Theme : Dreams

Dorsal Raphe Nucleus Administration of 5-HT1A Receptor Agonist and Antagonists: Effect on Rapid Eye Movement Sleep in the Rat

Jaime M. Monti, Héctor Jantos, Daniel Monti and Fernando Alvariño
Department of Pharmacology and Therapeutics, Clinics Hospital, Montevideo, Urugray
Abstract
The effect of flesinoxan, a selective 5-HT1A receptor agonist, WAY 100635, a selective 5-HT1A receptor antagonist, and (±)pindolol, a mixed ß-adrenoceptor and 5-HT1A/B receptor antagonist, on spontaneous sleep was studied in adult rats implanted for chronic sleep recordings. Drugs were infused directly into the dorsal raphe nucleus (DRN). Direct application of flesinoxan (25.0 and/or 50.0 ng) into the DRN induced a significant increment of REM sleep (REMS) during the second and third 2 h period of recording. On the other hand, microinjection into the DRN of (±)pindolol (100.0 and/or 200.0 ng), and WAY 100635 (12.5, 25.0 and 50.0 ng) significantly reduced REMS during the first and/or second 2 h recording period . Our findings support previous studies indicating that microdialysis perfusion of the 5-HT1A receptor agonist 8-OHDPAT into the DRN increases REMS. In addition, they favor the proposal that microinjection of 5-HT1A receptor antagonists into the DRN would suppress 5-HT inhibition and reduce REMS.

Current Claim: Direct administration of the 5-HT1A receptor agonist flesinoxan into the dorsal raphe nucleus increases REMS, whereas microinjections of the 5-HT1A receptor antagonists pindolol and WAY 100635 suppresses REMS.

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The proposal that identifies cholinergic neurons in the laterodorsal (LDT) and pedunculopontine (PPT) tegmental nuclei as promoting REM sleep (REMS), and their inhibition by serotonergic afferents from the dorsal raphe nucleus (DRN) and by noradrenergic afferents from the locus coeruleus (LC), is the one that best corresponds to the experimental evidence concerning REMS (McCarley, 1993; Thakkar et al., 1998; Strecker et al., 1999). Current lesion, electrophysiological, neurochemical, and pharmacological data strongly support the hypothesis that serotonin (5-HT) can inhibit REMS (Hobson et al., 1998).

The 5-HT1A receptor is located on the soma and the dendrites of 5-HT neurons within the DRN and at postsynaptic sites. Stimulation of the somatodendritic 5-HT1A receptor inhibits the firing rate of serotonergic neurons, whereas activation of the postsynaptic receptor induces inhibitory responses on target structures (Andrade et al., 1986; Blier et al., 1989).

Flesinoxan is a phenylpiperazine derivative that binds potently and selectively to central 5-HT1A receptors (Ki=1.7 nM). Slightly larger values (Ki=2.8 nM) have been described for 8-OHDPAT (Olivier et al., 1992). Direct administration of flesinoxan (10 nM-1 µM) into the median raphe nucleus of freely moving guinea pigs induced a concentration-dependent decrease of 5-HT levels to (maximally) 47% at central sites, and this effect was prevented by WAY 100635 (N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl-N-(2-pyridinyl) cyclohexanecarboxamide trihydrochloride), a selective 5-HT1A receptor antagonist (Van der Heyden et al., 1996). Moreover, systemic administration of flesinoxan to anesthetized rats dose-dependently (ID50=19.5 µg/kg, i.v.) inhibited the firing rate of DRN serotonergic neurons. This action was abolished by WAY 100635 (31 µg/kg, i.v.) (Gobert et al., 1995; Lejeune and Millan, 1998).

Mixed ß-adrenoceptor and 5-HT1A/B receptor antagonist pindolol acts at both pre- and postsynaptic sites, as does WAY 100635, a selectively high affinity silent antagonist. Variable effects of systemic WAY 100635 on the activity of serotonergic DRN nucleus have been reported. In this respect, Fornal et al. (1996), and Mundey et al. (1996), showed a dose-related increase of the basal firing rate of 5-HT neurons after WAY 100635 administration in the DRN of the guinea pig and the cat. The effect was evident during waking (W) but not during sleep. On the other hand, in the study by Gartside et al. (1995), WAY 100635 failed to increase serotonergic neuronal activity.

Portas et al. (1996, 1998), provided direct evidence that suppression of DRN serotonergic activity increases REMS. To this purpose, the authors measured extracellular 5-HT in the DRN and behavioral state changes by simultaneous polygraphic recordings. Microdialysis perfusion of 8-OHDPAT into the DRN decreased 5-HT levels across the sleep/wake cycle and significantly increased REMS.

If activation of somatodendritic 5-HT1A receptor by 8-OHDPAT is responsible for the increase of REMS, then 5-HT1A antagonists injected into the DRN should block local serotonergic inhibition in periods in which there is endogenous 5-HT release, and trigger a series of events that would culminate in the reduction of REMS. In the present study, we tested this proposal. To this purpose, we analyzed the effect of direct microinjection of flesinoxan, pindolol, and WAY 100635 into the DRN of the rat.

