5,7-Dihydroxitryptamine toxicity to serotonergic neurons in serum free raphe cultures

5,7-Dihydroxytryptamine (5,7-DHT), is an experimentally widely used selective serotonergic neurotoxin, though the mechanisms of toxicity remain to be fully elucidated. In the present study, we evaluated 5,7-dihydroxitryptamine (5,7-DHT) induced serotonergic neurotoxicity in foetal raphe serum free cultures from the rat. For this purpose, a model of foetal raphe serum free neuronal cultures from the rat was established, containing about 16% serotonergic neurons and studied up to 3 months. Two weeks old raphe cultures were exposed to the serotonergic neurotoxin 5,7-DHT (concentration range 10-100 microM) for 72 h, after which the medium was replaced and neurotoxicity was evaluated by immunocitochemistry 1 week later. Lactate dehydrogenase release into the medium, 72 h after exposure to 5,7-DHT, showed a concentration-dependent neurotoxicity. To access morphologically the serotonergic toxicity tryptophan hydroxylase (TPH) was used as a specific marker of these neurons. Immunocitochemistry using TPH antisera showed a concentration-dependent serotonergic neurotoxicity induced by 5,7-DHT. Serotonergic neurons showed the typical pattern of "pruning" accompanied by axon terminals and dendrites loss, which were either partial or total. The axotomy induced by the neurotoxin was morphologically characteristic of retrograde axonal degeneration. Fluoxetine (0.1 microM) pre-treatment reduced 5,7-DHT-induced serotonergic neurotoxicity. These results indicate that the mechanism by which 5,7-DHT-induces serotonergic neurotoxicity is, at least partially, dependent on the toxin uptake by the serotonin transporter. Finally, we have established a robust model of primary raphe neuronal culture to evaluate serotonergic neurons development and the mechanisms of toxicity involving this neuronal population.     

Efflux of 3H-5-hydroxytryptamine from rat hypothalamic slices by continuous electrical stimulation: Frequency-dependent responses to serotonergic antagonists and 5-hydroxytryptamine

Rat hypothalamic slices were incubated with 3H-5-hydroxytryptamine and superfused in the presence of paroxetine to inhibit 5-hydroxytryptamine (5-HT) reuptake. The slices were continuously stimulated electrically with rectangular pulses at varying frequencies. Continuous stimulation for up to 42 min at 1 Hz or at 3 Hz evoked a steady efflux of tritium that slowly decayed with time. The efflux produced by continuous stimulation at 5 Hz declined more rapidly with time. Continuous stimulation at 1 Hz in the presence of increasing concentrations of unlabelled 5-HT produced a concentration-dependent decrease in tritium efflux. The presence of methiothepin (0.5 mumol/l), quipazine (10 mumol/l) and (-)- but not (+)-propranolol (1 mumol/l) attenuated this response to 5-HT. From these data, the apparent pA2 values were calculated and found to be in agreement with published values. Frequency-dependent responses were determined using a "cumulative stimulation" protocol whereby the slices were subjected to three consecutive 14 min periods of stimulation at increasing frequencies (1, 3 and then 5 Hz). Unlabelled 5-HT (1 mumol/l) inhibited electrically-evoked tritium efflux more at 1 than at 5 Hz. Methiothepin (0.5 mumol/l) and quipazine (10 mumol/l) enhanced the stimulated efflux in a manner inversely related to the frequency of stimulation. Neither (+)- nor (-)-propranolol enhanced stimulated tritium efflux at any of the three frequencies tested. It is concluded that continuous electrical stimulation of rat hypothalamic slices at a low frequency provides a rapid means of obtaining apparent affinities and intrinsic activities of drugs that modify the serotonergic autoreceptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of the two antidepressant drugs mianserin and indalpine on the serotonergic system: Single-cell studies in the rat

