 500 micrograms of estradiol benzoate (EB) on day 1 of life, or their corresponding sham-treated controls, were divided on day 75 into the following groups: (1) orchidectomized; (2) injected intraventricularly with 5,7-dihydroxytryptamine (5,7-DHT); (3) orchidectomized and treated with 5,7-DHT, and (4) sham operated. 15 days later, the animals were decapitated and their FHS, LH and prolactin plasma values measured by specific RIA systems. After the treatment with 5,7-DHT, control animals showed a decline in basal prolactin levels but no modification in basal LH and FSH values. After castration, 5,7-DHT-treated animals showed a reduced LH increase and a more marked prolactin decrease. In neonatal estrogen-treated animals, the 5,7-DHT injection did not change FSH, LH or prolactin levels but did partially or completely abolish the post-castration rise in FSH and LH levels, respectively. These data seem to indicate that neonatal estrogenization induced a modification of the serotoninergic role in the control of LH, FSH and prolactin.     

Effects of luteolin on learning acquisition in rats: involvement of the central cholinergic system

The study was conducted to investigate the ameliorating effects of luteolin on memory acquisition in rats. The effects of luteolin on scopolamine-induced impairment of passive avoidance response were evaluated primarily, as well as the role of the central nervous system through the use of central neurotoxins and central nervous antagonists. Luteolin was not reversed by scopolamine N-methylbromide (M-SCOP) but blocked the impairment of learning acquisition induced by cholinergic neurotoxin (ethylcholine aziridinium, AF64A) and muscarinic (scopolamine hydrobromide, SCOP) and nicotinic (mecamylamine, MECA) receptor antagonists. However, it did not block dopaminergic neurotoxin (6-hydroxydopamine, 6-OHDA)-induced and serotonergic neurotoxin (5,7-dihydroxytryptamine, 5,7-DHT)-induced impairments. From these results, we suggest that the attenuating effect of luteolin (10 mg/kg, i.p.) on the deficits of passive avoidance performance induced by SCOP may be related to the increases in the activities of central muscarinic and nicotinic receptors.     

Effects of 5,7-DHT Injection into the Optic Lobe on the Circadian Locomotor Rhythm in the Cricket, Gryllus bimaculatus

The effect of direct 5,7-dihydroxytryptamine (5,7-DHT) injection into the medulla region of the optic lobe on the locomotor activity was investigated in the adult male cricket, Gryllus bimaculatus. After a 6 hr phase advance of a light-dark cycle, the 5,7-DHT injected animals needed significantly longer time for resynchronization to the new cycle (6.55 +/- 0.62 days) than the control, Ringer's solution injected animals (3.17 +/- 0.15 days; P < 0.001, t-test). Light induced a bout of activity (i.e., masking effect) when light-dark cycle was phase advanced by 6 hr and the duration of the masking effect was significantly longer in 5,7-DHT injected animals. An initial bout of the nocturnal activity was significantly greater in the 5,7-DHT injected animal. Under constant darkness, the freerunning periods of both groups were not significantly different. Under constant light, a significantly higher percentage of 5,7-DHT injected animals showed arrhythmicity compared with the control group. An analysis carried by high-pressure liquid chromatography with electro-chemical detection (HPLC-ECD) revealed that the serotonin content in the optic lobe was significantly reduced to less than 50% in the 5,7-DHT injected animals, even one month after the injection. These results suggest that serotonin plays important roles in the regulation of circadian locomotor rhythms of the cricket mainly by regulating the sensitivity of the photoreceptive system.     

Actions of avermectin B1a on the gamma-aminobutyric acidA receptor and chloride channels in rat brain

