Orexin A and B (OXA/B, also known as hypocretin 1 and 2, respectively) identified in the rat hypothalamus are endogenous neuropeptide agonists for the G-protein coupled orexin-1 (OX1) and orexin-2 (OX2) receptor.1 Orexins (OXs) and their receptors are widely distributed in the brain and spinal cord including the brainstem. Eighty percent of extracellular norepinephrine (NE) in the prefrontal cortex (PFC) is derived from the locus coeruleus (LC),2 which regulates the sleep-wake cycle and is densely innervated with orexinergic neurons.3–5 We have previously reported that OXs selectively evoked NE release from rat cerebrocortical slices, mainly via OX1 receptors, as release was sensitive to SB-334867-A, an OX1 receptor antagonist.6 Similarly, Soffin et al.7 reported that SB-334867-A antagonizes LC activation induced by OXs. These data strongly suggest that orexinergic neurons may interact with noradrenergic neurons in the brain.
Previous studies indicate that brain noradrenergic neuronal activity is also related to the mechanism of general anesthesia. It has been suggested that noradrenergic neurons of the LC may be involved in the production of the anesthetic state.8–11 The N-methyl-d-aspartate (NMDA) receptor antagonist, ketamine, markedly increased NE release in the PFC to induce anesthesia in the rat.12 Physostigmine significantly reduced ketamine anesthesia time and ketamine-evoked NE release in rat PFC.13 These data suggest that NE neuronal activity could involve ketamine anesthesia. Moreover, it is possible that there is some interaction between OX and NMDA receptors in other brain regions. OXA and B decreased the amplitude of NMDA currents in isolated neurons of the nucleus accumbens.14 Application of OXA caused an increase in masseter muscle tone when applied to the V motor nucleus, a response that was blocked by pretreatment with a NMDA receptor antagonist.15 We therefore hypothesized that orexinergic neurons may play an important role in ketamine anesthesia via an interaction with noradrenergic neurons. To test this hypothesis, we studied 1) in vivo effects of OXA and SB-334867-A on ketamine-induced anesthesia time, 2) in vivo effects of OXA on ketamine-induced increase in NE release from the PFC assessed using microdialysis, and 3) in vitro effects of ketamine on OXA-evoked NE release from rat cerebrocortical slices.
With the approval of the institutional committee on animal research of the University of Hirosaki School of Medicine, 57 male Sprague Dawley rats weighing 340–400 g (for in vivo experiments) and another six male Sprague Dawley rats weighing 300–350 g (for in vitro experiments) were used. All animals were housed for at least 1 wk before experimentation and were maintained in a 12-h light/dark cycle environment (lights on 8:00–20:00 h) at a temperature of 24°C–26°C and 40% humidity. Animals had access to food and water ad libitum except on the day of the experiment.
OXA was purchased from Peptide Institute (Osaka, Japan) and dissolved by artificial cerebrospinal fluid before use. Ketamine hydrochloride was from Sankyo Pharmaceutical Co. (Tokyo, Japan). Pentobarbital sodium was from Abbott Pharmaceutical Co. (Tokyo, Japan). Pyrogen-free saline was from Otsuka Pharmaceutical Co. (Tokyo, Japan). Pargyline and nomifensin were from Sigma Aldrich (Tokyo, Japan) and HEPES from Dojin Laboratories (Kumamoto, Japan). All other chemicals used were of the highest purity available. SB-334867-A was a gift from SmithKline Beecham Pharmaceuticals (Harlow, UK).
In Vivo Experiments
We performed in vivo experiments, an anesthesia time study and a microdialysis study, based on established methods reported previously.16
Intracerebroventricular catheterization and microdialysis for measurement of NE release in the PFC.
Rats were surgically implanted with a lateral intracerebroventricular (ICV) cannula and microdialysis probe under pentobarbital (50 mg/kg) anesthesia. On the day before the experiment, a microdialysis probe (A-I-4-03, Eicom, Kyoto, Japan) was inserted into the guide cannula. The tip of the probe extended 3.0 mm beyond the tip of the guide to reach the medial PFC.17 Twenty-four hours after microdialysis probe insertion, rats were placed in a custom-built plastic box in which they could move freely (Free Moving Unit CMA/125, BAS, Tokyo, Japan). To measure NE release in the PFC, the probe was perfused at a flow rate of 1.3 μL/min with artificial cerebrospinal fluid. Dialysis samples were collected at 10-min intervals.
