State-dependent effects of orexins on the serotonergic dorsal raphe neurons in the rat

The serotonergic dorsal raphe (DR) neurons play an important role in sleep-wakefulness regulation. Orexinergic neurons in the lateral hypothalamus densely project to the brainstem sites including the DR. To test the effects of orexins on the serotonergic DR neurons, we applied orexin A (0.1 mM) by pressure to these neurons in unanesthetized and urethane anesthetized rats. Orexin A caused excitation in 10 of 15 neurons under unanesthetized condition. The excitation was characterized by slow onset (0-18 s), long lasting duration (15-150 s) and state-dependency. Orexin A applied during REM sleep or slow wave sleep induced significant excitation while during wakefulness, the similar amount of orexin A did not increase the firing rate any more. In the anesthetized animals, orexin A induced excitation in four of eight neurons. The excitation had slow onset and was long lasting. These results suggest that orexinergic neurons exert excitatory influence on the serotonergic DR neurons to maintain tonic activity of them, thereby participating in regulation of sleep-wakefulness cycles and other functions.     

Both indoleamine and phenylethylamine hallucinogens increase serotonin in both dorsal and median raphe neurons

Administration of either indoleamine or phenylethylamine hallucinogens selectively increased the level of serotonin within cell bodies of both the dorsal and median raphe nuclei. Changes in intracellular serotonin levels were quantitated by a new cytofluorimetric technique which relies on a measure of fluorescence fading to detect changes in serotonin and distinguish them from changes in catecholamines.     

Disruption of model-based decision making by silencing of serotonin neurons in the dorsal raphe nucleus

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Single-cell activity and network properties of dorsal raphe nucleus serotonin neurons during emotionally salient behaviors

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Synaptic relations of neurotensinergic neurons in the dorsal raphe nucleus

The ultrastructure and synaptic relations of neurotensinergic neurons in the rat dorsal raphe nucleus (DRN) were examined. The neurotensin-like immunoreactive (NT-LI) neurons in the DRN were fusiform or spherical. The NT-LI perikarya could only be detected in colchicine-treated animals whereas the immunoreactive axon terminals could only be found in the anirnals not treated with colchicine. Although many NT-LI dendrites received synapses from nonimmunoreactive axon terminals, the NT-LI perikarya received few synapses. NT-LI axon terminals also made synapses on nonimmunoreactive dendrites. Occasionally, synapses were found between the NT-LI axon terminals and NT-LI dendrites in the cases in which the animals were not treated with colchicine.     

Dopamine and serotonin effects on neurons of dorsal root ganglion isolated from rats

The effects of 1×10^–8–1×10^–5 M dopamine (DA) and serotonin (HT) on membrane potential, input resistance (R_M), and action potential (AP) when added to the superfusing fluid for 0.5 min were investigated in perfused dorsal root ganglia (DRG) neurons isolated from 30–36-day old rats during experiments using intracellular recording techniques. Application of DA induced reversible changes in membrane potential in 48 out of 52 test cells as compared with 38 out of 44 for HT. Distribution of different patterns of response to DA and HT was similar: depolarization was recorded in 64.6 and 73.7% and hyperpolarization in 16.7 and 15.8%; two-stage response occurred in 18.7 and 10.5% of responding cells, respectively. Both monoamines induced reversible change in the AP and R_M pattern in a number of cells. Depolarization was accompanied by a decline and hyperpolarization by a rise in R_M. Both substances were found to affect mainly those neurons with electrophysiological properties characteristic of small cells. The possibility of afferent spike train modulation at the level of primary sensory neurons is suggested.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian USSR, Kiev. Translated from Neirofiziologiya, Vol. 21, No. 5, pp. 644–651, September–October, 1989.     

