Short-term lithium treatment enhances responsiveness of postsynaptic 5-HT1A receptors without altering 5-HT autoreceptor sensitivity: an electrophysiological study in the rat brain

Short-term lithium administration to rats has previously been shown to enhance 5-HT neurotransmission through a modification of 5-HT neuron properties. In the first part of the present study, the effect of lithium on the function of terminal 5-HT autoreceptors was assessed by comparing in controls and lithium-treated rats the differential effect of two frequencies of stimulation (0.8 and 5 Hz) and that of methiothepin, a terminal 5-HT autoreceptor antagonist, on the effectiveness of the electrical activation of the ascending 5-HT pathway in suppressing dorsal hippocampus pyramidal neuron firing activity. Both procedures produced similar effects in controls and lithium-treated rats. In the second part of the study, the function of somatodendritic 5-HT autoreceptors was studied. The effect of intravenous LSD, an agonist of the somatodendritic 5-HT autoreceptor, on the firing activity of 5-HT neurons was not modified by the lithium treatment, whereas that of intravenous 8-OH-DPAT, a 5-HT1A receptor agonist, was increased two-fold. However, lithium did not alter the responsiveness of 5-HT neurons to direct microiontophoretic applications of 8-OH-DPAT as well as of LSD and 5-HT. It is concluded that short-term lithium treatment does not alter the function of terminal and somatodendritic 5-HT autoreceptors and that it enhances the sensitivity of a subset of postsynaptic 5-HT1A receptors involved in controlling 5-HT neuron firing activity, presumably through a feedback loop.     

Pre- and post-synaptic effects of the 5-HT3 agonist 2-Methyl-5-HT on the 5-HT system in the rat brain

Microiontophoretic applications of 5-HT and of the 5-HT₃ agonist 2-methyl-5-HT produced a current-dependent suppression of firing activity of both hippocampal (CA₁ and CA₃) and cortical neurons in anesthetized rats. Concomitant microiontophoretic applications of the 5-HT₃ antagonists BRL 46470A and S-zacopride, as well as their intravenous injection, did not antagonize the inhibitory effect of 5-HT and 2-methyl-5-HT. In contrast, the 5-HT_1A antagonist BMY 7378, applied by microiontophoresis or administered intravenously, significantly reduced the inhibitory action of 5-HT and 2-methyl-5-HT. The firing activity of dorsal raphe 5-HT neurons was also reduced by 5-HT, 2-methyl-5-HT and the 5-HT_1A agonist 8-OH-DPAT applied by microiontophoresis. While BRL 46470A (0.1 and 1 mg/kg, i.v.) did not antagonize the inhibitory effect of the three 5-HT agonists on 5-HT neuronal firing activity, only that of 8-OH-DPAT was attenuated by the 5-HT_1A antagonist (+) WAY 100135. R-zacopride significantly reduced the duration of suppression of firing activity of CA₃ pyramidal neurons induced by the electrical stimulation of the ascending 5-HT pathway, and this reducing effect was prevented by the three 5-HT₃/5-HT₄ antagonists renzapride, S-zacopride and tropisetron, but not by BRL 46470A. Finally, in in vitro superfusion experiments, both BRL 46470A and S-zacopride antagonized the enhancing action of 2-methyld-HT on the electrically-evoked release of [³H]-5-HT in both rat frontal cortex and hippocampus slices. These findings suggest that, in vivo, the suppressant effect of 2-methyl-5-HT on the firing activity of dorsal hippocampus pyramidal, somatosensory cortical, and dorsal raphe 5-HT neurons is not mediated by 5-HT₃ receptors, but rather by 5-HT_1A receptors. The attenuating effect of R-zacopride on the effectiveness of the stimulation of the ascending 5-HT pathway is not mediated by 5-HT₃ receptors. In contrast, in vitro, the enhancing action of 2-methyl-5-HT on the electrically-evoked release of [³H]5-HT in both frontal cortex and hippocampus slices is mediated by 5-HT₃ receptors. © 1995 Wiley-Liss, Inc.     

Effect of neurokinin-I receptor antagonists on the function of 5-HT and noradrenaline neurons

Substance P antagonists have been proposed to be a new class of antidepressants. The present study aimed to determine the effect of the selective non-peptide rat neurokinin-1 (NK1) receptor antagonists WIN 51,708 and CP-96,345 on the firing activity of rat dorsal raphe serotonin (5-HT) and locus coeruleus noradrenaline (NA) neurons. While WIN51,708 (2mg/kg, i.v.) and CP-96,345 (0.15 mg/kg, i.v.) did not modify the firing activity of 5-HT and NA neurons, both antagonists attenuated the suppressant effect of the alpha2-adrenoceptor agonist clonidine on the firing activity of both types of neurons. In contrast, the responsiveness of 5-HT neurons to the i.v. administration of the 5-HT autoreceptor agonist LSD and the 5-HT1A receptor agonist 8-OH-DPAT remained unchanged. These findings suggest that NK1 receptor antagonists affect markedly the NA system via an attenuation of the function of alpha2-adrenoceptors on the cell body of NA neurons and, consequently, may also modulate 5-HT neurotransmission.     