Effects of OXA on ketamine-induced anesthesia time.
We performed a crossover study with an interval of 5 days on the same rats. Rats were given intraperitoneal (IP) ketamine with ICV saline (4 μL) or ICV OXA 1 nmol (4 μL), and 5 days later, the same rats received IP ketamine with ICV OXA 1 nmol or ICV saline, respectively. Four doses of the ketamine were used: 50 mg/kg (n = 7), 100 mg/kg (n = 9), 125 mg/kg (n = 8), and 150 mg/kg (n = 9). Anesthesia time was defined as the duration from the loss of the righting reflex to recovery of the ability to perform three successive rightings. In addition, another experiment using nine rats was performed to determine whether SB-334867-A (OX1 receptor antagonist, 10 nmol ICV) could reverse the effects of OXA (1 nmol ICV) on ketamine (100 mg/kg IP)-induced anesthesia time.
In vivo effects of ketamine on OXA-evoked NE release.
Rats were subdivided into three groups: ICV OXA 1 nmol (4 μL) and IP saline 10 mL/kg (Group OX, n = 5), simultaneous ICV saline (4 μL) and IP ketamine 100 mg/kg Group K, n = 5), and ICV OXA 1 nmol (4 μL) and IP ketamine 100 mg/kg (Group OX + K, n = 5). After obtaining five consecutive stable NE samples, agents were administered and then 18 additional samples were obtained over 180 min for each rat. NE mass in the in vivo release samples (10 μL) was determined directly by high-performance liquid chromatography with electrochemical detection, as described previously.16
In Vitro Experiments
We performed invitro experiments based on the established methods reported previously.6,18
Six rats were decapitated, and the brains were quickly removed and immersed in ice-cold Krebs–Ringer bicarbonate buffer solution (KRBS) oxygenated with 95% O2 and 5% CO2 of the following composition: NaCl 133, KCl 4.8, KH2PO4 1.2, MgSO4 1.2, CaCl2 1.5, glucose 11.1, HEPES 10 (in mM, pH 7.4). Cerebrocortical tissue was dissected from its internal structures and crosschopped using a tissue chopper to produce slices (350 × 350 μm). The slices were then washed three times in ice-cold KRBS and transferred (1-mL aliquots of slices: equivalent to about 7 mg tissue) to polypropylene tubes. Cerebrocortical slices from one rat were used for one experiment (i.e., one concentration-response curve for an agent was constructed).
In vitro effects of ketamine on OXA-evoked NE release from rat cerebrocortical slices.
After discarding the supernatant, the slices were resuspended in 1 mL of fresh KRBS and incubated for 10 min at 37°C. This procedure was repeated again to obtain stable basal release. Immediately after this incubation, the slices were resuspended and incubated with ketamine (10−9 to 10−4 M, n = 6) for 10 min in the absence (basal release) and presence of 10−7 M OXA (evoked release). All buffers used in release studies contained pargyline (10 μM), and the reuptake inhibitor, nomifensin (10 μM). NE mass in the in vitro release samples (20 μL) was determined directly by high-performance liquid chromatography with electrochemical detection as described previously.18
All data are presented as mean ± sem. Anesthesia time data per dose were compared with corresponding control groups using Student’s paired t-test. The area under the curve of the NE concentration time course from 10 to 180 min after agent injection was analyzed by repeated measures analysis of variance followed by Scheffé tests (Stat View version 5.0 SAS Institute, Cary, NC). Concentrations of agents producing 50% (I/EC50) of the maximal inhibition/stimulation (I/Emax) in vitro were estimated by nonlinear regression analysis (Graphpad-Prism, version 1.0). P < 0.05 was considered significant.
Effects of OXA on Ketamine-Induced Anesthesia Time
OXA significantly decreased ketamine-induced anesthesia time at 50, 100, and 125 mg/kg doses of ketamine (Fig. 1A). SB-334867-A (10 nmol ICV) fully reversed this reduction (Fig. 1B).
