Injections of kainic acid into the raphe dorsal nucleus in freely moving cats: electroencephalographic, behavioral and histopathological consequences.

Results are presented concerning the functional and morphological changes occurring after kainic acid injections into raphe dorsal nucleus in freely moving cats. The effects of kainic acid at two doses, 4 and 12 nmol, on electroencephalographic activity, behavior and cell morphology were examined on the day of injection, and on the 1st, 3rd, 6th, 15th, 30th and 65th day. A well-pronounced dose-dependent effect of kainic acid was established: in 50% of the animals injected with 4 nmol of kainic acid and in 85.7% of the animals injected with 12 nmol electroencephalographic and behavioral seizures appeared. The strength of the seizures in the animals receiving 12 nmol of kainic acid was different--from focal single epileptiform patterns first in raphe dorsal nucleus and then in other brain regions to generalized high-voltage ictal activity and in some animals to epileptic state. In all animals receiving 4 nmol of kainic acid and in 75% of the animals receiving 12 nmol of kainic acid, the electrical activity and behavior returned to normal several hours after or within two days following the kainic acid injection. The histopathological data obtained by light microscopy showed a dose-dependent cytoarchitectural disorganization (mainly chromatolytic changes) not only at the injection site but also in many other brain structures. The present results confirm our previous studies on cats in acute conditions showing enhancement of brain excitability level up to hyperexcitation (seizures) after kainic acid injection into the raphe dorsal nucleus.     

Arousal induced by injection of triazolam into the dorsal raphe nucleus of rats

In order to explore the possible sites at which benzodiazepines alter sleep, we have performed sleep studies following administration of 0.5 microgram of triazolam into the dorsal raphe nucleus of rats. Triazolam significantly increased sleep latency and decreased non-rapid eye movement (REM) sleep, with an effect greatest in the first 2 hours after injection. Total REM sleep time was not significantly affected, although there was a modest trend toward reduction in the first 2 hours. In contrast to the decreased sleep resulting from injection into the dorsal raphe nucleus, triazolam did not significantly alter sleep in animals in whom it was injected into surrounding areas. Similarly, the low dose employed in this study did not significantly affect sleep when injected into the lateral ventricle. These data are reminiscent of studies showing transient decreases in sleep following lesions of the dorsal raphe nucleus.     

Anxiogenic effects of methyl-beta-carboline-3-carboxylate in a light/dark choice situation

Doses of benzodiazepine, clorazepate, and also of the inverse agonist of the benzodiazepine receptor, beta-CCM, which failed to present sedative or postictal depressive effects, were at first determined in a free exploratory situation. Then, the effects of clorazepate dosed at 1.0, 2.0 and 4.0 mg/kg and beta-CCM dosed at 1.0 and 2.5 mg/kg were studied in the light/dark box choice procedure. Clorazepate tended to produce an increase of the time spent by mice in the lit box as well as of the number of transitions between the two boxes, whereas the dose of 1.0 mg/kg of beta-CCM had opposite effects. The benzodiazepine antagonist RO 15-1788 completely counteracted the anxiolytic effects of clorazepate dosed at 2.0 mg/kg and the anxiogenic effects of beta-CCM.     

Conditioned place preference induced by microinjection of 8-OH-DPAT into the dorsal or median raphe nucleus