Effects of short-term serotonin depletion on the efficacy of serotonin neurotransmission: electrophysiological studies in the rat central nervous system

The effects of short-term serotonin (5-HT) depletion by p-chlorophenylalanine (PCPA) on the firing activity of dorsal raphe nucleus 5-HT neurons, on the responsiveness of dorsal hippocampus pyramidal neurons to microiontophoretically applied 5-HT and on the efficacy of the electrical stimulation of the ascending 5-HT pathway in suppressing the firing activity of CA3 dorsal hippocampus pyramidal neurons were assessed in chloral hydrate-anesthetized rats. PCPA (250 mg/kg/day i.p. for 2 days) reduced the 5-HT content of the dorsal hippocampus by 90%. However, the number of spontaneously active 5-HT neurons per microelectrode trajectory through the dorsal raphe or their average rate of firing was unaltered. The effect of afferent 5-HT pathway stimulation was reduced in only 40% of treated rats, whereas the sensitivity of CA3 pyramidal neurons to microiontophoretic 5-HT was not modified. The function of the terminal 5-HT autoreceptor was assessed using methiothepin, an autoreceptor antagonist. Methiothepin (1 mg/kg, i.v.) significantly enhanced the efficacy of the stimulation in PCPA-treated rats, although the degree of enhancement was much less than in controls. A greater reduction of the effectiveness of the stimulation was obtained by increasing the dose of PCPA (350 mg/kg/day i.p. for 2 days). This regimen reduced the 5-HT content of the dorsal hippocampus by 95%. In these rats, the sensitivity of the terminal 5-HT autoreceptor was assessed by increasing the frequency of the stimulation from 1 to 5 Hz. This procedure reduced to a similar extent the efficacy of the stimulation in treated and control rats, suggesting that the reduced effectiveness of methiothepin in enhancing 5-HT synaptic transmission in PCPA-treated rats is due to a lower degree of activation of the terminal 5-HT autoreceptor. The present results showing that the 350 mg/kg/day regimen of PCPA, but not the 250 mg/kg/day regimen, reduced the efficacy of the stimulation of the ascending 5-HT pathway suggest that a greater than 90% depletion is required to affect 5-HT neurotransmission significantly. The reduced level of activation of terminal 5-HT autoreceptors in rats treated with the lower dose of PCPA may facilitate the release of the remaining 5-HT per stimulation-triggered action potential, ensuring a virtually unaltered synaptic efficacy.     

Effects of sustained (+/-)pindolol administration on serotonin neurotransmission in rats

OBJECTIVE: Given reports that (+/-)pindolol, a beta-adrenergic-5-HT1A/1B receptor antagonist, accelerates the onset of the therapeutic effect of certain antidepressant drugs in major depression, the aim of this study was to assess the effect of sustained (+/-)pindolol administration on the sensitivity of pre- and postsynaptic 5-HT1A receptors, terminal 5-HT1B autoreceptors and on overall 5-HT neurotransmission. DESIGN: Prospective animal study. ANIMALS: Sprague-Dawley rats. OUTCOME MEASURES: Modifications of the sensitivity of somatodendritic and postsynaptic 5-HT1A receptors using in vivo electrophysiological paradigms in animals treated with vehicle or (+/-)pindolol (20 mg/kg/day, subcutaneously) through osmotic minipumps for 2 weeks. RESULTS: (+/-)Pindolol attenuated the suppressant effect of the 5-HT autoreceptor agonist lysergic acid diethylamide (LSD) on the firing activity of 5-HT neurons, suggesting that (+/-)pindolol antagonized somatodendritic 5-HT1A autoreceptors in the dorsal raphe nucleus. However, following a 2-day washout period, the suppressant effect of LSD was still attenuated, indicating rather a desensitization of 5-HT1A autoreceptors had occurred. In the CA3 region of the dorsal hippocampus, (+/-)pindolol treatment did not modify the responsiveness of postsynaptic 5-HT1A receptors to microiontophoretic applications of 5-HT. Moreover, such a treatment modified neither the effectiveness of the electrical stimulation of 5-HT fibers nor the function of terminal 5-HT autoreceptors. Finally, the administration of the selective 5-HT1A receptor antagonist WAY 100635 (100 micrograms/kg, intravenously) did not increase the firing activity of dorsal hippocampus CA3 pyramidal neurons in rats treated with (+/-)pindolol, thus failing to reveal the enhanced tonic activation of postsynaptic 5-HT1A receptors associated with major classes of antidepressant treatments. CONCLUSION: Prolonged administration of (+/-)pindolol by itself is not sufficient to enhance overall 5-HT neurotransmission; pindolol should therefore not be endowed with intrinsic antidepressant activity. Although pindolol is capable of antagonizing the 5-HT1A autoreceptor upon the initiation of a 5-HT reuptake-blocker treatment, it also induces a desensitization of this 5-HT1A autoreceptor, which could explain why patients do not relapse upon its discontinuation when they continue taking a 5-HT reuptake blocker.     