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Male Wistar rats, each weighing 320-350 g, were implanted with Nichrome® electrodes (200 µm diameter) for chronic sleep recordings of electroencephalogram and electromyogram activities by means of placement on frontal and occipital cortex for the former, and on dorsal neck musculature for the latter. In addition, a guide cannula was inserted and maintained in the DRN according to the technique of Dib (1994). The final coordinates for the guide cannula implantation into the DRN were posterior-anterior: 3.4 mm; lateral: 0.0; vertical 6.4 mm below the top of lobule 5 of cerebellum. The tubular guide (gauge 26) for drug injection was implanted 2 mm above the DRN (V: 4.4 mm). Drug or vehicle was injected into the DRN with an injection cannula (29 gauge), which extended 2 mm beyond the guide, in a 0.4 µl volume over a 2 min period. On completion of the study, rats were sacrificed and cannulae placements were defined histologically. Correct cannula/injection sites were assessed using the atlas of Paxinos and Watson (1986) following a 0.4 µl injection of Fast-green dye into the DRN. All data presented in this report are derived from animals whose injection site was within the limits of the DRN. The animals were housed individually in a temperature-controlled room (23±1°C) under a 12 h light/12 h dark cycle (lights went on at 7:00 a.m.) and with food and water ad libitum. Ten days after surgery, the animals were habituated for four days to a soundproof chamber fitted with slip-rings and cable connectors. Thereafter, they were given a control solution or the drugs to be tested. Drugs were always administered during the light phase of the 12 h light/12 h dark cycle, at approximately 7:30 a.m. A balanced order of drug and control injections was used during the experimental procedures.

Electrographic activity of 25 s epochs was analyzed and assigned to the following categories based on the waveform: waking (W), light sleep (LS), slow wave sleep (SWS), and REM sleep (REMS). In addition, REM sleep latency and the number of REM periods were determined.

The effects of the 5-HT1A agonist and antagonists were studied following three different protocols, in three different groups of animals.

Experiment 1

Flesinoxan (Solvay-Duphar, The Netherlands) 12.5, 25.0 or 50.0 nanograms (ng) (0.057, 0.11 or 0.23 nmols), disolved in an isotonic NaCl solution, or vehicle (saline) was infused into the DRN. There were six animals in the experimental group. They received four injections each. At least three days were allowed to elapse between injections to avoid long-lasting effects on sleep.

Experiment 2

In the second set of experiments, (±)pindolol (Sigma, USA) 50.0, 100.0 or 200.0 ng (0.2, 0.4 or 0.8 nmols), or saline was injected into the DRN. Six rats were in the experimental group. They received four injections each.

Experiment 3

In the third set of experiments, WAY 100635 (Wyeth Research, UK) 12.5, 25.0 or 50.0 ng (0.05, 0.1 or 0.2 nmols), or saline was infused into the DRN. There were seven animals in the experimental group, and each rat received four microinjections.

A two-way analysis of variance (ANOVA) with time and treatment as between factors was performed, with multiple post hoc comparisons carried out with the Scheffé test when the ANOVA indicated significance.

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Figure 1

Figure 2

Figure 3
Effects of Microinjection of Flesinoxan into the DRN

Following the microinjection of 25.0 ng flesinoxan into the DRN, REMS was significantly increased (F=46.97, p<0.0001, and F=5.89, p<0.001 for variable time and treatment, respectively) during the second 2 h period after treatment (p<0.02). The 50.0 ng dose of the serotonin agonist induced a significant increase of REMS during the second and third 2 h period of recording (p<0.004). The smallest dose of flesinoxan (12.5 ng) did not show any remarkable effect on desynchronized sleep (Figure 1). Values corresponding to W, LS and SWS showed slight but inconsistent changes that did not attain significance. There were no significant changes in REMS latency. On the other hand, the number of REM periods was significantly increased (F=24.76, p<0.0001, and F=5.52, p<0.002 for variable time and treatment, respectively) after the two largest doses of the 5-HT1A receptor agonist during the third 2 h period of recording (Table 1).

Effects of Microinjection of (±)Pindolol into the DRN

In the rats recorded after receiving (±)pindolol, REMS showed a significant decrease (p<0.01) (F=15.58, p<0.0001, and F=8.01, p<0.0001 for variable time and treatment, respectively) after the 100.0 ng dose during the second 2 h period of recording, and the 200.0 ng dose during the first and second 2 h recording period (p<0.0006). The time spent in W, LS and SWS was slightly but not significantly modified (Figure 2). (±)Pindolol did not significantly modify REMS latency or the number of REM periods.