The interaction of avermectin B1a (AVM) with the gamma-aminobutyric acid (GABA) receptor of rat brain was studied using radioactive ligand binding and tracer ion flux assays. Avermectin potentiated the binding of [3H]flunitrazepam and inhibited the binding of both [3H]muscimol and [35S]t-butylbicyclophosphorothionate to the GABAA receptor. Inhibition of muscimol binding by AVM suggested competitive displacement. Two kinds of 36chloride (Cl) flux were studied. The 36Cl efflux from preloaded microsacs was potentiated by AVM and was highly inhibited by the Cl-channel blocker 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS). However, it was not potentiated by GABA nor was it sensitive to the convulsants picrotoxin or bicuculline. On the other hand, 36Cl-influx measurement in a different microsac preparation of rat brain was very sensitive to GABA and other GABA-ergic drugs. Avermectin induced 36Cl influx into these microsacs in a dose-dependent manner, but to only 35% of the maximal influx induced by GABA. The AVM-induced 36Cl influx was totally blocked by bicuculline. It is suggested that AVM opens the GABAA-receptor Cl channel by binding to the GABA recognition site and acting as a partial receptor agonist, and also opens a voltage-dependent Cl channel which is totally insensitive to GABA but is very sensitive to DIDS.     

Chronic alterations in serotonin function: dynamic neurochemical properties in agonistic behavior of the crayfish, Orconectes rusticus

The biogenic amine serotonin [5-hydroxytryptamine (5-HT)] has received considerable attention for its role in behavioral phenomena throughout a broad range of invertebrate and vertebrate taxa. Acute 5-HT infusion decreases the likelihood of crayfish to retreat from dominant opponents. The present study reports the biochemical and behavioral effects resulting from chronic treatment with 5-HT-modifying compounds delivered for up to 5 weeks via silastic tube implants. High performance liquid chromatography with electrochemical detection (HPLC-ED) confirmed that 5,7-dihydroxytryptamine (5,7-DHT) effectively reduced 5-HT in all central nervous system (CNS) areas, except brain, while a concurrent accumulation of the compound was observed in all tissues analyzed. Unexpectedly, two different rates of chronic 5-HT treatment did not increase levels of the amine in the CNS. Behaviorally, 5,7-DHT treated crayfish exhibited no significant differences in measures of aggression. Although treatment with 5-HT did not elevate 5-HT content in the CNS, infusion at a slow rate caused animals to escalate more quickly while 5-HT treatment at a faster rate resulted in slower escalation. 5,7-DHT is commonly used in behavioral pharmacology and the present findings suggest its biochemical properties should be more thoroughly examined. Moreover, the apparent presence of powerful compensatory mechanisms indicates our need to adopt an increasingly dynamic view of the serotonergic bases of behavior like crayfish aggression.     

Immediate and Long-Term Effects of 5,7-Dihydroxytryptamine on Rat Striatal Serotonergic Neurons Measured Using In Vivo Voltammetry

The immediate and long-term effects of the selective serotonergic neurotoxin 5,7-dihydroxytryp-tamine (5,7-DHT) on rat striatal serotonergic neurons were examined after its intracerebroventricular administration using in vivo voltammetry. Extracellular concentration of 5-hydroxyindoles increased immediately following intracerebroventricular 5,7-DHT injection (200 g in 24 l, 18 min), peaked at 1.5-2 h, and returned to normal by 4 h. 5,7-DHT diffused to the contralateral striatum in detectable amounts 9 to 12 min after the start of injection and returned to basal levels by 1.5 h. Three to 6 days after 5,7-DHT lesions, 5-hydroxytryptophan administration produced an increase in striatal 5-hydroxyindoles that was greater than that produced in pre-lesioned rats. This effect was maximal at 14 to 17 days post-lesion, and remained even after 50 days. The short-term effect of 5,7-DHT may be attributable to increased serotonin release, inhibition of uptake, or monoamine oxidase inhibition. The long-term effect of 5,7-DHT lesions may attributable to increased synthesis of serotonin or decreased reuptake in remaining serotonergic neurons.     

Recovery of brain noradrenaline after 5,7-dihydroxytryptamine-induced axonal lesions in the rat

