Anesth Analg 2007;104:338-340
© 2007 International Anesthesia Research Society
doi: 10.1213/01.ane.0000252413.62821.2e
ANESTHETIC PHARMACOLOGY
The Effects of Benzodiazepines on Orexinergic Systems in Rat Cerebrocortical Slices
Ying He, MD*
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Mihoko Kudo, PhD*,
Tsuyoshi Kudo, PhD*,
Tetsuya Kushikata, MD*,
Enyou Li, MD, and
Kazuyoshi Hirota, MD, FRCA*
From the *Department of Anesthesiology, University of Hirosaki School of Medicine, Hirosaki, Japan; and Department of Anesthesiology, First Clinical College of Harbin Medical University, Harbin, China.
Address correspondence and reprint requests to K. Hirota, Department of Anesthesiology, University of Hirosaki School of Medicine, Hirosaki 036-8562, Japan. Address e-mail to hirotak{at}cc.hirosaki-u.ac.jp.
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Abstract
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BACKGROUND: As orexinergic (OXergic) neurons have been reported to mediate emotional changes, benzodiazepines might interact with OXergic neurons.
METHODS: We examined the interactions between OXergic neurons and benzodiazepine receptors in orexin-A (100 nM) and K+ (25 mM)-evoked norepinephrine release from rat cerebrocortical slices.
RESULTS: Midazolam, diazepam, and flunitrazepam concentration-dependently inhibited both OX-A- and K+-evoked norepinephrine release. The IC50 of midazolam for orexin-A-evoked release (0.87 µM, P < 0.01), which was insensitive to flumazenil, was significantly lower than that of diazepam and flunitrazepam (around 60 µM), whereas the IC50s for K+-evoked release were not different among the benzodiazepines.
CONCLUSION: There may be no interaction between OXergic neurons and central benzodiazepine receptors.
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Introduction
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Emotional stress markedly increases norepinephrine release, especially in the locus coeruleus (LC), hypothalamus, and amygdala (1). All noradrenergic projections to the cerebrocortex originate from the LC (2) and activation of the LC by emotional stress induces norepinephrine release from the prefrontal cortex (3). The LC regulates the sleep/wake cycle and is densely innervated with orexinergic (OXergic) neurons (4–6). Hagan et al. (5) reported that orexin-A (OX-A) stimulates LC cell firing and increases arousal in rats. Moreover, we (7) previously found that OX-A significantly evoked norepinephrine but not other neurotransmitter release from rat cerebrocortical slices, in marked contrast to high-K+ that stimulated all transmitters release. As OXergic neurons also project to the cerebrocortex (4,6), there may be OX-activating noradrenergic neurons, which project from the LC to the cerebrocortex. Recent articles (9,10) strongly suggest that OXergic neurons may contribute to acute stress responses and emotional changes.
Benzodiazepines, used as hypnotics and anxiolytics in clinical practice, decrease norepinephrine release from the cerebral cortex in rats (11,12). In addition, we (13) previously found that anesthetic barbiturates significantly inhibit OX-A-evoked norepinephrine release from rat cerebrocortical slices. Therefore, we hypothesized that benzodiazepines would interact with OXergic neurons.
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METHODS
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With the approval of the University animal research committee, we used in this study 48 male Wistar rats weighing 250_300 g. Experimental procedures have been previously reported in detail (7). Briefly, rats were decapitated, the brains quickly removed and immersed in ice-cold Krebs–Ringer bicarbonate buffer solution oxygenated with 95% O2 and 5% CO2 (pH = 7.4). Cerebrocortical tissue was dissected from its internal structures and cross-chopped using a tissue chopper to produce 350 µm x 350 µm slices. The slices were washed three times in ice-cold Krebs–Ringer bicarbonate buffer solution 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 a drug was constructed). After obtaining a stable baseline, the slices were resuspended and incubated with the following drugs: midazolam (10–9–10–3 M, n = 6 each) or diazepam (10–9–10–3 M, n = 6 each) or flunitrazepam (10–9–10–3 M, n = 6 each) for 4 min in the absence (basal release) and presence of 25 mM K+ (evoked release) or for 10 min in the absence and presence of 10–7 M OX-A. In another experiment, the slices were resuspended and incubated with these benzodiazepines in the absence or presence of flumazenil (= 10 x midazolam dose). Slices were then challenged with K+ for 4 min or OX-A for 10 min. All buffers used in release studies contained the monoamine oxidase inhibitor pargyline (10 µM), and the reuptake inhibitor nomifensin (10 µM). Norepinephrine was determined directly by high-performance liquid chromatography with electrochemical detection (ESA Coulochem Model5100A) with 3.3% of the intra-assay maximal coefficient of variation.
All data are presented as mean ± sem. The concentrations (IC50) of benzodiazepines producing 50% of the maximal inhibition (Imax) were estimated from individual curves by nonlinear regression analysis (GRAPHPAD_PRISM3.0). Statistical analysis was performed by one-way repeated measures or factorial ANOVA. P < 0.05 was considered significant.