Experiments were conducted to examine the ability of the selective 5-hydroxytryptamine (5-HT)_1A agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) to induce a conditioned place preference following peripheral injection, and direct microinjection into the dorsal or median raphe nuclei. An unbiased place preference paradigm was used in which control animals showed no preference for either of two compartments differing in terms of colour (white versus black), floor texture (rough versus smooth) and olfactory cues (no odour versus acetic acid odour). Drug treatments were paired with access to either of the two compartments, and saline injections were paired with access to the other compartment. Rats experiencing a low dose of 8-OH-DPAT (125 µg/kg) with a specific compartment demonstrated a significant preference for that compartment over one paired with saline injections. The magnitude of this effect was similar to that observed in rats treated with 1.5 mg/kgd-amphetamine. A significant place preference was found in animals receiving injections of 8-OH-DPAT in the dorsal raphe at 0.1 µg but not 1 µg. Animals also displayed a preference for the compartment paired with 1 µg 8-OH-DPAT injected into the median raphe; lower doses were not effective. These results indicate that the mechanism by which 8-OH-DPAT induces a conditioned place preference involves activation of raphe somatodendritic 5-HT_1A autoreceptors, leading to a reduction in 5-HT neurotransmission. This demonstration of the rewarding properties of 8-OH-DPAT, together with previous results showing increased feeding and sexual behaviour following 8-OH-DPAT treatment, strongly suggests an important role for brain 5-HT systems in reward and reinforcement processes.     

Behavioural consequences following infusion of selective neurokinin agonists into the median raphe nucleus of the rat.

The locomotor activity induced after infusion of selective neurokinin (NK) agonists into the median raphé nucleus of rats was investigated. In photocell cages, the NK-2-agonist, GR64349, and the NK-3 agonist, senktide, both increased motor activity in a dose-dependent manner. However, the NK-1 agonist, GR73632, had little effect over a range of doses. In the open field, the motor effect of all three NK agonists was identical to that observed in the photocell cages. In addition, senktide induced straub-tail, hind-limb splaying and various oral movements. Such effects were not noted with the other two agonists. These results suggest that activation of NK-2 or NK-3 receptors by the neurokinins, in the median raphe nucleus of the rat, leads to an increase in locomotor activity.     

Microinjections of 5-HT1A agonists into the dorsal motor vagal nucleus produce a bradycardia in the atenolol-pretreated anaesthetized rat.

1. The effects of microinjections (100 nl) into the dorsal motor vagal nucleus of the 5-HT1A receptor agonists 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) and flesinoxan, the 5-HT2 receptor agonist (+-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI), the 5-HT3 receptor agonist phenylbiguanide (PBG), the alpha 2-adrenoceptor agonist clonidine and the excitatory amino acid glutamate on heart rate, blood pressure, tracheal pressure and phrenic nerve activity were investigated in atenolol-pretreated rats anaesthetized with sodium pentobarbitone. 2. Microinjections of glutamate (2.5 nmol) caused decreases in blood pressure, heart rate and phrenic nerve activity. In contrast, microinjections of 5-HT (1.2 nmol), 8-OH-DPAT (1.2 nmol) and flesinoxan (1.3 nmol) all caused a bradycardia but had no effect on blood pressure. In addition, 8-OH-DPAT and flesinoxan caused an increase in phrenic nerve activity. 3. Microinjections of DOI, PBG and clonidine had no significant effect on any of the variables recorded. None of the drugs used had any significant effect on tracheal pressure. 4. These results support the hypothesis that activation of 5-HT1A receptors causes excitation of cardiac vagal motoneurones and suggest that these receptors are also important in the control of central respiratory drive.     

Neuropeptide FF receptors control morphine-induced analgesia in the parafascicular nucleus and the dorsal raphe nucleus