In Vivo Effects of Ketamine on OXA-Evoked NE Release
Basal NE release was stable and similar among groups (Fig. 2). In each group, NE content significantly increased after OXA ICV, reaching a maximum of 204% of baseline at 60 min, after ketamine IP a maximum of 390% and after OX and ketamine with 271% of baseline at 70 min, respectively. Maximum release in Group OX and Group OX + K were significantly smaller than that in Group K (K versus OX + K, P = 0.029; K versus OX, P = 0.015; OX + K versus OX, P = 0.254). When considering the area under the curve from 10 to 180 min in Group OX + K and Group OX, they were significantly smaller than that in Group K (K versus OX + K, P = 0.039; K versus OX, P = 0.024; OX + K versus OX, P = 0.30, Fig. 2).
In Vitro Effects of Ketamine on OXA-Evoked NE Release from Rat Cerebrocortical Slices
Ketamine inhibited OXA-evoked NE release in a concentration-dependent manner (Fig. 3) with pIC50 (IC50) and Imax of 4.75 ± 0.07 (17.8 μM) and 80.6% ± 2.6%, respectively. Ketamine did not affect basal NE release (data not shown).
In this study, we have shown that 1) OXA decreased ketamine-induced anesthesia time, 2) OXA decreased ketamine-evoked NE release in the rat PFC, and 3) ketamine inhibited OX-evoked NE release from rat cerebrocortical slices.
Our in vivo studies used the well-characterized measure of anesthesia time: the loss of righting reflex. This is a simple and sensitive whole animal assay capable of determining both time to produce loss of righting response (a correlate of induction time) and also total anesthesia time. We have shown that OXA decreased ketamine-induced anesthesia time, and the SB-334867-A fully reversed this effect. This model has been used extensively to examine the effects of a range of anesthetics and sedatives. Indeed, we have used this model to study the effect of α-2 adrenergic agents on propofol-induced anesthesia time where clonidine increased anesthesia time after IP administration.16
Using in vivo microdialysis, NE release evoked from the rat PFC by simultaneous administration of OXA and ketamine was significantly lower than by ketamine alone. It has been suggested8–11 that noradrenergic neurons of the LC may be involved in the production of the anesthetic state. Several reports have demonstrated that OXs activate the LC noradrenergic system and that this activation may increase arousal and locomotor activity.4,19 Icv OXA significantly increased NE release in the PFC. These data agree with our previous invitro study demonstrating that OXs selectively evoked NE release from rat cerebrocortical slices.6 The OX1 receptor is strongly expressed in the LC.20
Ketamine inhibited OXA-evoked NE release in a concentration-dependent manner, but it did not affect basal NE release. We previously found that NE release from rat cerebrocortical slices was almost fully antagonized by SB-334867-A, an OX1 receptor antagonist6; therefore, OX increased NE release with OX1 receptor in rat cerebrocortical slices. The present in vitro study showed that the IC50 of ketamine for inhibition of OXA-evoked NE release from cerebrocortical slices was 17.8 μM, which is within the clinically relevant concentration range seen during general anesthesia (about 20 μM).21 Victor and Hugh22 also showed that ketamine did not affect NE release from isolated rat cortical nerve terminals. It should be noted that in these preparations (cerebrocortical slices) the connection between important groups of neurons (e.g., LC-cortex), and hence a degree of complexity, will be lost. Ketamine interacts with many receptors: opioid, monoamines, and muscarinic in addition to NMDA.23 Ketamine might activate LC-noradrenergic neurons and increased NE release in rat PFC. OX and their receptors are widely distributed in the brain, and therefore, it is possible that ketamine directly affected the OX receptor in rat cerebrocortical slices and inhibited NE release.
