Search tips
Search criteria 


Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Anesthesiology. Author manuscript; available in PMC 2010 November 1.
Published in final edited form as:
PMCID: PMC2784127

Halothane-induced Hypnosis Is Not Accompanied by Inactivation of Orexinergic Output in Rodents

Heinrich Gompf, Ph.D., Research Fellow,* Jingqiu Chen, M.S., Research Specialist, Yi Sun, M.D. Ph.D., Research Associate,|| Masashi Yanagisawa, M.D. Ph.D., Professor, Gary Aston-Jones, Ph.D., Professor,ξ and Max B. Kelz, M.D. Ph.D., Assistant Professor[theta]



One underexploited property of anesthetics is their ability to probe neuronal regulation of arousal. At appropriate doses, anesthetics reversibly obtund conscious perception. However, individual anesthetic agents may accomplish this by altering the function of distinct neuronal populations. Previously we showed that isoflurane and sevoflurane inhibit orexinergic neurons, delaying reintegration of sensory perception as denoted by emergence. Herein we study the effects of halothane. As a halogenated alkane, halothane differs structurally, has a nonoverlapping series of molecular binding partners, and differentially modulates electrophysiologic properties of several ion channels when compared with its halogenated ether relatives.


c-Fos immunohistochemistry and in vivo electrophysiology were used to assess neuronal activity. Anesthetic induction and emergence were determined behaviorally in narcoleptic orexin/ataxin-3 mice and control siblings exposed to halothane.


Halothane-induced hypnosis occurred despite lack of inhibition of orexinergic neurons in mice. In rats, extracellular single-unit recordings within the locus coeruleus showed significantly greater activity during halothane than during a comparable dose of isoflurane. Microinjection of the orexin-1 receptor antagonist, SB-334867-A during the active period slowed firing rates of locus coeruleus neurons in halothane-anesthetized rats, but had no effect on isoflurane-anesthetized rats. Surprisingly, orexin/ataxin-3 transgenic mice, which develop narcolepsy with cataplexy due to loss of orexinergic neurons, did not show delayed emergence from halothane.


Coordinated inhibition of hypothalamic orexinergic and locus coeruleus noradrenergic neurons is not required for anesthetic induction. Normal emergence from halothane-induced hypnosis in orexin-deficient mice suggests that additional wake-promoting systems likely remain active during general anesthesia produced by halothane.


A central tenet of neuroscience states that behaviors are dependent upon the common output of the appropriate neuronal circuit. Recent studies suggest that the hypnotic properties of general anesthetics are produced by specific interactions of anesthetics with thalamic, hypothalamic, and brainstem arousal nuclei.16 Isoflurane and sevoflurane are prototypical halogenated ethers and are among the most commonly used general anesthetics. Halothane is the prototype halogenated alkane anesthetic that together with the halogenated ethers and small molecule anesthetics such as xenon and nitrous oxide comprise the inhaled class of general anesthetics.

Although isoflurane and halothane both inhibit thalamocortical gating and impair midline reticular formation structures,1 the two show differences in their effects on the cortical electroencephalogram with isoflurane generally causing more cortical depression than halothane.79 The molecular and cellular mechanisms responsible for this isoflurane-halothane difference are unknown but may reflect differences in protein binding targets,10 in a drug's ability to modulate currents at voltage gated ion channels11 or G-protein coupled receptors,12,13 or in larger network level activation and inhibition of individual sleep- or wake-active centers in brain implicated in mediating anesthetic hypnosis.3,4,14 Exploiting the differences in regional activation and inhibition of various arousal centers produced by different anesthetics should help identify the minimal subset of neuroanatomic substrates whose function must be altered to produce anesthetic-induced unconsciousness (hereafter referred to as hypnosis) and whose function must be restored to permit emergence from general anesthesia.