The ability of (1DMe)Y8Fa (D.Tyr–Leu–(NMe)Phe–Gln–Pro–Gln–Arg–Phe–NH₂), a selective neuropeptide FF analog resistant to enzymatic degradation, to control morphine-induced analgesia was investigated in rat after microinfusion into the dorsal raphe nucleus and the nucleus parafascicularis of the thalamus. Infusion of (1DMe)Y8Fa (2.5 nmol) in the nucleus raphe dorsalis did not modify the animal response in the tail-immersion test but significantly reversed analgesia induced by coinjected morphine (27 nmol). Similarly, (1DMe)Y8Fa (5 nmol) inhibited morphine effects in the hot-plate test after co-injection into the parafascicular nucleus. Furthermore, (1DMe)Y8Fa injected into the parafascicular nucleus attenuated analgesia induced by morphine injected into the nucleus raphe dorsalis and similarly, the neuropeptide FF analog in the nucleus raphe dorsalis decreased the effects of 27 nmol morphine injected in the parafascicular nucleus. The density of neuropeptide FF receptors did not decrease in the nucleus raphe dorsalis after lesion of serotonergic neurons by 5,7-dihydroxytryptamine. However, after this lesion, (1DMe)Y8Fa injected in the nucleus raphe dorsalis was no longer able to modify analgesic effects of morphine in hot-plate and tail-immersion tests. Similarly, the serotonin (5-HT) depletion induced by a systemic administration of para-chlorophenylalanine did not modify morphine analgesia microinjected into the nucleus raphe dorsalis and the parafascicular nucleus but blocked the ability of (1DMe)Y8Fa to reverse morphine effects in both nuclei. These data show that neuropeptide FF exerts anti-opioid effects directly into both the nucleus raphe dorsalis and the parafascicular nucleus and acts also at distance on opioid functions. Furthermore, anti-opioid effects of neuropeptide FF require functional serotonergic neurons although neuropeptide FF receptors are not carried on these neurons.     

Benzodiazepines: potentiation of a GABA inhibitory response in the dorsal raphe nucleus.

Based on evidence that the dorsal raphe nucleus (DR) has specific and independent receptors for 5HT, GABA and glycine (Gallager and Aghajanian, 1976; Wang and Aghajanian, 1977), alterations in the firing rate of DR neurons following the administration of benzodiazepines (BZ) were evaluated to determine whether they were the result of a direct interaction with 5HT receptors or due to interactions of these drugs with GABA and/or glycine. The effects of BZs after both direct and systemic application were tested in rats using microiotophoretic and single-cell recording techniques. Although the BZs did not alter the spontaneous firing rate of the DR, both the systemic and iontophoretic administration of these drugs were found to potentiate the inhibitory response produced by GABA. The data suggest that this potentiation is mediated postsynaptically. Since the effects of BZs on the spontaneous activity of the DR are only apparent following pretreatments with AOAA, it is speculated that these drugs may only have pronounced effects when GABAergic input is prominent.     

Effects of morphine on the single unit activity of nucleus dorsal raphe

Effects of morphine were studied in 36 gallamine triethiodide immobilized adult cats under light N2O anesthesia. Single units were recorded from the nucleus dorsal raphe using a stainless steel microelectrode. Dorsal raphe neurons were divided into two types; one was a clock-like (CL) neuron which was typically slow in rate, rhythmic and stable throughout recording time, and the other was a non-clock like (NCL) neuron which was relatively irregular in pattern as compared with CL neuron. Out of 36 neurons recorded in this experiment, 13 were CL neurons and 23 were NCL neurons. Mean discharge rates (spikes per sec) of CL neuron and NCL neuron were 2.84 (range of 2.01 approximately 3.68) and 4.11 (range of 0.10 approximately 35.09), respectively. None of the 13 CL neurons responded to the nociceptive (pinch and/or brdaykinin) and non-nociceptive (hair bending and/or tapping) stimuli. On the contrary, out of 23 NCL neurons, 13 responded to both nociceptive and non-nociceptive stimuli, 4 were responsive to only non-nociceptive stimuli and 6 were not responsive to these stimuli. Receptive fields of NCL neurons were wide with various somatic modalities. The latency for bradykinin (3 microgram) was 6.46 +/- 0.77 sec. Effects of morphine were examined in 6 CL neurons and 12 NCL neurons. After morphine, no nociceptive neuron responded to nociceptive stimuli, although there was no appreciable change in the responsiveness to non-nociceptive stimuli. Firing frequency and pattern of all CL neurons were unaffected by morphine. Out of 12 NCL neurons, 7 were unaffected in firing frequency and 5 were decreased after morphine.     