Differential responsiveness of the rat dorsal and median raphe 5-HT systems to 5-HT1 receptor agonists and p-chloroamphetamine

The dorsal and median raphe 5-HT neurons give rise to projections that differ in axon morphology and in vulnerability to certain amphetamine derivatives. The present study was undertaken to determine if these two 5-HT systems possess different functional properties. To this end, we studied the effects of selective 5-HT1A or 5-HT1A/5-HT1B receptor agonists and of p-chloroamphetamine on extracellular levels of indoleamines, as measured by differential pulse voltammetry with extracellular levels of indoleamines, as measured by differential pulse voltammetry with electrochemically pretreated carbon fiber electrodes, in cell body and nerve terminal regions of these subsets of 5-HT neurons in the rat brain. The selective 5-HT1A agonist 8-OH-DPAT produced a gradual decrease in the height of the 300 mV oxidation peak in the dorsal raphe and in the frontal cortex, reaching a maximum of 60% 3 h after the i.v. injection of 30 micrograms/kg. However, the same dose of 8-OH-DPAT was ineffective in the median raphe and in the dentate gyrus that receives its 5-HT innervation exclusively from the median raphe. A higher dose of 8-OH-DPAT (150 micrograms/kg, i.v.) produced a 60% decrease in the height of the 300 mV oxidation peak in the median raphe, whereas only a 20% decrease was obtained in the dentate gyrus. In contrast, the non-selective 5-HT1 agonist RU 24,969 (10 mg/kg, i.p.) caused a 70% reduction of the 300 mV peak height in both the dorsal and median raphe and a 50% decrease in both the frontal cortex and the dentate gyrus. Moreover, although a high dose of 8-OH-DPAT (150 micrograms/kg, i.v.) given alone reduced by 20% the amplitude of the oxidative peak in the dentate gyrus, subsequent administration of RU 24,969 (10 mg/kg, i.p.) caused a further 30% diminution of the oxidative peak height. The greater responsiveness of dorsal as compared to median raphe 5-HT systems to 5-HT1A receptor agonists was confirmed in two further series of experiments. First, the microiontophoretic application of 8-OH-DPAT directly onto 5-HT neurons was three times more potent in suppressing the firing rate of dorsal raphe 5-HT neurons than that of their median raphe congeners. Second, 8-OH-DPAT and buspirone were ten and four times, respectively, more potent in decreasing 5-HT synthesis in the frontal cortex than in the hippocampus.(ABSTRACT TRUNCATED AT 400 WORDS)     

Partial agonistic activity of R- and S-enantiomers of 8-OH-DPAT at 5-HT1A receptors.

In this study, the 5-HT1A agonistic activity of R- and S-enantiomers of the prototypical 5-HT1A agonist 8-OH-DPAT was investigated using in vivo microiontophoresis and the hypothermic response in rats. Both the R- and S-enantiomers suppressed current-dependently the firing activity of dorsal hippocampus CA3 pyramidal neurons. The number of spikes suppressed/nA of R-(+)-OH-DPAT was about 2-fold greater than that of S-(-)-OH-DPAT, which indicates greater agonistic activity of the R-enantiomer. The determination of the effectiveness of 5-HT in suppressing the firing activity of CA3 pyramidal neurons prior to and during application of either the R- or S-enantiomer showed that both compounds antagonized the effect of 5-HT, thus demonstrating their partial agonistic activity. Racemic 8-OH-DPAT produced a dose-dependent hypothermia which was attenuated by the 5-HT1A antagonist pindolol, but not by the nonselective 5-HT antagonist methysergide. Similarly, both R- and S-enantiomers induced a dose-dependent hypothermia, which was greater and longer lasting in the case of R-(+)-OH-DPAT when compared to S-(-)-OH-DPAT. In conclusion, R-(+)-OH-DPAT, displayed a greater agonistic activity at 5-HT1A receptors than S-(-)-OH-DPAT, both in suppressing firing activity of CA3 pyramidal neurons and in decreasing body temperature. Nevertheless, both compounds behaved as partial agonists.     