Isoflurane and sevoflurane are known to inhibit orexin (also known as hypocretin) signaling.15 Based upon the attenuated cortical depression observed at hypnotic doses of halothane as compared to equipotent doses of isoflurane and sevoflurane, we hypothesized that halothane administration might not impair orexin signaling in brain. Moreover, as the noradrenergic locus coeruleus (LC) receives the densest efferent orexin innervation outside of the hypothalamus,16 expresses the orexin-1 receptor,17 and is depolarized by orexin-A,18 we chose to study the effects of different anesthetics on LC neuronal firing in intact animals. Despite delivery of equipotent concentrations of halothane and isoflurane to rodents, we demonstrate that during halothane-induced hypnosis orexinergic neurons remain active as do their noradrenergic target neurons in the LC. This pattern of activity contrasts with isoflurane-induced hypnosis in which both wake-active groups are inhibited.15

Materials and Methods


All studies were approved by the Institutional Animal Care and Use Committee at the University of Pennsylvania (Philadelphia, Pennsylvania) and were conducted in accordance with the National Institutes of Health guidelines. Adult C57BL6/J male mice (Jackson Labs, Bar Harbor, ME) aged 8–12 weeks were placed on a 12 h light dark cycle and given food and water ad libidum. On experimental days, mice were placed in loss of righting reflex chambers19 and exposed to either 1.0% halothane dissolved in 100% oxygen (halothane group, n=8) or in 100% oxygen (wake control group, n=8) in the 2 h just after lights out (denoted by “ZT12–ZT14,” where ZT is Zeitgeber time—a 24-h clock defined by the onset of light, which occurs at ZT0) during their period of maximal activity.20 Twenty-six orexin/ataxin-3 mice aged 70 ± 5 days and 13 sibling controls aged 65 ± 9 days of both genders were used in righting reflex behavioral assays. For the electrophysiology experiments, 37 adult male Sprague Dawley rats (250–300 g, Harlan, Indianapolis, IN) were kept on a 12-h light-dark cycle (lights on at 0800) for at least one week after arriving in the lab before experiments, and were given food and water ad libidum.

Immunohistochemistry and cell counting

All mice were immediately sacrificed by cervical dislocation followed by rapid brain harvest, postfixation in 4% paraformaldehyde, and paraffin embedding. Sections were cut at 10 μm and stained for c-Fos (rabbit polyclonal antibody PC05, Calbiochem, corresponding to amino acids 4–17 of the human c-Fos, at 1:1000) and prepro-Orexin (mouse monoclonal antibody MAB763, Chemicon corresponding to amino acids 34–66 of the human orexin precursor, at 1:4000) according to standard protocols as previously described.15 Fluorescent secondary antibodies were an Alexa 594-labeled goat anti-rabbit (Invitrogen, Carlsbad, CA, at 1:200) for detection of c-Fos and an Alexa 488 goat anti-mouse (Invitrogen at 1:200) for detection of prepro-orexin. Three to six sections centered on the perifornical hypothalamus were counted per brain. Counting was performed by one blinded and confirmed by a second blinded experimenter.

Electrophysiological recordings

Before electrophysiological recordings, animals were anesthetized with halothane or isoflurane in air via spontaneous respiration. Animals were intubated with a tracheal cannula, continuously anesthetized with either 0.9 – 1.2% halothane (for subsequent LC recordings under halothane) or 1.0–1.4% isoflurane (for subsequent recordings under isoflurane) using a Riken FI21 refractometer (AM Bickford, Wales Center, NY) to confirm delivered anesthetic concentration, and placed in a stereotaxic frame with the incisor bar lowered to place the skull approximately 12° from the horizontal plane. Body temperature was maintained at 34 – 36°C with a thermistor-controlled heating pad. Animals recorded during their dark period had their eyes covered with black tape to avoid any light input during set-up, surgery or recording.

During LC recordings and microinjections, animals were maintained under stable levels of either 0.8–0.9% halothane or 1.0–1.1% isoflurane. These equipotent doses correspond to 1.3 times the ED50 dose required for loss of righting reflex in rats21,22 and were sufficient to permit in vivo recordings in the stereotaxic frame without movement artifact. For recording of baseline firing frequencies during the active or rest periods, animals were maintained exclusively under the influence of one or the other anesthetic. To examine the effects of the orexin-1 receptor antagonist SB-334867-A, animals were initially anesthetized with either halothane or isoflurane and then switched to the alternative anesthetic. In animals initially anesthetized with halothane (n=4), LC neurons were recorded until a neuron displaying inhibition to SB-334867-A was found. Subsequently while recording from the same neuron, the inhaled anesthetic was switched to isoflurane. Rats initially anesthetized with isoflurane underwent a similar switch to halothane while continuously recording from an LC neuron (n=2 animals). A minimum of 1.5 h with fresh gas flow exceeding minute ventilation passed to allow the rats to equilibrate with the new inhaled anesthetic.