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RESULTS
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OX-A and K+ depolarization produced 10.5 ± 0.6 and 23.7 ± 0.7 pg/mg protein of norepinephrine release, respectively.
Midazolam, diazepam, and flunitrazepam concentration-dependently inhibited both OX-A- and K+-evoked norepinephrine release (Fig. 1) with pIC50 and Imax shown in Table 1. These benzodiazepines did not change basal norepinephrine release. The pIC50 of midazolam for OX-A-evoked release was significantly higher than that of diazepam and flunitrazepam. In contrast, there was no significant difference in benzodiazepine pIC50 for K+-evoked release.
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Figure 1. Inhibitory effects of benzodiazepines on orexin A (100 nM) (Panel A) and K+(25 mM) (Panel B)-evoked norepinephrine release from rat cerebrocortical slices. All data are mean ± sem (n = 6, each).
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Table 1. pIC50 (Mean IC50) and Imax Values for Benzodiazepine Inhibition of Orexin A (OX-A) and K+-Evoked Norepinephrine Release from Rat Cerebrocortical Slices
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The inhibitory effects of these benzodiazepines on OX-A-evoked release were not affected by flumazenil, a central benzodiazepine antagonist (Table 2). However, flumazenil significantly antagonized inhibition of K+-evoked norepinephrine release by midazolam (30 µM) from 55.8 ± 1.4% to 36.0 ± 1.4%. In addition, flumazenil per se did not change either OX-A- or K+-evoked release.
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Table 2. % Inhibition of Benzodiazepines with or without Flumazenil for Orexin A (100 nM)-Evoked Norepinephrine Release
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DISCUSSION
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In the present study, all benzodiazepines tested significantly inhibited OX-A-evoked norepinephrine release from rat cerebrocortical slices (Imax = 50%–60%). Kokare et al. (14) reported that subeffective doses of the 
-aminobutyric acid (GABA)A-receptor agonist muscimol and diazepam significantly enhanced the hyperphagic effect of OX-A. Thus, the GABAA/benzodiazepine-receptor complex may interact with OXergic neurons. As clinical concentrations of midazolam, diazepam, and flunitrazepam have been reported to be <1, 5, and 0.5 µM, respectively (15,16), only midazolam produced a response at clinically relevant concentrations. However, surprisingly, the inhibitory effects of midazolam tested were not antagonized by flumazenil. Previous reports (17,18) show that the Ki of midazolam for GABAA/benzodiazepine-receptor complex is <10 nM, which is 100-fold lower than the IC50 of midazolam for inhibition of OX-A-evoked norepinephrine release. Thus, the GABAA/benzodiazepine-receptor complex may not interact with the OXergic neurons in the rat cerebrocortex. Similarly, we previously reported that the inhibitory effects of thiopental on OX-A-evoked norepinephrine release were not antagonized by a GABAA-receptor antagonist, bicuculline (13), whereas the inhibition of K+-evoked release was antagonized (19). Moreover, muscimol did not inhibit OX-A-evoked release (13). Therefore, the effect of midazolam on OX-A-evoked norepinephrine release is mediated by a nonspecific effect of midazolam.
In contrast to OX-A-evoked release, K+-evoked norepinephrine release from the cerebrocortical slices was almost fully inhibited by three benzodiazepines with similar IC50s (approximately 50 µM). However, these IC50s were higher than their clinically relevant concentrations. Koga et al. (20) found that these benzodiazepines equipotently relaxed guinea pig tracheal smooth muscles with about 20–30 µM of EC50, which is similar to our findings. We previously showed that K+-evoked neurotransmitter releases from rat brain slices are mediated via P/Q-type voltage-sensitive Ca2+ channels (VSCCs). (19,21) In addition, several articles (22,23) suggest that benzodiazepines depress VSCCs. Yoshimura et al. (23) reported that midazolam 30 µM reduced K+-increased intracellular Ca2+ concentration of porcine tracheal smooth muscle by approximately 50%. Nakae et al. (22) reported that diazepam and midazolam inhibited the spontaneous beating rate and amplitude of cultured rat ventricular myocytes and these IC50s were similar and between 10 and 100 µM. In addition, as the depressive effects of diazepam and midazolam were almost fully antagonized by Bay K8644 (L-type VSCC activator), these depressive effects could be mediated via L-type VSCCs. Therefore, these benzodiazepines may equipotently inhibit P/Q-type VSCCs in the cerebrocortex. Moreover, as the inhibitory effect of midazolam on K+-evoked release was flumazenil-sensitive, the inhibition may be partially mediated via the GABAA/benzodiazepine-receptor complex, similar to the inhibitory effect of thiopental that may be partially mediated via GABAA-receptors (19).
In conclusion, midazolam significantly inhibited OX-A-evoked norepinephrine release from rat cerebrocortex. However, this inhibition may not have been mediated via the GABAA/benzodiazepine-receptor complex.
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Footnotes
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Accepted for publication October 16, 2006.
Supported, in part, by the Ministry of Education, Science and Culture, Tokyo, Japan Grant 17390423.
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Abstract
Introduction