Sustained blockade of neurokinin-1 receptors enhances serotonin neurotransmission.

BACKGROUND: Antagonists of neurokinin-1 (NK(1)) receptors, through which substance P acts, have been proposed to belong to a new class of antidepressants with a unique mode of action. It was postulated that they exert this putative therapeutic effect independently of the serotonin (5-HT) neurons. METHODS: The aim of the present study was to assess, using in vivo electrophysiological paradigms, the effects of sustained administration of the nonpeptidic NK(1) antagonist CP-96,345 on the firing activity of rat dorsal raphe 5-HT neurons, the responsiveness of pre- and postsynaptic 5-HT(1A) receptors, and overall 5-HT neurotransmission in the hippocampus. RESULTS: Both short- and long-term treatments with CP-96,345 significantly increased the spontaneous firing activity of dorsal raphe 5-HT neurons, and this increase was associated with an attenuation of somatodendritic 5-HT(1A) autoreceptor responsiveness. In contrast, the inactive enantiomer of CP-96,345 at NK(1) receptors, CP-96,344, did not alter these parameters after short-term administration. Because 5-HT(1A) receptor activation inhibits the firing activity of dorsal hippocampus CA(3) pyramidal neurons, the degree of disinhibition produced by the selective 5-HT(1A) receptor antagonist WAY 100635 was determined to assess the net change in 5-HT neurotransmission. Intravenous injection of WAY 100635 did not disinhibit CA(3) pyramidal neuron firing in rats given saline, CP-96,345 for 2 days, or CP-96,344 for 14 days, but produced a significant enhancement of firing in rats treated with CP-96,345 for 2 weeks. Therefore, only long-term treatment with CP-96,345 enhanced the tonic activation of postsynaptic 5-HT(1A) receptors. CONCLUSIONS: Similar to all other major types of antidepressant treatments, these data indicate that substance P antagonists might alleviate anxiety and major depression, at least in part, by enhancing the degree of activation of some 5-HT receptors in the forebrain.     

In vivo electrophysiological evidence for the regulatory role of autoreceptors on serotonergic terminals

The present in vivo studies were undertaken to evaluate electrophysiologically the modulatory role of the terminal 5-HT autoreceptor on 5-HT neurotransmission. In a first series of experiments, the effect of the electrical activation of the ascending 5-HT pathway on the firing activity of CA3 hippocampal pyramidal neurons was measured before and after the intravenous administration of methiothepin, a terminal 5-HT autoreceptor antagonist. Methiothepin significantly increased the duration of the suppression of firing activity of these neurons by the electrical stimulation of the 5-HT pathway, without modifying their responsiveness to microiontophoretically applied 5-HT. This suggests that endogenously released 5-HT activates the 5-HT terminal autoreceptor and that methiothepin enhances the efficacy of 5-HT synaptic transmission by blocking this activation. In a second series of experiments, further evidence for the activation of terminal 5-HT autoreceptors by 5-HT released by the electrical stimulation was sought by assessing the effectiveness of 2 series of stimulations of the ascending 5-HT pathway delivered at different frequencies while recording the same postsynaptic neuron. Increasing the frequency of stimulation (from 0.8 to 5 Hz) significantly reduced the duration of suppression of firing activity of the postsynaptic neurons. This difference between the 0.8 and 5 Hz stimulations was decreased by intravenous methiothepin, suggesting that the reduced effectiveness of the stimulations delivered at the higher frequency is attributable to a greater activation of the terminal 5-HT autoreceptor. These results provide direct electrophysiological evidence for the modulatory role of the 5-HT terminal autoreceptor on 5-HT neurotransmission.     

Assessment of the serotonin reuptake blocking property of YM992: electrophysiological studies in the rat hippocampus and dorsal raphe.