Ghrelin postsynaptically depolarizes dorsal raphe neurons in rats in vitro

Ghrelin promotes growth hormone (GH) secretion and feeding. Recent studies further showed that ghrelin displayed a defending effect against the depressive-like symptoms and affected sleep in animals and humans. Serotonergic system is considered to be implicated in feeding, depression and other mood disorders, and sleep. The dorsal raphe nucleus (DRN) utilizes serotonin (5-HT) as its major neurotransmitter and expresses GH secretagogue receptors (GHS-Rs). Therefore, the present study was carried out to examine the electrophysiological effect of ghrelin on rat DRN neurons in vitro and determine the ionic mechanism involved. Whole-cell recording revealed that ghrelin depolarized DRN neurons dose-dependently in tetrodotoxin-containing artificial cerebrospinal fluid (TTX ACSF). Pretreatment with [d-Lys³]-GHRP-6, a selective antagonist for GHS-Rs, antagonized the ghrelin-induced depolarization. The depolarization was significantly reduced in a low-Na⁺ TTX ACSF and in a high-K⁺ TTX ACSF and was abolished in the combination of both ACSFs, suggesting that the ghrelin-induced depolarization is mediated by a dual ionic mechanism including an increase in nonselective cationic conductance and a decrease in K⁺ conductance. The experiments on the reversal potential also supported an involvement of the dual ionic mechanism in the ghrelin-induced depolarization. On the basis of their electrophysiological and pharmacological properties, approximately 80% of DRN neurons were classified as putative 5-HT-containing neurons and ghrelin depolarized 75% of them. These results suggest that DRN neurons, especially 5-HT-containing neurons, might be involved in the neural mechanisms through which ghrelin participates in the development and/or regulation of feeding behavior, sleep-wake states and depressive-like symptoms.     

Role of norepinephrine in regulating the activity of serotonin-containing dorsal raphe neurons

Previous studies have yielded conflicting results concerning the role of noradrenergic afferents to the dorsal raphe nucleus in regulating the activity of serotonergic neurons. In the present study, we recorded the activity of serotonin-containing dorsal raphe neurons in mouse brain slices $\text{in vitro}$ under the following conditions: (a) no treatment, (b) phenylephrine added to the incubation medium, (c) in tissue obtained from mice that were anesthetized with halothane, (d) same condition as c, with phenylephrine added to the incubation medium, and (e) same as condition c, with the addition of bicuculline to the incubation medium. The data revealed that the neurons recorded with no treatment exhibited a spontaneous discharge rate of 3.40 ± 0.29 spikes/sec and a cell/tract ratio of 1.15, while cells recorded from tissue slices obtained from halothane anesthetized mice exhibited a discharge rate of 2.01 ± 0.27 spikes/sec and a cell/track ratio of 0.58. Addition of phenylephrine to the incubation media in slices obtained from anesthetized mice increased both the discharge rate (4.23 ± 0.30 spikes/sec) and cell/tract ratio (1.28). Similarly, addition of bicuculline to the incubation media increased both the discharge rate (4.09 ± 0.46 spikes/sec) and cell/tract ratio (1.21) in mouse brain slices obtained from anesthetized animals. Thus, we conclude that a noradrenergic input (which is removed in the tissue slice preparation) is not necessary to maintain the spontaneous activity of serotonergic dorsal raphe units. Halothane anesthesia depressed the activity of these neurons, presumably by releasing GABA from interneurons. Finally, while dorsal raphe neurons are not dependent upon an excitatory noradrenergic input to maintain their spontaneous activity, these neurons can be excited by noradrenergic a fferents under certain conditions.     

Altered serotonin and norepinephrine metabolism in rat dorsal raphe nucleus after drug-induced hypertension

The effect of drug-induced hypertension on neurotransmitter release from dorsal raphe nucleus was studied by in vivo electrochemical electrodes in urethane anesthetized male Sprague-Dawley rats. Carbon paste electrodes were stereotaxically placed into dorsal raphe nucleus and neurotransmitter release was estimated electrochemically. Blood pressure was recorded from a femoral arterial catheter. Voltammograms taken from dorsal raphe nucleus showed two distinct peaks corresponding to norepinephrine and 5-hydroxyindole acetic acid (5-HIAA). After basal blood pressure and neurotransmitter release were monitored for 30 min, blood pressure was raised 50 mmHg by continuous intravenous infusion of L-phenylephrine hydrochloride. Drug infusion was discontinued after 50 min, but blood pressure and neurotransmitter release were measured for an additional 2 hr. Results showed that the 5-HIAA response increased immediately after the initiation of hypertension and remained elevated. By contrast, norepinephrine release initially decreased, then returned to the basal level and then rose in parallel with 5-HIAA to a level above baseline as drug-induced hypertension was discontinued. The same experimental protocol was used to study the electrochemical response to drug-induced hypotension. Blood pressure was lowered 20 mmHg by intravenous infusion of sodium nitroprusside dihydrate. During hypotension, no changes were seen in either transmitter response. However, as reflex hypertension appeared following discontinuation of the sodium nitroprusside infusion, the 5-HIAA response increased and the norepinephrine response decreased. These results show that drug-induced and reflex hypertension reduce norepinephrine release and increase serotonin turnover in dorsal raphe nucleus in anesthetized normotensive rats. These reciprocal changes appear to be a part of the neural response to hypertension.     