LC recordings and microinjections were performed using standard methods as previously described.23,24 In brief, a 5-mm-diameter hole was drilled in the skull above the LC and the dura was reflected. A glass recording micropipette (tip diameter, 2 – 4 μm; 10 – 20 MΩ impedance) was filled with 2% pontamine sky blue dye in 0.5 M Na-acetate and aimed at the LC (4 mm caudal to lambda, and 1.2 mm lateral to midline). Signals were amplified and continuously displayed on an oscilloscope as unfiltered and filtered (0.3 – 10 kHz bandpass). LC neurons (5.8 – 6.5 mm ventral to the skull surface) were tentatively identified during recording using well established criteria: an entirely positive, notched waveform 2 ms or more in duration in unfiltered records; a slow, tonic spontaneous discharge (0.5 – 6.0 Hz); and a strong phasic activation followed immediately by long-duration inhibition (0.5 – 1.0 s) in response to noxious stimuli (electrical foot shock stimulation).25 Following identification of putative LC neurons, spike amplitude was continuously monitored to ensure stable recordings. Footpad stimulation was delivered through paired 26-gauge needles inserted in the medial aspect of the contralateral hindpaw and was given as monophasic 0.5 ms pulses of 90 V. Stimuli were given once every 2 s for a total of 50 repetitions. Spikes of single neurons were discriminated, and digital pulses were led to a computer for on-line data collection using a laboratory interface and software (CED 1401, SPIKE2; Cambridge Electronic Design, Cambridge, England). Response magnitudes to foot shock stimulation were calculated as the total number of spikes recorded from 20 to 100 ms following stimulation minus the expected number of spikes (average number of spikes / sec recorded 1 min prior to stimulation).

Injection pipettes (tip diameter 12 – 20 μm) were fashioned from microdispensing capillary tubes as described previously.26,27 The thin lumen of these pipettes allowed accurate measurement of microinjection volume (accuracy within 15 nl). The shank of the pipette was bent and attached to the recording pipette using ultraviolet light-curing dental resin (Filtek Supreme, Scotchbrand multi purpose adhesive, 3M, St. Paul, MN), with its tip recessed approximately 100 μm.

Microinjections were made by pneumatic pressure through the infusion barrel of the compound recording/infusion microelectrode (Picospritzer III, Parker Instruments, Cleveland, OH). After isolating a single LC neuron, baseline firing frequencies were recorded for at least 150 s. Following this baseline period, solutions were administered by applying pressure pulses of 30 – 100 ms duration at 40 psi every 15 s for a total of 150 – 225 s. With this procedure, each pulse delivered approximately 12 nl and the total volume applied was 120 – 180 nl. Care was taken to ensure that microinjection did not interfere with unit recordings by constantly monitoring spike amplitude during delivery. Typically, neurons could be held long enough to observe firing frequencies return to baseline (washout) within 30 – 300 s.

SB-334867-A (100 μM, Tocris, Ellisville MO) was dissolved at its final concentration in artificial cerebrospinal fluid, consisting of (in mM): 112 NaCl, 3.1 KCl, 1.2 MgSO4, 0.4 NaH2PO4, and 25 NaHCO3, and was either used fresh or stored at −20 °C for no longer than one week before use. This dose of 100μM was chosen based upon previously published in vivo electrophysiological studies.28,29 At the end of recordings, micropipette penetrations were marked by iontophoretic ejection of dye from the recording pipette (−20 μA, 50 % duty cycle for 15 min). Brains were snap-frozen in a solution of isopentane at −80 °C and coronal sections through the LC (40 μm thick) were cut on a cryostat, mounted on gelatinized glass slides, and stained with neutral red.

Loss and return of righting reflex studies in mice

Induction and emergence from halothane was defined behaviorally using the respective loss and return of the righting reflex. Mice were placed in cylindrical gas-tight controlled environment chambers arrayed in parallel. After 90 min of habituation with 100% oxygen each day on two successive days, anesthesia was induced with a Dräger model 19.1 halothane vaporizer (Draeger Medical, Inc Telford, PA) using 12 incremental increases in halothane dissolved in 100% oxygen. Halothane gas concentration was determined in triplicate during the last 2 min of each step. Initial concentration was 0.41%. After 15 minutes at each concentration to allow for equilibration of the mouse with the anesthetic vapors, the concentration of halothane was increased by 8 ± 3% of the preceding value. Peak halothane concentration was 1.06%. At the end of each 15-min interval, the cylindrical chambers were rotated 180°. A mouse was considered to have lost the righting reflex if it did not turn itself prone onto all four limbs within 2 min. After the last mouse lost its righting reflex, halothane concentration was increased one more time before measurements of emergence time, which was defined as the duration that elapsed until each mouse regained its righting reflex by turning prone onto all four feet. Mouse temperature was maintained between 36.6± 0.6°C by submerging the controlled environment chambers in a 37°C water bath. In an effort to minimize both the number of mice used and the number of anesthetic exposures, induction and emergence from halothane in orexin/ataxin-3 mice and sibling controls were performed during the same experiment. Sensitivity to anesthetic induction and emergence from isoflurane and sevoflurane has been previously reported.15