YM992 is a selective serotonin (5-HT) reuptake inhibitor and a 5-HT(2A) antagonist with potential antidepressant activity. As expected from a 5-HT reuptake inhibitor, which induces an accumulation of 5-HT in the dorsal raphe, YM992 inhibited the firing activity of these 5-HT neurons (ED50: 2.0+/-0.2 mg/kg, i.v.). This effect was reversed by the 5-HT(1A) antagonist WAY 100635. YM992 also dose-dependently prolonged the time for CA3 neurons to recover 50% of their firing rate following microiontophoretic applications of 5-HT, a reliable index of the function of the 5-HT reuptake carrier. In a second series of experiments, the adaptative properties of 5-HT neurons were examined during sustained administration of YM992 (20 mg/kg/day, s.c., delivered by osmotic minipumps) after 2 days of treatment. YM992 decreased by more than 60% the firing activity of the 5-HT neurons. There was a partial recovery of firing after 7 days and a complete one after 14 days of treatment in the presence of the minipump still delivering the drug. In a third series of experiments, the sensitivity of pre- and postsynaptic 5-HT(1A) receptors in the dorsal raphe and the dorsal hippocampus were assessed. The results showed that YM992 attenuated the inhibitory effect of intravenous administration of LSD and the 5-HT(1A) agonist 8-OH-DPAT on the firing activity of 5-HT neurons. As did the selective 5-HT reuptake inhibitor fluvoxamine, YM992 markedly increased the effectiveness of the electrical stimulation of ascending 5-HT fibres on firing activity of the postsynaptic hippocampus pyramidal neurons. This enhancement of 5-HT neurotransmission by YM992 was attributable to a desensitization of the terminal 5-HT(1B) autoreceptors since the postsynaptic 5-HT(1A) receptors in the hippocampus remained normosensitive.     

5-HT1A and 5-HT1B receptors control the firing of serotoninergic neurons in the dorsal raphe nucleus of the mouse: studies in 5-HT1B knock-out mice

The characteristics of the spontaneous firing of serotoninergic neurons in the dorsal raphe nucleus and its control by serotonin (5-hydroxytryptamine, 5-HT) receptors were investigated in wild-type and 5-HT1B knock-out (5-HT1B-/-) mice of the 129/Sv strain, anaesthetized with chloral hydrate. In both groups of mice, 5-HT neurons exhibited a regular activity with an identical firing rate of 0.5-4.5 spikes/s. Intravenous administration of the 5-HT reuptake inhibitor citalopram or the 5-HT1A agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) induced a dose-dependent inhibition of 5-HT neuronal firing which could be reversed by the selective 5-HT1A antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohe xane carboxamide (WAY 100635). Both strains were equally sensitive to 8-OH-DPAT (ED50 approximately 6.3 microgram/kg i.v.), but the mutants were less sensitive than wild-type animals to citalopram (ED50 = 0.49 +/- 0.02 and 0.28 +/- 0.01 mg/kg i.v., respectively, P < 0.05). This difference could be reduced by pre-treatment of wild-type mice with the 5-HT1B/1D antagonist 2'-methyl-4'-(5-methyl-[1,2,4]oxadiazol-3-yl)-biphenyl-4-carbox yli c acid [4-methoxy-3-(4-methyl-piperazine-1-yl)-phenyl]amide (GR 127935), and might be accounted for by the lack of 5-HT1B receptors and a higher density of 5-HT reuptake sites (specifically labelled by [3H]citalopram) in 5-HT1B-/- mice. In wild-type but not 5-HT1B-/- mice, the 5-HT1B agonists 3-(1,2,5, 6-tetrahydro-4-pyridyl)-5-propoxypyrrolo[3,2-b]pyridine (CP 94253, 3 mg/kg i.v.) and 5-methoxy-3-(1,2,3, 6-tetrahydropyridin-4-yl)-1H-indole (RU 24969, 0.6 mg/kg i.v.) increased the firing rate of 5-HT neurons (+22.4 +/- 2.8% and +13.7 +/- 6.0%, respectively, P < 0.05), and this effect could be prevented by the 5-HT1B antagonist GR 127935 (1 mg/kg i.v.). Altogether, these data indicate that in the mouse, the firing of 5-HT neurons in the dorsal raphe nucleus is under both an inhibitory control through 5-HT1A receptors and an excitatory influence through 5-HT1B receptors.     

Is there a role for 5-HT1A agonists in the treatment of depression?

The role of serotonin (5-hydroxytryptamine; 5-HT) in the treatment of depressive and anxiety disorders is underscored by the therapeutic action of selective 5-HT reuptake inhibitors acting to enhance the degree of activation of various 5-HT receptor subtypes. The 5-HT1A receptors are particularly relevant to the antidepressant and anxiolytic responses in human beings. They are located presynaptically in the raphe nuclei, where they act as cell body autoreceptors to inhibit the firing rate of 5-HT neurons, and are located postsynaptically in limbic and cortical regions, where they also attenuate firing activity. The azapirones are full agonists at 5-HT1A autoreceptors and are generally, but not exclusively, partial agonists at postsynaptic 5-HT1A receptors. Some of these drugs, including gepirone and other 5-HT1A agonists such as buspirone, have been reported to exert anxiolytic and antidepressive activity in double-blind, placebo-controlled, and comparative trials. Their delayed therapeutic activity is believed to result from increased activation of postsynaptic 5-HT1A receptors occurring only after 5-HT neurons regain their normal firing activity. The recovery of this parameter, which is attributable to 5-HT1A autoreceptor desensitization, also restores 5-HT release. At this point, the summed effects of a normalized level of synaptic 5-HT and the exogenous 5-HT1A agonist can be exerted on postsynaptic 5-HT1A receptors. The widespread recognition of the clinical efficacy of such agents has largely been hampered by their undesirable pharmacokinetic properties. Most 5-HT1A agonists are indeed readily absorbed but are also rapidly eliminated, thereby often producing either suboptimal therapeutic responses at low doses, or cumbersome adverse effects at higher doses. Extended-release formulations allow once-daily dosing regimens, thus avoiding sharp peak plasma concentrations. This improves compliance and permits the use of higher dosages, which may be associated with enhanced efficacy and better tolerability relative to the immediate-release formulations. In sum, 5-HT1A receptor agonism represents a valuable and efficacious therapeutic approach to major depression.     