Time-dependent changes in regional CNS noradrenaline (NA) concentration, 3H-NA uptake and fluorescence morphology of CNS NA neurons were analysed in the adult rat up to 6 months after intraventricular injection of 5,7-dihydroxytryptamine (5,7-DHT), and compared with the time-course of changes in brain and spinal cord indolamine neurons. Following a substantial depletion of both amines in all CNS regions (telodiencephalon, brainstem and spinal cord) at 10 days after 150 mug 5,7-DHT, brain NA--but not 5-HT--levels recovered to near-normal values in brainstem and forebrain (35% below the age-matched controls) within 4 months. This was accompanied by a total restoration of the initially decreased capacity of the brain tissue to accumulate 3H-NA in vitro. Within 10 days after 5,7-DHT, there was a disappearance of NA terminals from many telencephalic, diencephalic and lower brain stem nuclei, from the cerebral and cerebellar cortices, and the grey matter of the spinal cord, concomitant with the appearance of numerous distorted, highly fluorescent swellings along the non-terminal axons of the major noradrenergic projection pathways. The recovery of the NA levels was paralleled by a re-appearance of fluorescent fibres, signifying an intense sprouting and regrowth of the drug-lesioned axons, which eventually re-innervated some of the previously denervated telodiencephalic regions. Except for a permanent loss of some surface-near perikarya in group A1 (the main source of the bulbospinal projections) there was no evidence of a retrograde degeneration of noradrenergic cell bodies in the rat CNS. The results are compatible with the idea that 5,7-DHT mainly causes a lesion of NA axons at a distance from the cell bodies, and this is followed by sprouting and regrowth of axons from the lisioned neurites, and formation of new terminal-like fibres in some previously denervated telodiencephalic regions. These findings indicate that chemical axotomy of central NA neurons induced by 5,7-DHT is--in contrast to that induced by 6-hydroxydopamine--followed by extensive axonal regeneration.     

The molecular aspects of the functional heterogeneity of the GABA receptors

It is generally accepted that gamma-aminobutyric acid (GABA) is one of the main inhibitory transmitter in the mammalian brain. There are three types of GABA receptors in the vertebrata central nervous system: the GABAA, GABAB and GABAC receptors. The GABAA receptor is a GABA-gated Cl- channel and is the tetramer ore the pentamer made of some classes of subunit (alpha, beta, gamma, delta). GABAB receptors are not affiliated with Cl(-) ionophore. GABAB receptors appear to be coupled to Ca2+ and K+ channels of presynaptic membranes. It seems they regulate the release of neurotransmitters release. The structural and functional properties of GABA receptors are discussed.     

Effects of imipramine and serotonin-2 agonists and antagonists on serotonin-2 and beta-adrenergic receptors following noradrenergic or serotonergic denervation

The effects of chronic (14 day) administration of the tricyclic antidepressant imipramine, the serotonin-2 (5-HT2) antagonist ketanserin, and the serotonin agonist quipazine on 5-HT2 receptor binding parameters and 5-HT2-mediated behavior were examined in rats with or without prior serotonergic denervation [via 5,7-dihydroxytryptamine (5,7-DHT)] or noradrenergic denervation [via N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4)]. Chronic administration of imipramine, ketanserin, or quipazine produced a marked reduction in the number of 5-HT2 binding sites which was accompanied by reductions in the 5-HT2-mediated quipazine-induced head shake response. In animals receiving DSP4 or 5,7-DHT lesions and continuous vehicle treatment, beta-adrenergic receptor binding sites were significantly up-regulated while 5-HT2 receptor binding sites did not change. Imipramine normalized the lesion-induced increases in beta-adrenergic binding observed in DSP4 and 5,7-DHT-lesioned rats but failed to down-regulate beta-adrenergic binding sites below non-lesioned control levels. Chronic imipramine, ketanserin, and quipazine reduced quipazine-induced head shakes and down-regulated 5-HT2 binding sites in rats with noradrenergic denervation. While imipramine, ketanserin, and quipazine all down-regulated 5-HT2 binding sites in animals with serotonergic denervation, only imipramine's ability to reduce quipazine-induced head shakes was attenuated in 5,7-DHT-lesioned rats. The present results suggest that imipramine-induced down-regulation of 5-HT2 receptors may not involve presynaptic 5-HT mechanisms, and imipramine-induced alterations in 5-HT2 sensitivity as reflected in the quipazine-induced head shake may, in part, be influenced by beta-adrenergic receptors.     