Modulation of electrically evoked serotonin release in cultured rat raphe neurons

Electrically evoked release of serotonin (5-HT) and its modulation via 5-HT autoreceptors and alpha(2)-heteroreceptors was studied in primary cell cultures prepared from the embryonic (ED 15) rat mesencephalic brain region comprising the raphe nuclei. Cultures were grown for up to 3 weeks on circular glass coverslips. They developed a dense network of non-neuronal and neuronal cells, some of which were positive for tryptophan hydroxylase. To measure 5-HT release, the cultures were pre-incubated with [(3)H]5-HT (in the presence of the selective noradrenaline reuptake inhibitor oxaprotiline [1 micromol/L]), superfused with modified Krebs-Henseleit medium containing 6-nitroqipazine [1 micromol/L] and electrically stimulated using two conditions. Condition A: 360 pulses, 3 Hz, 0.5 ms, 90 mA, or condition B: 4 pulses 100 Hz, 0.5 ms, 90 mA (a condition which diminishes interactions with endogenously released transmitters during ongoing stimulation). After only 1 week in culture, the electrically evoked overflow of [(3)H] was Ca(2+) dependent and tetrodotoxin sensitive, suggesting an action-potential-induced exocytotic release of 5-HT. Using stimulation condition A in cultures grown for 2 weeks, both basal and evoked 5-HT release were strongly enhanced by methiotepine (1 micromol/L) but unaffected by the 5-HT(1B) autoreceptor agonist CP-93, 129 (1 micromol/L) and the alpha(2)-adrenoceptor agonist UK-14, 304 (1 micromol/L). Conversely, using stimulation condition B, not only CP-93, 129 (IC(50) 8.1 +/- 1.4 nmol/L) and UK-14, 304 (IC(50) 14.9 +/- 1.6 nmol/L) had inhibitory effects on cells grown for 2 weeks, but also the 5-HT(1A) agonist 8-hydroxy-2-(di-n-propylamino)tetralin. In conclusion, we describe for the first time electrically evoked release of 5-HT from primary cultures of fetal raphe cells and its modulation via 5-HT(1B) and 5-HT(1A) auto- and alpha(2)-heteroreceptors. Such cultured raphe cells may represent a suitable model to study expression and development of presynaptic receptors on serotonergic neurons in-vitro.     

Inter- and intracellular relationship of substance P-containing neurons with serotonin and GABA in the dorsal raphe nucleus: combination of autoradiographic and immunocytochemical techniques

Double-labeling experiments were performed at the electron microscopic level in the dorsal raphe nucleus of rat, in order to study the inter- and intracellular relationship of substance P with gamma-aminobutyric acid (GABA) and serotonin. Autoradiography for either [3H]serotonin or [3H]GABA was coupled, on the same tissue section, with peroxidase-antiperoxidase immunocytochemistry for substance P in colchicine-treated animals. Intercellular relationships were represented by synaptic contacts made by [3H]serotonin-labeled terminals on substance P-containing somata and dendrites, and by substance P-containing terminals on [3H]GABA-labeled cells. Intracellular relationships were suggested by the occurrence of the peptide within [3H]serotonin-containing and [3H]GABA-containing cell bodies and fibers. Doubly labeled varicosities of the two kinds were also observed in the supraependymal plexus adjacent to the dorsal raphe nucleus. The results demonstrated that, in addition to reciprocal synaptic interactions made by substance P with serotonin and GABA, the dorsal raphe nucleus is the site of intracellular relationships between the peptide and either the amine or the amino acid.     