Selective 5-HT receptor inhibition of glutamatergic and GABAergic synaptic activity in the rat dorsal and median raphe

The dorsal (DR) and median (MR) raphe nuclei contain 5-hydroxytryptamine (5-HT) cell bodies that give rise to the majority of the ascending 5-HT projections to the forebrain. The DR and MR have differential roles in mediating stress, anxiety and depression. Glutamate and GABA activity sculpt putative 5-HT neuronal firing and 5-HT release in a seemingly differential manner in the MR and DR, yet isolated glutamate and GABA activity within the DR and MR has not been systematically characterized. Visualized whole-cell voltage-clamp techniques were used to record excitatory and inhibitory postsynaptic currents (EPSC and IPSC) in 5-HT-containing neurons. There was a regional variation in action potential-dependent (spontaneous) and basal [miniature (m)] glutamate and GABAergic activity. mEPSC activity was greater than mIPSC activity in the DR, whereas in the MR the mIPSC activity was greater. These differences in EPSC and IPSC frequency indicate that glutamatergic and GABAergic input have distinct cytoarchitectures in the DR and MR. 5-HT(1B) receptor activation decreased mEPSC frequency in the DR and the MR, but selectively inhibited mIPSC activity only in the MR. This finding, in concert with its previously described function as an autoreceptor, suggests that 5-HT(1B) receptors influence the ascending 5-HT system through multiple mechanisms. The disparity in organization and integration of glutamatergic and GABAergic input to DR and MR neurons and their regulation by 5-HT(1B) receptors may contribute to the distinction in MR and DR regulation of forebrain regions and their differential function in the aetiology and pharmacological treatment of psychiatric disease states.     

Electrophysiological evidence for excitatory 5-HT2 and depressant 5-HT1A receptors on neurones of the rat midbrain tectum.

It has been claimed that the aversive behaviour induced by electrical stimulation of the midbrain tectum (MT) has validity as an animal model of panic attack. A great deal of evidence obtained from behavioural studies suggests that 5-HT2 mechanisms phasically inhibit the substrates of aversion in the MT. In order to test this hypothesis we employed the technique of microiontophoresis of drugs onto neurons of the MT to assess the identity of the receptors mediating the effects of 5-hydroxytryptamine (5-HT). The results obtained show that the majority of 5-HT responsive cells in MT are cells excited by 5-HT (72%). These cells were silent or showed very low spontaneous firing activity, whereas cells depressed by 5-HT showed high spontaneous firing activity at baseline. The 5-HT1A receptor agonists, 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT), buspirone and gepirone caused consistent reduction in the firing rate of cells depressed by 5-HT while they did not change the firing activity of cells excited by 5-HT. The excitatory effects induced by 5-HT on MT neurones were clearly attenuated by concomitant application of ketanserin, a highly specific 5-HT2 antagonist. Excitatory responses to DL-homocysteic acid were not affected by ketanserin. Previous administration of zimelidine, a selective 5-HT uptake inhibitor, caused a significant enhancement of the excitatory effects of 5-HT while similar application of gepirone did not affect the size of the excitatory responses to 5-HT.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sex hormone-dependent desensitization of 5-HT1A autoreceptors in knockout mice deficient in the 5-HT transporter