Several antidepressant treatments enhance serotonergic neurotransmission. The present electrophysiological studies were undertaken to assess the effect of mianserin and indalpine, two antidepressant drugs with different pharmacological profiles, on serotonergic neurotransmission. In a first series of experiments, the responsiveness of hippocampal pyramidal neurons to microiontophoretic applications of serotonin (5-HT), norepinephrine (NE) and -aminobutyric acid (GABA) was assessed following mianserin, imipramine (5 mg/kg/day IP) or saline administration for 14 days. At 48 h after the last dose of mianserin, responsiveness to 5-HT was increased whereas that to NE and GABA was not modified. The degree of sensitization to 5-HT was the same as that produced by imipramine. Acute IV administration of mianserin (up to 10 mg/kg) did not decrease the firing rate of dorsal raphe 5-HT neurons. In a second series of experiments, long-term administration of indalpine (5 mg/kg/day IP for 14 days) did not modify the responsiveness of hippocampal pyramidal neurons to microiontophoretically applied 5-HT, NE and GABA whereas imipramine treatment (5 mg/kg/day IP) increased selectively their sensitivity to 5-HT when compared to indalpine-treated rats. In keeping with its potent reuptake-blocking property, acute IV indalpine produced a marked decrease in the firing rate of dorsal raphe 5-HT neurons (ED₅₀ 0.33 mg/kg). The firing rate of dorsal raphe 5-HT neurons was assessed following 2-, 7- and 14-day treatments with indalpine (5 mg/day IP). After 2 days, the firing rate of 5-HT neurons was greatly reduced, after 7 days it had recovered partially and after 14 days it had returned to normal. At this point, the responsiveness of 5-HT neurons to IV LSD, an agonist of the 5-HT autoreceptor, and to microiontophoretically-applied 5-HT was decreased twofold, indicating desensitization of the autoreceptor. In conclusion, it is proposed that long-term treatment with mianserin, as with tricyclic antidepressant drugs and electroconvulsive shocks, increases 5-HT neurotransmission via sensitization of postsynaptic neurons to 5-HT whereas long-term treatment with indalpine, as with zimelidine, results in the same final effect via its presynaptic effect on 5-HT neurons presumably by blocking 5-HT reuptake. These data further support the notion that enhancing 5-HT neurotransmission might have an antidepressant effect.     

Distinct electrophysiological effects of paliperidone and risperidone on the firing activity of rat serotonin and norepinephrine neurons

Rationale Paliperidone (9-OH-risperidone) is the main metabolite of the atypical antipsychotic risperidone. While both drugs are potent dopamine (D)₂ antagonists, they have quantitative differential affinities for serotonin (5-HT) and norepinephrine (NE) receptor binding sites. Objectives The present study aimed to determine if paliperidone exerts distinct effects on 5-HT and NE neuronal activity from those of risperidone. Materials and methods Risperidone and paliperidone were administered to Sprague–Dawley rats. Neuronal activity of 5-HT and NE neurons was assessed using in vivo electrophysiology. Results Acute administration of risperidone but not paliperidone inhibited the firing of 5-HT neurons, as previously reported. This inhibition was partially antagonized by the NE reuptake inhibitor desipramine, by the 5-HT_1A receptor antagonist WAY 100635, and completely reversed when both drugs were given consecutively. Risperidone inhibited the firing of 5-HT neurons after 2 and 14 days of administration, with or without escitalopram. Paliperidone did not alter the firing rate of NE neurons by itself, but it reversed the suppression of NE neurons induced by escitalopram, as it was previously reported for risperidone. Conclusion These results indicate that although risperidone and paliperidone share a qualitatively similar receptor binding profile in vitro, they differentially alter the firing of 5-HT and NE neurons in vivo. The capacity of paliperidone to reverse the selective serotonin reuptake inhibitor (SSRI)-induced inhibition of NE neuronal firing, without interfering with the effect of SSRIs of 5-HT neuronal activity, suggests that paliperidone may be a very effective adjunct in SSRI-resistant depression.     

Neurotensin excitation of serotonergic neurons in the rat nucleus raphe magnus: ionic and molecular mechanisms

To understand the cellular and molecular mechanisms by which neurotensin (NT) induces an analgesic effect in the nucleus raphe magnus (NRM), whole-cell patch-clamp recordings were performed to investigate the electrophysiological effects of NT on acutely dissociated NRM neurons. Two subtypes of neurons, primary serotonergic and secondary non-serotonergic cells, were identified from acutely isolated NRM neurons. During current-clamp recordings, NT depolarized NRM serotonergic neurons and evoked action potentials. Voltage-clamp recordings showed that NT excited serotonergic neurons by enhancing a voltage-insensitive and non-selective cationic conductance. Both SR48692, a selective antagonist of subtype 1 neurotensin receptor (NTR-1), and SR 142948A, a non-selective antagonist of NTR-1 and subtype 2 neurotensin receptor (NTR-2), failed to prevent neurotensin from exciting NRM serotonergic neurons. NT-evoked cationic current was inhibited by the intracellular administration of GDP-beta-S. NT failed to induce cationic currents after dialyzing serotonergic neurons with the anti-G(alphaq/11) antibody. Cellular Ca(2+) imaging study using fura-2 showed that NT induced the calcium release from the intracellular store. NT-evoked current was blocked after the internal perfusion of heparin, an IP(3) receptor antagonist, or BAPTA, a fast Ca(2+) chelator. It is concluded that neurotensin enhancement of the cationic conductance of NRM serotonergic neurons is mediated by a novel subtype of neurotensin receptors. The coupling mechanism via G(alphaq/11) proteins is likely to involve the generation of IP(3), and subsequent IP(3)-evoked Ca(2+) release from intracellular stores results in activating the non-selective cationic conductance.     