Relationship between levels and uptake of serotonin and high affinity [3H]imipramine recognition sites in the rat brain

High affinity [3H]imipramine binding, endogenous levels of serotonin and noradrenaline, and serotonin uptake were determined in brain regions of rats with selective destruction of serotonergic neurons by 5,7-dihydroxytryptamine (5,7-DHT), of adrenergic neurons by 6-hydroxydopamine (6-OHDA), and of rats treated with reserpine. Neonatal treatment with 5,7-DHT resulted in a significant decrease of both serotonin levels and density (Bmax) of high affinity [3H]imipramine binding sites in the hippocampus. In contrast, an elevation of serotonin levels and an increase in Bmax of [3H]imipramine binding were noted in the pons--medulla region. No changes were observed in the noradrenaline content in either of these regions. Intracerebral 6-OHDA lesion produced a drastic suppression of noradrenaline levels in cerebral cortex but failed to alter the binding affinity (KD) or density (Bmax) of [3H]imipramine recognition sites. A single injection of reserpine (2.5 mg/kg) resulted in marked depletion of both serotonin (by 57%) and noradrenaline (by 86%) content and serotonin uptake (by 87%) in the cerebral cortex but had no significant influence of the parameters of high affinity [3H]imipramine binding in this brain region. The results suggest that high affinity [3H]imipramine binding in the brain is directly related to the integrity of serotonergic neurons but not to the magnitude of the uptake or the endogenous levels of the transmitter, and is not affected by damage to noradrenergic neurons or by low levels of noradrenaline.     

Polarographic Measurements of Spontaneous and Mitochondria-Promoted Oxidation of 5,6-and 5,7-Dihydroxytryptamine

Abstract: Spontaneous oxygen consumption by 5,6- and 5,7-DHT (dihydroxytryptamine), related indoleethylamines, and 6-hydroxydopamine and oxygen consumption by these compounds in the presence of rat liver mitochondria were measured by the polarographic oxygen electrode technique. 5,6- and 5,7-DHT react with oxygen at very different rates (2.7 nmol O₂/min and 33.4 nmol O₂/min, respectively) when incubated in buffer, pH 7.2, at a concentration of 1 mm and with different kínetic characteristics. While the oxidation of 5,7-DHT obeys a reaction of second-order type, the oxidation of 5,6-DHT is more complex and characterized by autocatalytic promotion. Coloured quinoidal oxidation products appeared during the degradation of both indoleamines. Glutathione, ascorbate, dithiothreitol, cysteine, albumin, and superoxide dismutase partially prevented 5,6- and 5,7-DHT from oxidative destruction. Catalase saved oxygen only in the case of 5,6-DHT by recycling of O₂ released from near-stoichiometrically formed H₂O₂ during oxidation of 5,6-DHT: 5,7-DHT did not generate H₂O₂ in measurable amounts. Oxygen consumption rates of 5,6- and 5,7-DHT were enhanced after addition of rat liver mitochondria to the incubation medium; this resulted in an accelerated formation of quinoidal products. This stimulatory effect on the oxidation rates of both 5,6- and 5,7-DHT was blocked by cyanide, but not rotenone, and was abolished by boiling of the mitochondria fraction. The observed increase in oxygen consumption in the presence of mitochondria was found not to be influenced by monoamine oxidase-dependent deamination of 5,6- and 5,7-DHT. It is postulated that 5,6- and 5,7-DHT are capable of participating in the electron transfer of the mitochondrial respiration chain beyond complex III. Results obtained in determinations of ADP:0 ratios in respiratory control experiments exclude a possible interference of 5,6-DHT, 5,7-DHT, and 6-OH-DA with phosphorylating sites. During the activated state of respiration, no signs of electron transfer inhibition by 5,6- and 5,7-DHT were detectable. A comparison and evaluation of the autoxidation rates of various hydroxylated indoleethylamines, of their affinity to the 5-HT transport sites, and their neurotoxic potency in vivo reveals that interaction of these compounds with oxygen at restricted reaction velocity is a prerequisite for efficient toxicity in monoaminergic neurons following active accumulation in these neurons via the high-affinity uptake systems.     