The serotonin transporter (5-HTT) is the target of most antidepressant drugs, whose therapeutic action is related to their facilitatory influence on 5-HT neurotransmission. In this study, we investigated the functional adaptive properties of 5-HT1A autoreceptors, which regulate serotonergic neuronal firing, in knockout mice deficient in 5-HTT. Neurons of the dorsal raphe nucleus (DRN) were recorded extracellularly under chloral hydrate anaesthesia in male and female knockout 5-HTT mice and their wild-type counterparts. The inhibitory response of DRN neurons to intravenous injection of the 5-HT1A agonist 8-OH-DPAT was dramatically reduced in knockout 5-HTT compared with wild-type mice, especially in females. Changes in 8-OH-DPAT-induced hypothermia and autoradiographic labelling of 5-HT1A sites in the DRN confirmed a greater level of desensitization/down-regulation of 5-HT1A autoreceptors in female than in male knockout 5-HTT mice. After gonadectomy, the functional status of 5-HT1A autoreceptors was unchanged in wild-type mice, whereas in knockout 5-HTT, castrated males exhibited a down-regulation, and ovariectomized females an up-regulation of these receptors, as shown by electrophysiological recording and autoradiographic labelling in the DRN, as well as by changes in 8-OH-DPAT-induced hypothermia. Finally, in gonadectomized knockout 5-HTT mice, treatment with testosterone or estradiol restored the DRN neuronal firing sensitivity to 8-OH-DPAT back to sham control level in males or females, respectively. These data indicate that sexual hormones participate in the mechanisms responsible for the desensitization of 5-HT1A autoreceptors in knockout 5-HTT mice. The differential effects of testosterone and estradiol on 5-HT1A-mediated control of 5-HT neurotransmission might be related to the well-established gender differences in the vulnerability to depression.     

Altered regulation of the 5-HT system in the brain of MAO-A knock-out mice

Genetic deficiency of monoamine oxidase-A (MAO-A) induces major alterations of mood and behaviour in human. Because serotonin (5-HT) is involved in mood regulation, and MAO-A is responsible for the catabolism of 5-HT, we investigated 5-HT mechanisms in knock-out mice (2-month-old) lacking MAO-A, using microdialysis, electrophysiological, autoradiographic and molecular biology approaches. Compared to paired wild-type mice, basal extracellular 5-HT levels were increased in ventral hippocampus (+202%), frontal cortex (+96%) and dorsal raphe nucleus (DRN, +147%) of MAO-A mutant mice. Conversely, spontaneous firing rate of 5-HT neurons in the DRN (recorded under chloral hydrate anaesthesia) was approximately 40% lower in mutants. Acute 5-HT reuptake blockade by citalopram (0.2 and 0.8 mg/kg i.v.) produced a much larger increase in extracellular 5-HT levels (by approximately 4 fold) and decrease in DRN neuronal firing (with a approximately 4.5 fold decrease in the drug's ED50) in MAO-A knock-out mice, which expressed lower levels of the 5-HT transporter throughout the brain (-13 to -34% compared to wild-type levels). The potency of the 5-HT1A agonist 8-OH-DPAT to produce hypothermia and to reduce the firing of DRN serotoninergic neurons was significantly less in the mutants, indicating a desensitization of 5-HT1A autoreceptors. This was associated with a decreased autoradiographic labelling of these receptors (-27%) in the DRN. Altogether, these data indicate that, in MAO-A knock-out mice, the enhancement of extracellular 5-HT levels induces a down-regulation of the 5-HT transporter, and a desensitization of 5-HT1A autoreceptors which allows the maintenance of tonic activity of 5-HT neurons in the DRN.     

Activation of postsynaptic 5-HT(1A) receptors by fluoxetine despite the loss of firing-dependent serotonergic input: electrophysiological and neurochemical studies

Systemic doses of fluoxetine slow dorsal raphe cell firing by blocking the reuptake carrier located in the cell body region with the surplus 5-HT thus generated activating inhibitory autoreceptors. The concurrent actions of fluoxetine on postsynaptic receptors in raphe projection areas has not been as thoroughly investigated, although it is presumed that a reduction in cell firing should curtail these targeted effects. The goal of the present studies was to assess the degree of postsynaptic receptor activation obtained with fluoxetine and relate it to cell body autoreceptor activation and the level of extracellular 5-HT obtained at the nerve endings. Changes in firing rates of CA3 hippocampal neurons following systemic administration of fluoxetine were used as a marker of SSRI-dependent changes in postsynaptic 5-HT receptor activation; monitoring of unit activity of neurons in the dorsal raphe nucleus served to gauge the degree of serotonergic input in a parallel series of animals. Estimates of the corresponding changes in terminal 5-HT release in the CA3 region were analyzed by microdialysis. The results indicate that fluoxetine inhibits hippocampal cell firing in a dose-dependent manner (ED(50) = 4.4 mg/kg i.v.) and one sensitive to pretreatment with the 5-HT(1A) antagonist WAY-100,635. Within the same dose range, increases in hippocampal extracellular 5-HT approaching 300% above basal levels were achieved. Both the changes in hippocampal neuronal activity and extracellular 5-HT are evident at doses of fluoxetine in excess of that required to inhibit dorsal raphe cell firing (ED(50) = 1.1 mg/kg i.v.). Taken together, these data suggest that increases in extracellular levels of 5-HT on the order of that observed are sufficient to alter postsynaptic excitability and that this accumulation of synaptic 5-HT and the subsequent activation of postsynaptic 5-HT(1A) receptors are achievable despite loss of firing-dependent 5-HT release.     