Additive deficits in the choice accuracy of rats in the delayed non-matching to position task after cholinolytics and serotonergic lesions are non-mnemonic in nature

The role of serotonin (5-HT) and its interaction with the muscarinic or nicotinic receptor-mediated mechanisms in the modulation of working memory and motor activity was investigated by assessing the effects of 5-HT lesion and cholinergic receptor blockade on the performance of rats in a working memory (delayed non-matching to position, DNMTP) task. A global serotonergic lesion was induced by the intracerebroventricular adminstration of 5,7-dihydroxytryptamine (5,7-DHT). Post-mortem neurochemical analysis revealed that serotonin and 5-hydroxyindoleacetic acid (5-HIAA) levels were reduced in frontal and parieto-occipital cortices and in hippocampi of 5,7-DHT lesioned rats. 5-HIAA levels were also reduced in striatum. 5,7-DHT lesion slightly impaired choice accuracy of rats in the DNMTP task and also transiently reduced motor activity in rats. Even the lower dose of scopolamine (0.075 mg/kg), a muscarinic receptor antagonist, impaired the choice accuracy already at the shortest delay (i.e. not indicative of a working memory impairment per se), and caused a marked disruption of motor activity (lengthened response latencies, increased probability of omissions and decreased trials completed). Furthermore, the quaternary analogue, N-methylscopolamine (0.150 mg/kg), affected the motor activity of rats to the same extent as scopolamine. Mecamylamine (1.0; 3.0 mg/kg) also interfered with motor activity and it slightly decreased the choice accuracy, which was not dependent on the delay. Although mecamylamine disrupted the performance of rats in the DNMTP task, the disruption was not as severe as that seen with scopolamine. Moreover, both scopolamine and mecamylamine augmented the slight impairment on the choice accuracy of 5,7-DHT lesioned rats, but this was non-mnemonic in character. We conclude that there is no evidence for any major interaction between the serotonergic system and muscarinic or nicotinic cholinergic mechanisms in working memory per se, but muscarinic and nicotinic receptor antagonists may act additively with the 5,7-DHT lesion to disrupt the choice accuracy of rats. Received: 22 November 1995 / Final version: 25 November 1996     

Ipsapirone and 8-OH-DPAT reduce ethanol preference in rats: involvement of presynaptic 5-HT1A receptors

The selective serotonin(5-HT)_1A receptor agonists 8-OH-DPAT and ipsapirone were tested in selectively inbred Wistar rats, with high preference [70–90%: defined as the ratio of ethanol (EtOH) to total fluid intake] for EtOH (10% v/v) over water in a two-bottle free choice situation. Rats were injected shortly before the overnight test session (8:00p.m.–8:00a.m.). EtOH and water consumption were determined in 20-min intervals; food consumption after the session. 8-OH-DPAT (ED₅₀: 2.4 mg/kg, SC) and ipsapirone (ED₅₀: 12.5 mg/kg, SC) reduced EtOH preference in a dose-dependent manner. In addition, 8-OH-DPAT increased total fluid intake, whereas ipsapirone enhanced total food intake. The EtOH preference reduction was time-dependent and reached a maximum within the second 4 h after application of 8-OH-DPAT (–73%) and ipsapirone (–72%). The preference reducing effect of ipsapirone (20 mg/kg, PO) was completely blocked by the nonselective 5-HT_1A antagonist spiperone (0.05 mg/kg, SC). Local application of 8-OH-DPAT (10 µg, 0.5 µl) into the dorsal raphe nucleus (DRN, a brain area rich in somatodendritic 5-HT_1A autoreceptors), reduced the EtOH preference significantly as compared to the saline injection in the same animal (–12%, 8:00–12:00p.m.). Only marginal effects on ingestion behavior were observed after micro-injection into the nucleus accumbens. Reduction of brain 5-HT levels by pretreatment with the 5-HT synthesis inhibitor pCPA (2×150 mg/kg, IP) resulted in a short lasting, marked reduction (–54%) and a long lasting, small attenuation of the EtOH preference. Total food consumption was strongly decreased but returned soon to normal; total fluid intake was only slightly decreased. The EtOH preference reducing effect of ipsapirone (5 and 20 mg/kg, SC) was attenuated in pCPA-pretreated rats. The present data suggest that 5-HT_1A receptor ligands reduce EtOH preference via stimulation of 5-HT_1A receptors in the DRN. The possibility of additional mechanism(s) is discussed.