Effects of 5,7-dihroxytryptamine administered supraspinally or spinally on the blood glucose level in D-glucose-fed and immobilization stress models

5,7-Dihydroxytryptamine (5,7-DHT) is a neurotoxin which causes the depletion of serotonin. Moreover, the serotonergic system is the regulator of the blood glucose level. However, the role of centrally located serotonergic system in blood glucose regulation after D-glucose feed and immobilization (IMO) stress was not clearly characterized yet. Thus the present study was designed to examine the effect of 5,7-DHT administered intracerebroventricularly (i.c.v.) or intrathecally (i.t.) on the blood glucose level in D-glucose-fed and immobilization stress models. Mice were pretreated once i.c.v. or i.t. with 5,7-DHT (from 10 to 40?µg) for 3 days and D-glucose (2?g/kg) was fed orally. The blood glucose level was measured at 0, 30, 60 and 120?min after D-glucose feeding and immobilization stress initiation. We found that i.c.v. or i.t. pretreatment with 5,7-DHT attenuated the blood glucose level in both animal models. D-glucose feeding causes an increase in plasma insulin level, whereas the plasma corticosterone level was downregulated in the D-glucose-fed model. The i.c.v. or i.t. pretreatment with 5,7-DHT alone slightly increased the plasma corticosterone level. In addition, the i.c.v. or i.t. pretreatment with 5,7-DHT caused a reversal of the downregulation of plasma corticosterone level induced by D-glucose feeding, whereas immobilization stress causes an increase in plasma corticosterone and insulin levels. The i.c.v or i.t. pretreatment with 5,7-DHT attenuated the immobilization stress-induced plasma corticosterone and plasma insulin levels. Our results suggest that supraspinal and spinal depletion of serotonin appears to be responsible for the downregulation of blood glucose level in both D-glucose-fed and immobilization stress models.     

GABA-stimulated 36Cl- influx into reconstituted vesicles with purified GABAA/benzodiazepine receptor complex

Solubilized and Purified gamma-aminobutyric acid (GABA)A receptors from membrane vesicles of the bovine cerebral cortex were reconstituted into phospholipid vesicles and 36Cl- influx into the vesicles was examined. GABA induced a significant stimulation of the 36Cl- influx into reconstituted vesicles with 1.5% CHAPS/0.15% asolectin solubilized receptor and flunitrazepam further enhanced the GABA-stimulated influx. The purification of GABAA/benzodiazepine receptor complex and Cl- channel solubilized by 1.5% CHAPS/0.15% asolectin from membrane vesicles was achieved by 1012-S affinity column chromatography. The reconstituted vesicles with the purified receptor complex and Cl- channel also exhibited GABA-stimulated 36Cl- influx. This GABA-stimulated influx of 36Cl- was also enhanced by flunitrazepam, while suppressed by bicuculline, a GABAA receptor antagonist. These results strongly suggest that GABAA receptor is directly coupled with Cl- channel, whereas benzodiazepine receptor may be functionally coupled with GABAA receptor and modulates the GABA-stimulated Cl- influx through GABAA receptor. The present results also indicate that the purified GABAA receptor complex is coupled with Cl- channel and possesses functional characteristics as GABAA receptor.     

Molecular mechanism of biological action of the serotonergic neurotoxin 5,7-dihydroxytryptamine

5,7-Dihydroxytryptamine (5,7-DHT) is a selective serotonergic neurotoxin by virtue of its selective uptake into 5-hydroxytryptamine neurons and its ability to undergo autoxidation. The mechanism by which 5,7-DHT induces neurodegenerative effects remains enigmatic. The mechanism of autoxidation of 5,7-DHT, which has been recently discovered, is unique among the autoxidizable neurotoxins and involves incorporation of oxygen to produce the 4-hydroperoxy-5-keto derivative of 5,7-DHT and thence the (4,7) p-quinone of 4,5,7-trihydroxytryptamine (4,5,7-THTQ), a relatively unreactive quinone. In addition, no reduced oxygen species such as hydrogen peroxide, superoxide and hydroxyl radical are produced during autoxidation of 5,7-DHT. Yet, there is evidence to suggest that both the covalent modification of endogenous macromolecules by 5,7-DHT derived products and the toxic effects of reduced oxygen species are, at least in part, responsible for the neurodegenerative effects of 5,7-DHT. Here we propose that (1) the 4-hydroperoxy-5-keto derivative of 5,7-DHT may serve as a substrate for glutathione peroxidase to eventually produce reduced oxygen species and 4,5,7-THTQ, (2) 4,5,7-THTQ may undergo redox cycling thereby generating reduced oxygen species and lowering the reducing equivalents of the neuron, (3) rapid oxygen consumption by 5,7-DHT and the products derived from it may lead to hypoxia, and (4) the product of autoxidation of 5,7-dihydroxyindole-3-acetaldehyde, the monoamine oxidase metabolite of 5,7-DHT, may serve as an alkylating (crosslinking) agent of proteins.     