中缝背核5-羟色胺能神经元在睡眠调节中的作用研究

目的：研究中缝背核(DRN)5-羟色胺(5-HT)能神经元在睡眠中的调节作用。方法：运用脑立体定位、核团微量注射和多导睡眠描记(PSG),观察DRN 5-HT能神经元对大鼠睡眠的影响。结果：DRN微量注射谷氨酸钠(L-Glu),大鼠睡眠减少,特别是深慢波睡眠(SWS2)明显减少,觉醒(W)增加;DRN微量注射海人酸(KA)和对氯苯丙氨酸(PCPA),大鼠SWS2和异相睡眠(PS)增加,W减少。结论：DRN 5-HT能神经元参与睡眠的调节,兴奋DRN 5-HT能神经元睡眠时间减少,抑制DRN 5-HT能神经元则具有促进睡眠的作用。     

Chloral hydrate anesthesia alters the responsiveness of dorsal raphe neurons to psychoactive drugs

The effects of several psychoactive drugs on raphe unit activity in freely moving cats was compared with drug-induced effects in chloral hydrate anesthetized cats. The anesthesia greatly potentiated the depressant effects of LSD, phenoxybenzamine, clonidine, methiothepin, clozapine, and chlorimipramine on raphe units, but partially antagonized the depressant effects of diazepam. These results demonstrate that apparently discrepant reports of the affects of these drugs on raphe neurons in anesthetized rats versus freely moving cats are attributable to the use of anesthesia in rat studies. These data underscore the importance of conducting such drug studies in awake, freely moving animals, for which the results would be far more relevant to the issue of human drug use.     

Spontaneous unit activity of neurons within the dorsal raphe nucleus of the neonatal rat

Extracellular recordings were made of spontaneously active neurons in the dorsal raphe nucleus (DRN) of neonatal rats. The firing pattern and rate of these neurons were similar to those characterized for 5-HT-containing cells in the DRN of adult rats. Neonatal DRN cells were also inhibiteby small systematic doses of LSD, as previously described for 5-HT-containing DRN neurons in adult rats. These results indicate that DRN neurons in neonatal rats are physiologically active and display many characteristics similar to mature 5-HT-containing DRN neurons.     

Biophysical properties and computational modeling of calcium spikes in serotonergic neurons of the dorsal raphe nucleus

Serotonergic neurons of the dorsal raphe nuclei, with their extensive innervation of nearly the whole brain have important modulatory effects on many cognitive and physiological processes. They play important roles in clinical depression and other psychiatric disorders. In order to quantify the effects of serotonergic transmission on target cells it is desirable to construct computational models and to this end these it is necessary to have details of the biophysical and spike properties of the serotonergic neurons. Here several basic properties are reviewed with data from several studies since the 1960s to the present. The quantities included are input resistance, resting membrane potential, membrane time constant, firing rate, spike duration, spike and afterhyperpolarization (AHP) amplitude, spike threshold, cell capacitance, soma and somadendritic areas. The action potentials of these cells are normally triggered by a combination of sodium and calcium currents which may result in autonomous pacemaker activity. We here analyse the mechanisms of high-threshold calcium spikes which have been demonstrated in these cells the presence of TTX (tetrodotoxin). The parameters for calcium dynamics required to give calcium spikes are quite different from those for regular spiking which suggests the involvement of restricted parts of the soma-dendritic surface as has been found, for example, in hippocampal neurons.     

The orexinergic synaptic innervation of serotonin- and orexin 1-receptor-containing neurons in the dorsal raphe nucleus

Orexin/hypocretin has been well demonstrated to excite the serotonergic neurons in the dorsal raphe nucleus (DRN). We studied the morphological relationships between orexin-containing axon terminals and serotonin- as well as orexin-receptor-containing neurons in the dorsal raphe nucleus. Using immunohistochemical techniques at the light microscopic level, orexin A (OXA)-like immunoreactive neuronal fibers in the DRN were found to make close contact with serotonergic neurons, while some of the serotonergic neurons also expressed the orexin 1 receptor (OX1R). At the electron microscopic level, double-immunostaining experiments showed that the orexin A-like immunoreactive fibers were present mostly as axon terminals that made synapses on the serotonin- and orexin 1-receptor-containing neurons. While only axodendritic synapses between orexin A-containing axon terminals and serotonergic neurons were detected, the synapses made by orexin A-containing axon terminals on the orexin 1-receptor-containing neurons were both axodendritic and axosomatic. The present study suggests that excitation effect of orexin A on dorsal raphe serotonergic neurons is via synaptic communication through orexin 1 receptor.