Effects of Microinjection of WAY 100635 into the DRN

Figure 3 shows that intra-DRN injection of WAY 100635 (12.5-50.0 ng) induced a significant decrease of REMS during the second 2 h recording period (p<0.001) (F=21.98, p<0.0001, and F=4.53, p<0.005 for variable time and treatment, respectively). REM sleep values were also significantly reduced (p<0.02) after the 50.0 ng dose during the first 2 h period of recording. Waking, LS and SWS were not significantly different as compared to the control.

WAY 100635 (50.0 ng) increased REMS latency (p<0.01) (F=19.17, p<0.0001, and F=4.52, p<0.005 for variable time and treatment, respectively). Moreover, the number of REM periods was significantly reduced after the whole range of doses during the second 2 h recording period (Table 2).

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The main finding of this study is that direct application of the 5-HT1A receptor agonist, flesinoxan, into the DRN induced an increment of REMS, and the number of REM periods. On the other hand, microinjections of the 5-HT1A receptor antagonists, (±)pindolol and WAY 100635, into the DRN reduced REMS. In addition, WAY 100635 increased REMS latency and decreased the number of REM periods.

In spite of the animals being thoroughly adapted to the microinjection procedure, relatively high amounts of W were observed during the first 2 h period. As mentioned above, all injections were at least three days apart, and rats were not readapted to the recording procedure between the microinjection experiments. This could tentatively explain the presence of relatively high amounts of W during the first 2 h of the recording sessions.

Flesinoxan took 3 to 4 h to show a significant effect on REMS. This could be partly related to its slow diffusion from the injection site to other subdivisions of the DRN. In the study by Bjorvatn et al. (1997), where 8-OHDPAT was perfused continuously for 6 h into the DRN of rats, a significant increase of REMS was apparent only during the third 2 h period. Although the authors did not address this issue in the discussion, a slow diffusion of the 5-HT1A agonist from the perfusion area cannot be discarded. Moreover, in either study, rats were moved from the animal quarters to a different recording chamber, which may have disturbed their normal sleeping behavior and increased the time necessary for the 5-HT1A receptor agonist to significantly augment REMS.

REM sleep increase during the second 2 h period after 25.0 ng flesinoxan was related to a nonsignificant increment of REM period frequency. On the other hand, REM period duration remained almost unchanged. Moreover, 25.0 ng flesinoxan significantly increased REM period frequency during the third 2 h period, but REM period duration was reduced. As a result, REMS values remained unchanged.

The data of the present study support previous findings indicating that direct administration into the DRN of a selective 5-HT1A receptor agonist increases REMS. Accordingly, Portas et al. (1996, 1998), described that microdialysis perfusion of 8-OHDPAT into the DRN decreases 5-HT release and increases REMS in the freely moving cat and rat. The authors hypothesized that inhibition of DRN activity following stimulation of somatodendritic 5-HT1A receptors suppressed 5-HT inhibition of mesopontine cholinergic neurons and increased REMS. In this respect, Vertes and Kocsis (1994), and Honda and Semba (1994) described moderate to dense projections of the DRN to the PPT/LDT nuclei of the rat. However, only 12% of all anterogradely labeled DRN axons seem to synapse with cholinergic neurons of the PPT-pars compacta and the LDT of the rat; the remaining 88% synapse with unlabeled terminals, presumptively glutamatergic neurons (Steiniger et al., 1997). Whether inhibition of this weak projection from DRN to PPT/LDT nuclei is fully responsible of REMS increase is a matter of debate.

It is currently accepted that the REMS induction region of the medial pontine reticular formation (mPRF) predominantly includes glutamatergic neurons, which are in turn activated by acetylcholine (Hobson et al., 1998). In addition to cholinergic projections from the PPT/LDT nuclei, the REMS induction zone receives various aminergic inputs. According to Semba (1993), serotonergic afferents represent a mean of 44% of all aminergic/cholinergic projections to the REMS induction zone, the heaviest projections arising from the DRN and the medial raphe nucleus. Thus, the possibility remains that release of glutamatergic neurons in the REMS induction zone from the inhibitory influence of 5-HT contributes to the increase of desynchronized sleep. However, further studies are needed to resolve this issue.

The inhibitory effect of (±)pindolol and WAY 100635 on REMS supports our prediction that locally administered 5-HT1A antagonists would block serotonergic inhibition and decrease desynchronized sleep. At the cellular level, WAY 100635 has been shown to produce a dose-related increase in the basal firing of 5-HT neurons in the DRN during W but not during sleep, and to restore the firing of DRN cells previously inhibited by 8-OHDPAT in the guinea pig and the cat (Fornal et al., 1996; Mundey et al., 1996). In addition, it has been shown that (±)pindolol and/or WAY 100635 markedly potentiate the citalopram, clomipramine and phenelzine-induced rise of extracellular 5-HT levels (Hjorth et al., 1996; Romero et al., 1996), and this effect in all probability reflects the ability of the serotonergic antagonists to block 5-HT1A somatodendritic receptors in the DRN.

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The authors do not wish to include any acknowledgments.

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