Our previous study showed that 1 nmol and 5 nmol OXA significantly decreased pentobarbital, thiopental, and phenobarbital anesthesia times.18 Pentobarbital decreases NE release from PFC in conscious rats.12 These findings suggest that the increase in activity of noradrenergic neurons by exogenous OX counterbalanced the decrease by pentobarbital anesthesia. We previously demonstrated that wakefulness may be maintained in some range of brain activity and that sleep or anesthesia may occur if brain conditions were out of that range, i.e., suppression or overexcitation of brain activity.23 For example, physostigmine, which antagonize ketamine anesthesia, decreased ketamine anesthesia time while suppressing ketamine-induced NE release in rat PFC.13 If there is no interaction between orexinergic neurons and NMDA receptors, NE release should be additive. We think that OXA adjusts noradrenergic neurons in LC modulated by anesthetics and produces a state of wakefulness. As a result, OXA decreased NE effects by anesthetics. In the current study, ketamine increased NE release in rat PFC, and OXA attenuated this effect in LC. Of course, there is now good agreement that no single mechanism of anesthetic action can account for the anesthetic state. There are several neurochemical modulatory sites for arousal states, of which OXA is an example. OXA increases cortical acetylcholine release and electroencephalographic activation during isoflurane anesthesia.24 However, even if acetylcholine were interactive, this does not explain how OX could suppress ketamine-induced NE release in rat PFC.
The sedative actions of α2-agonist dexmedetomidine are diminished by lesions in the hypothalamic regions contributing to nonrapid-eye movement sleep, consistent with NE being relevant for both sleep and anesthesia.25 Thus, we hypothesized a significant interaction between α2-adrenoceptors and OX receptors in the cerebrocortex. However, as α2-ligand and OX receptor ligand-induced actions were not mutually antagonistic, there may be no interaction between these systems/neuronal groups in the cerebral cortex.26 Therefore, this suggests that the NE changes at the PFC induced by OXA administration were not because of α2-adrenoceptors.
Our study has several limitations. First, we did not measure NE release in LC. Therefore, we cannot determine whether NE change in PFC originated from LC. Second, we could not exclude the possibility that OXA affected other sites and decreased ketamine anesthesia time and NE release in rat PFC. Because we used freely moving rats in vivo experiments, it was difficult for us to directly inject OXA to the LC.
In conclusion, the present data consistently indicate that there is a significant interaction between orexinergic neurons and ketamine anesthesia in the rat. OXA reduced ketamine anesthesia time, but it should also be remembered that OXA, per se, produces “wakefulness,” possibly indicating that OXA interacted with ketamine to reverse anesthesia time rather than merely increasing arousal. Further studies will be required to elucidate the physiological relevance of this interaction.
1.Sakurai T, Amemiya A, Ishii M, Matsuzaki I, Chemelli RM, Tanaka H, Williams SC, Richarson JA, Kozlowski GP, Wilson S, Arch JR, Buckingham RE, Haynes AC, Carr SA, Annan RS, McNulty DE, Liu WS, Terrett JA, Elshourbagy NA, Bergsma DJ, Yanagisawa M. Orexins and orexin receptors: a family of hypothalamic neuropeptides and G protein-coupled receptors that regulate feeding behavior. Cell 1998;92:573–85
2.Van Gaalen M, Kawahara H, Kawahara Y, Westerink BH. The locus coeruleus noradrenergic system in the rat brain studied by dual-probe microdialysis. Brain Res 1997;763:56–62
3.Peyron C, Tighe DK, Van den pol AN, De leces L, Heller HC, Sutcliffe JG, Kilduff TS. Neurons containing hypocretin (orexin) project to multiple neuronal systems. J Neurosci 1998;18:9996–10015
4.Hagan JJ. Orexin A activates locus coeruleus cell firing and increases arousal in the rat. Proc Natl Acad Sci USA 1999; 96:10911–16