Cardiovascular effects of 5-HT1A receptor agonists injected into the dorsal raphe nucleus of conscious rats

The aim of this study was to investigate the cardiovascular effects of the 5-HT1A receptor agonists, 8-OH-DPAT (8-hydroxy-2-(di-n-propylamino)tetralin), flesinoxan and 5-carboxamidotryptamine (5-CT) following injection into the dorsal raphe nucleus of conscious rats. 8-OH-DPAT (0.5-2.5 micrograms), hypotension, bradycardia and flat body posture. In contrast, injection of 8-OH-DPAT (0.5 microgram) into the median raphe nucleus caused no cardiovascular changes or flat body posture. (-)Pindolol (0.5 microgram dorsal raphe nucleus) had little effect on cardiovascular parameters, but significantly attenuated the cardiovascular effects of 8-OH-DPAT (0.5 microgram dorsal raphe nucleus). N-Methylatropine (1 mg/kg i.v.) antagonised the cardiovascular effects of 8-OH-DPAT (0.5 microgram dorsal raphe nucleus), suggesting these were vagally mediated. Both pretreatments also appeared to reduce 8-OH-DPAT-induced flat body posture. The results suggest that 8-OH-DPAT activates 5-HT1A receptors in the dorsal raphe nucleus to cause hypotension and bradycardia.     

Intrinsic behavioural actions of n-propyl beta-carboline-3-carboxylate

The behavioural effects of n-propyl beta-carboline-3-carboxylate (beta-CCP) were assessed in the social interaction test of anxiety and in the holeboard test of exploratory behaviour. n-Propyl beta-carboline-3-carboxylate (2 mg/kg) significantly reduced the time spent in social interaction without affecting locomotor activity, indicating an anxiogenic action. This reduction was not significantly reversed by chlordiazepoxide (3 mg/kg). In the holeboard, beta-CCP (4 mg/kg) reduced exploratory head-dipping and rearing; neither of these effects was significantly reversed by chlordiazepoxide (5 mg/kg). The actions of beta-CCP in these two tests are similar to those of the structurally-related compound ethyl beta-carboline-3-carboxylate.     

Orexin A excites serotonergic neurons in the dorsal raphe nucleus of the rat

Orexin A (10-300 nM) strongly excited dorsal raphe serotonergic neurons maintained in vitro. The depolarization persisted in the presence of tetrodotoxin (TTX, 0.5 microM) and was associated with an increase in input resistance. These results have relevance in the context of food intake regulation and the disease, narcolepsy.     

Receptor-mediated regulation of serotonin output in the rat dorsal raphe nucleus: effects of risperidone

OBJECTIVES: The present study was undertaken to characterize the regulation of serotonin (5-HT) efflux and neuronal activity in the dorsal raphe nucleus (DRN) as well as to examine the potential ability of the antipsychotic drug risperidone to interfere with these mechanisms. METHODS AND RESULTS: By using microdialysis in freely moving rats, it was found that administration of the alpha2 adrenoceptor antagonist idazoxan (0.25 mg/kg, SC), the 5-HT1B/D receptor antagonist GR 127,935 (1.0 mg/kg, SC) and risperidone (0.6 or 2.0 mg/kg, SC) increased 5-HT output in the DRN. Local DRN perfusion with GR 127,935 or risperidone via reversed dialysis (100 or 10-100 microM, respectively) enhanced 5-HT efflux in this area, whereas idazoxan (10-100 microM) failed to affect this parameter. Both systemic administration and reversed DRN dialysis of the D2/3 and 5-HT2A receptor antagonists raclopride (2.0 mg/kg, SC or 10-100 microM) and MDL 100,907 (1.0 mg/kg, SC or 10-100 microM), respectively, were without effect. Intraraphe dialysis of the 5-HT1B/D receptor agonist CP 135,807 (0.2 microM) decreased the efflux of 5-HT in the DRN, an effect which was antagonized by co-administration of either GR 127,935 or risperidone (10 and 3.3 microM, respectively). By using single-cell recording, it was found that administration of GR 127,935 (50-400 microg/kg, IV) decreased, whereas CP 135,807 (2.5-20 microg/kg, IV) increased firing of 5-HT cells in the DRN. CONCLUSIONS: Our findings suggest a regulatory role of local 5-HT1B/D receptors on 5-HT efflux as well as cell firing in the DRN and indicate that risperidone may interfere with the regulation of 5-HT availability in this area primarily via blockade of 5-HT1D receptors.     