Effect of the selective noradrenergic reuptake inhibitor reboxetine on the firing activity of noradrenaline and serotonin neurons

Reboxetine is a non-tricyclic antidepressant with selective noradrenergic (NA) reuptake-blocking effects. The effects of acute and sustained administration of reboxetine, on the firing activity of locus coeruleus NA neurons and dorsal raphe 5-HT neurons, were assessed using in vivo extracellular unitary recording in rats anaesthetized with chloral hydrate. Reboxetine (0.1-1.25 mg/kg, i.v.) dose-dependently decreased the firing activity of NA neurons (ED50 = 480 +/- 14 microg/kg). A 2-day treatment with reboxetine at 1.25, 2.5, 5, or 10 mg/kg per day (using osmotic minipumps implanted subcutaneously) produced significant decreases of 52%, 68%, 81%, and 83%, respectively, of NA firing activity. When the reboxetine treatment (2.5 mg/kg per day) duration was prolonged to 7 days, a 66% decrease in NA firing activity was observed which further decreased to 80% after 21 days of treatment. In contrast, 5-HT neuron firing rate remained unaltered following short- and long-term reboxetine treatments. The suppressant effect of the alpha2-adrenoceptor agonist clonidine on the firing activity of NA neurons was unchanged in long-term reboxetine-treated rats, but its effect on the firing activity of 5-HT neurons was blunted. The enhancement of NA firing activity by the 5-HT1A agonist 8-OH-DPAT was abolished in long-term reboxetine-treated rats, whereas, the inhibitory effect of the 5-HT2 agonist DOI was attenuated by about three-fold. In conclusion, sustained NA reuptake blockade by reboxetine lead to profound alterations in the function of NA neurons and of 5-HT receptors modulating their firing activity.     

Electrophysiological responses of serotoninergic dorsal raphe neurons to 5-HT1A and 5-HT1B agonists

A direct comparison was made of the effects of serotonin 5-HT1A and 5-HT1B selective compounds on the spontaneous firing rate of dorsal raphe serotoninergic neurons in chloral-hydrate-anesthetized rats. Following intravenous administration, the 5-HT1A selective compounds ipsapirone (TVX Q 7821) and LY 165163 potently inhibited single-unit activity in a dose-dependent manner whereas the 5-HT1B selective compounds, m-chlorophenylpiperazine (mCPP) and trifluoromethylphenylpiperazine (TFMPP), displayed only weak or irregular actions. Low microiontophoretic currents of ipsapirone and LY 165163 were also effective in suppressing spontaneous firing; dose-response relationships for the 5-HT1A compounds were indistinguishable from that of 5-HT itself. In contrast, dorsal raphe neurons were only weakly responsive to microiontophoretic application of mCPP and TFMPP; dose-response relationships for the 5-HT1B compounds were significantly displaced from that of 5-HT. In intracellular studies, ipsapirone and LY 165163, when added to the media bathing brain slices, mimicked the actions of 5-HT in hyperpolarizing dorsal raphe cell membranes and decreasing input resistance; however, the maximal effects of the 5-HT1A compounds on these membrane properties exceeded those of 5-HT. In summary, dorsal raphe 5-HT neurons appear highly responsive to 5-HT1A, but not to 5-HT1B compounds; these findings are discussed with regard to the 5-HT receptor subtypes as candidates for the somatodendritic autoreceptor of dorsal raphe neurons.     

Role of cholinergic and GABAergic systems in the feedback inhibition of dorsal raphe 5-HT neurons

Several observations indicate that 5-HT1A receptors found on a long neuronal feedback loop, originating from the medial prefrontal cortex, regulate 5-HT neuronal firing. In the present study, the muscarinic (M) receptor antagonists atropine and scopolamine as well as the M2 receptor antagonist AF-DX 116, but not the preferential M1 receptor antagonist pirenzepine, reduced the suppressant effect of the 5-HT1A receptor agonist 8-OH-DPAT on the spontaneous firing activity of rat dorsal raphe 5-HT neurons. Moreover, AF-64A-induced lesions of cholinergic neurons directly in the medial prefrontal cortex and after its i.c.v. injection attenuated the effect of 8-OH-DPAT. Finally, the NMDA receptor antagonist (+)MK-801 and the GABA(B) receptor antagonist SCH-50911, but not the GABA(A) receptor antagonist (-)bicuculline, dampened the latter response. The present study unveiled a key role for the cholinergic and GABAergic systems in the feedback inhibition of dorsal raphe 5-HT neurons.