5.Date Y, Ueta Y, Yamashita H, Yamaguchi H, Matsukura S, Kanagawa K, Sakurai T, Yanagisawa M, Nakazato M. Orexins, orexinergic hypothalamic peptides, interact with autonomic, neuroendocrine and neuroregulatory systems. Proc Natl Acad Sci USA 1996;96:10911–16
6.Hirota K, Kushikata T, Kudo M, Kudo T, Lambert DG, Matsuki A. Orexin A and B evoke noradrenaline release from rat cerebrocortical slices. Br J Pharmacol 2001;134:1461–6
7.Soffin EM, Evans ML, Gill CH, Harries MH, Benham CD, Davies CH. SB-334867-A antagonizes orexin mediated excitation in the locus coeruleus. Neuropharmacology 2002;42:127–33
8.Mason ST, King RA, Banks P, Angel A. Brain noradrenaline and anaesthesia: behavioural and electrophysiological evidence. Neuroscience 1983;10:177–85
9.Mason ST, Angel A. Behavioral evidence that chronic treatment with the antidepressant desipramine causes reduced functioning of brain noradrenaline systems. Psychopharmacology 1983;81:73–7
10.Nacif-Coelho C, Correa-Sales C, Chang LL, Maze M. Perturbation of ion channel conductance alters the hypnotic response to the α2
-adrenergic agonist dexmedetomidine in the locus coeruleus of the rat. Anesthesiology 1994;81:1527–34
11.Chen CL, Yang YR, Chiu TH. Activation of rat locus coeruleus neuron GABAA receptors by propofol and its potentiation by pentobarbital or alphaxalone. Eur J Pharmacol 1999;386:201–10
12.Kubota T, Anzawa N, Hirota K, Yoshida H, Kushikata T, Matsuki A. Effect of ketamine and pentobarbital on noradrenaline release from the medial prefrontal cortex in rats. Can J Anaesth 1999;46:388–92
13.Kubota T, Hirota K, Anzawa N, Yoshida H, Kushikata T, Matsuki A. Physostigmine antagonizes ketamine-induced noradrenaline release from the medial prefrontal cortex in rats. Brain Res 1999;840:175–8
14.Gilles M, Veronique F, George R. Luis de Lecea. Interaction of the hypocretins with neurotransmitters in the nucleus accumbens. Regul Pept 2004;104:111–7
15.Peever J, Yuan-Yang L, Jerome MS. Excitatory effects of hypocretin-1 (orexin A) in the trigeminal motor nucleus are reversed by NMDA antagonist. J Neurophysiol 2003;89:2591–600
16.Kushikata T, Hirota K, Yoshida H, Kubota T, Ishihara H, Matsuki A. Alpha-2 adrenoceptor activity affects propofol-induced sleep time. Anesth Analg 2002;94:1201–6
17.Paxinos G, Watson C. The rat brain in stereotoxic coordinates. 2nd ed. San Diego, CA: Academic Press, 1986
18.Kushikata T, Hirota K, Yoshida H, Kudo M, Lambert DG, Smart D, Jerman JC, Matsuki A. Orexinergic neurons and barbiturate anesthesia. Neuroscience 2003;121:855–63
19.Horvath TL, Peyron C, Diano S, Ivanov A, Aston N, Jones G, Kilduff TS, van den Pol AN. Hypocretin (orexin) activation and synaptic innervation of locus coeruleus noradrenergic system. J Comp Neurol 1999;415:145–59
20.Hervieu GJ, Cluderay JE, Harrison DC, Roberts JC, Leslie RA. Gene expression and protein distribution of the orexin-1 receptor in the rat brain and spinal cord. Neuroscience 2001; 103:777–97
21.White PF, Ham J, Way WL, Trevor AJ. Pharmacology of ketamine isomers in surgical patients. Anesthesiology 1980; 52:231–9
22.Victor NP, Hugh CH. The effects of general anesthetics on norepinephrine release from isolated rat cortical nerve terminals. Anesth Analg 2002;95:1274–81
23.Hirota K, Kushikata T. Central noradrenergic neurons and the mechanism of general anaesthesia. Br J Anaesth 2001;87:811–12
24.Dong HL, Fukuda S, Murata E, Zhu Z, Higuchi T. Orexins increase cortical acetylcholine release and electroencephalographic activation through orexin-1 receptor in the rat basal forebrain during isoflurane anesthesia. Anesthesiology 2006;104:1023–32
25.Nelson LE, Lu J, Guo T, Saper CB, Franks NP, Maze MB. The alpha2-adrenoreceptor agonist dexmedetomidine converges on an endogenous sleep-promoting pathway to exert its sedative effects. Anesthesiology 2003;98:428–36
26.Hirota K, Kudo M, Tose R, Yoshida H, Kudo T, Kushikata T. Lack of interaction between orexinergic and alpha2-adrenergic neuronal systems in rat cerebrocortical slices. Neurosci Lett 2005;387:49–52