Collateral inhibition of serotonergic neurones in the rat dorsal raphe nucleus: pharmacological evidence.

Previous studies have shown that serotonergic (5-HT) neurones in the dorsal raphe are inhibited following electrical stimulation of the major ascending 5-HT fibre pathway in the ventromedial tegmentum (VMT); this effect appears to be mediated directly through 5-HT axon collaterals. This possibility was tested further in the present study by pharmacological manipulations of the 5-HT system. When rats were pretreated with either para-chlorophenylalanine (P-CPA, which inhibits trypto phan hydroxylase, the rate-limiting enzyme for the 5-HT synthesis) or reserpine (which is believed to block storage of biogenic amines) or both, the inhibitory responses of antidromically identified 5-HT neurones to VMT stimulation were either totally prevented or markedly reduced in the great majority of cells tested. Furthermore, the P-CPA effects were reversed by the injection of 5-hydroxytryptophan (the immediate precursor of 5-HT which bypasses the synthesis step inhibited by P-CPA); low doses of l-dihydroxyphenylalanine (a precursor of catecholamines) were not effective. In some cells after the drug treatment, prolonged periods of suppression of firing of 5-HT cells still occurred following VMT stimulation. In such cells, continuous stimulation of the VMT (at 10 Hz for 3 min). which presumably depleted residual transmitter, diminished the VMT-induced effect. Of some possible 5-HT antagonists tested, only metergoline (1–2 mg/kg, i.v.) was effective in antagonizing the VMT induced depressant effect on 5-HT neurones; however, the development of the effect was extremely delayed. The present results show that the VMT-induced depression of firing of 5-HT neurones depends on the availability of 5-HT. This finding is consistent with the view that the inhibitory responses of 5-HT neurones to VMT stimulation are mediated directly through 5-HT axon collaterals (or dendo-dendritic junctions).     

Blockade by naltrexone of analgesia produced by stimulation of the dorsal raphe nucleus

Naloxone blockade of stimulation-produced analgesia in the rat is partial and variable. In the present study the effectiveness of the long-acting narcotic antagonist naltrexone is examined. Bipolar stainless steel electrodes were implanted in the dorsal raphe nucleus or ventral periaqueductal gray matter of male rats. Analgesia produced by electrical stimulation was tested by the tail flick method before and twenty min following the administration of saline or naltrexone. Saline administered IP failed to alter the analgesic response. Following naltrexone the degree of analgesia was reduced by a mean of 79% for IV injection (3.7 mg/kg) and by means of 26%, 52%, 81% and 83% for IP administration of 0.3, 1.0, 3.0 and 10 mg/kg, respectively. These results confirm the participation of opiate mechanisms in stimulation-produced analgesia, and indicate that, under certain circumstances, only opiate mechanisms are involved.     

Ipsapirone depresses neuronal activity in the dorsal raphe nucleus and the hippocampal formation

Ipsapirone, a putative, novel anxiolytic with a high affinity for 5-HT1A binding sites, suppressed neuronal activity in both the dorsal raphe nucleus and hippocampal formation of urethane-anesthetized rats. In the hippocampus, dentate granule cells matched the responsiveness of dorsal raphe serotonin neurons, the median effective dose for 50% inhibition being 125.0 micrograms/kg in both areas. In contrast, the responses of CA1 pyramidal cells were related directly to baseline firing rates. Slow-firing neurons, for example, were inhibited by 31.3 micrograms/kg and fast-firing cells were unresponsive even up to a dose of 500.0 micrograms/kg. These results indicate a potent effect of ipsapirone on neuronal activity in sites with a high density of 5-HT1A receptors.     