IN VITRO STUDIES ON THE INTERACTION OF BRAIN MONOAMINE OXIDASE WITH 5,6- AND 5,7-DIHYDROXYTRYPTAMINE12

[¹⁴C]5,6-Dihydroxytryptamine ([¹⁴C] 5,6-DHT) and [¹⁴C]5,7-dihydroxytryptamine ([¹⁴C]5,7-DHT) were deaminated to toluene-isoamylalcohol extractable products when incubated with homogenates of rat hypothalamus or pons-medulla oblongata. [¹⁴C]5,6-Dihydroxyindole acetic acid ([¹⁴C]5.6-DHIAA) and [¹⁴C]5,7-dihydroxyindole acetic acid ([¹⁴C]5,7-DHIAA) were detected as MAO metabolites by TLC besides non-identified components. The conversion of [¹⁴C]5,6-DHT and [¹⁴C]5,7-DHT obeyed, at least initially, Michaelis-Menten kinetics (K_m 5,7-DHT: 0.5 × 10^?3M; K_m 5,6-DHT: 1.25 × 10^?3M). Inhibition of the reaction by the MAO A inhibitor, clorgyline, resulted in a typical double sigmoidal inhibition curve indicating that both amines are metabolized by both types of MAO (A and B). In deprenyl inhibition studies, however, 5,7- and 5,6-DHT seemed to be preferred substrates of MAO A. Incubation of rat brain homogenates with [¹⁴C]5,6-DHT and [¹⁴C]5,7-DHT or with the MAO metabolites [¹⁴C]5,6-DHIAA and [¹⁴C]5,7-DHIAA caused a time-dependent break-down of the dihydroxylated indole compounds with subsequent binding of radioactivity to perchloric acid insoluble tissue components. 5,6-DHT inactivated MAO in rat brain homogenates parallel to its decomposition and extensive protein binding. The inactivation of MAO by 5,6-DHT and the extensive binding of radioactivity to protein were antagonized by dithiothreitol (DTT), glutathione (GSH) and L-ascorbic acid. Reduction of [O₂] in the incubation medium slightly attenuated the inactivation of MAO by 5,6-DHT. Catalase or superoxide dismutase failed to prevent MAO from being inactivated by 5,6-DHT. The results suggest that oxidation products of 5,6-DHT, e.g. its corresponding o-quinone, are involved in the inactivation of MAO in vitro and mainly responsible for the binding of radioactivity to brain proteins in vitro. Similar mechanisms may also be operative in the in vivo neurotoxicity of 5,6-DHT. The lack of inactivation of MAO by 5,7-DHT in vitro correlated with a low degree of radioactivity binding (from [¹⁴C]5,7-DHT) to homogenate protein pellets; the binding to proteins was barely influenced by GSH, cysteine, DTT and l -ascorbic acid. These latter findings do not provide a plausible explanation for the mechanism(s) involved in the well known in vivo neurotoxicity of 5,7-DHT.     

Analysis of the Mechanism underlying the Rhythm Reversal from Diurnal to Nocturnal in the Cricket Gryllus bimaculatus, with Special Reference to the Role of Serotonin

The cricket, Gryllus bimaculatus, shows a rhythm reversal from diurnal to nocturnal in about a week after the imaginal molt. In the present study, we investigated the role of serotonin (5-HT) in the rhythm reversal. The 5-HT content in the brain measured by HPLC equipped with an electrochemical detector gradually increased after the imaginal molt, and in fully nocturnal adults it was about 2 times of nymphal level. We then examined the effects of 5,7-dihydroxytryptamine (5,7-DHT), a selective neurotoxine to serotonergic neurons, on the locomotor rhythm. In most animals with 5,7-DHT (25 muM or 250 muM, 32.2 nl) injected into the brain, daytime activity significantly increased even after the rhythm reversal, while nighttime activity was not significantly affected, forming rather diurnal pattern. The serotonin content in the brain of animals injected with 250 muM 5,7-DHT was reduced by about 30%. On the basis of these results, possible involvement of 5-HT in the neural mechanism controlling the locomotor rhythm is discussed.