Effects of corticotropin-releasing factor on neuronal activity in the serotonergic dorsal raphe nucleus.

The present study examined the regional localization of corticotropin-releasing factor (CRF)- and 5-hydroxytryptamine (5-HT)-immunoreactive (IR) fibers within the rat dorsal raphe nucleus (DRN) using immunohistochemistry. Additionally, the effects of CRF, administered intracerebroventricularly (0.1-3.0 micrograms) or intraraphe (0.3-30 ng), on discharge rates of putative 5-HT DRN neurons were quantified using in vivo single unit recording in halothane-anesthetized rats. CRF-IR fibers were present at all rostrocaudal levels of the DRN and exhibited a topographical distribution. CRF produced predominantly inhibitory effects on DRN discharge at lower doses and these effects diminished or became excitatory at higher doses. Inhibition of DRN discharge by CRF was attenuated by the nonselective CRF antagonist, DPheCRF12-41 and the CRF-R1-selective antagonist, antalarmin, implicating the CRF-R1 receptor subtype in these electrophysiological effects. The present findings provide anatomical and physiological evidence for an impact of CRF on the DRN-5HT system.     

Behavioural and microdialysis study after neurotoxic lesion of the dorsal raphe nucleus in rats

The study investigated the effects of a 5,7-dihydroxytryptamine (5,7-DHT) lesion of the dorsal raphe nucleus (DRN) on anxiety-related behaviour and neurochemical correlates in rats. Behaviour was assessed in the elevated plus maze test (X-maze). Lesion of the DRN reduced markedly 5-HT levels in projection areas by at least 60%. Destruction of the serotonergic neurons in the DRN changed neither anxiety-related behaviour on the elevated plus maze, nor aversion-induced 5-HT release in the brain. Exposure of the lesioned rats to the elevated plus maze increased extracellular 5-HT (148%) in the ventral hippocampus similar as in sham-lesioned (162%) and non-lesioned (160%) controls. The results demonstrate that lesioning of 5-HT neurons in the DRN does not abolish totally the control of anxiety-related behaviour.     

Effects of general anaesthetics on 5-HT neuronal activity in the dorsal raphe nucleus

The ascending 5-HT system has been and continues to be the subject of much research. The majority of in vivo electrophysiological and neurochemical studies of 5-HT function in rodents have been conducted in animals under anaesthesia – usually chloral hydrate or urethane. However, the effects of anaesthetics, on 5-HT function have not been systematically investigated. Here we used in vitro electrophysiology in dorsal raphe slices, to determine the effects of anaesthetically relevant concentrations of chloral hydrate (100 μM and 1 mM), urethane (10 and 30 mM), pentobarbitone (10 and 100 μM) and ketamine (10, 100 and 300 μM) on regulators of 5-HT firing activity. We examined i) basal firing (driven by α₁ adrenoceptors), ii) the excitatory response to N-methyl-d-aspartate (NMDA), iii) the 5-HT_1A autoreceptor-mediated inhibitory response to 5-HT and iv) the GABA_A receptor-mediated inhibitory response to 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridinyl-3-ol (THIP, gaboxadol).Pentobarbitone selectively enhanced the response to THIP. Ketamine decreased basal firing, attenuated the response to NMDA, and enhanced responses to both 5-HT and THIP. Chloral hydrate had marginal effects on basal firing, slightly attenuated the NMDA response, and enhanced both the 5-HT and THIP responses. Urethane increased basal firing, decreased the NMDA response, increased the response to THIP, but had no effect on the 5-HT response. Our data indicate that all anaesthetics tested significantly affect the regulators of 5-HT neuronal function. These findings will aid in the interpretation of previous reports of in vivo studies of the 5-HT system and will allow researchers to make a rational selection of anaesthetic for future studies.