Statistical analysis

All data were tested for normality. Statistical testing was performed using Prism 4.0c (Graph Pad software, La Jolla, CA). Fos immunoreactivity in orexin-positive neurons spanning the dorsomedial through the perifornical hypothalamus was analyzed with a Mann-Whitney test and is reported as a median plus interquartile range. Electrophysiological spike frequencies were averaged into 10-sec bins using recording software (SPIKE 2; Cambridge Electronic Design) and exported to an Excel spreadsheet. After stable recordings were established and before drug application, a 150 s window of time was chosen to average baseline impulse activity and compare with average frequencies during drug application. Normality of these data sets was confirmed using Kolmogorov-Smirnov, D'Agostino and Pearson omnibus, as well as Shapiro-Wilk normality testing. Significance was tested by paired two-tail t-tests before and during drug application. To obtain EC50 and Hill slope constants, the log of volatile anesthetic gas concentration versus the fraction of the population having lost the righting reflex plots were generated and fit with a nonlinear dose-response curve with a variable slope by using Prism 4.0c (Graph Pad). EC50 and Hill slope constants are reported as means of two independent trials with 95% confidence limits. Emergence time data generated immediately after each anesthetic exposure are reported as a mean ± standard error.


Orexinergic neurons remain active during halothane anesthesia

To determine whether anesthetic-induced hypnosis must be accompanied by inhibition of wake-active orexinergic neurons, we examined the effect of an obtunding dose of halothane on nuclear c-Fos expression as a surrogate of neuronal activity. Adult C57BL/6J mice were exposed to either an oxygen control, or to anesthetizing doses of halothane 1.0% in oxygen for 2 h beginning at lights out, the period of maximal wakefulness, and sacrificed for immunohistochemical analysis. During halothane 37.7% (28.5% – 45.7%) of dorsomedial and perifornical orexinergic neurons exhibited c-Fos positive nuclear staining. This was not significantly different from nonanesthetized circadian wakeful controls 42.4% (34.5% – 47.1%) (n = 4 animals / group with 4–5 slices / animal; U = 7.000, p = 0.89; by Mann Whitney test, fig. 1).

Hypnotic doses of halothane fail to depress activity of orexinergic neurons as determined by double label immunohistochemistry. Coronal sections through perifornical hypothalamus reveal characteristic c-Fos protein expression (red nucleus) in orexinergic ...

Isoflurane abolishes circadian rhythm of LC neuronal impulse activity

We tested whether LC firing rates were similar during the period when nocturnal rodents are most awake and active (ZT13 – ZT19, 1 – 7 h after lights-off). During general anesthesia produced by isoflurane, representative in vivo recordings demonstrate that LC neurons fired significantly slower than under a comparable dose of halothane (2.95 ± 0.27 Hz halothane, n=29 neurons, 12 animals, vs. 1.88 ± 0.17 Hz isoflurane, n= 42 neurons, 7 animals, unpaired two-tail t-test: p = 0.001, t = 3.989, df = 69, fig. 2), consistent with preserved LC orexinergic input under halothane but not under isoflurane anesthesia. Rest period (ZT 5 – 11) firing frequencies were not significantly different in LC neurons recorded from halothane and isoflurane anesthetized animals (1.7 ± 0.2 Hz halothane, n=16 neurons, 8 animals; vs. 1.38 ± 0.12 Hz isoflurane, n=31 neurons, 5 animals, unpaired two-tail t-test: p = 0.17, t = 1.262, df = 30, fig. 2).

Locus coeruleus impulse activity differs under general anesthesia produced by halothane (A, B) as compared with isoflurane (C, D). Representative recordings demonstrate that intrinsic circadian fluctuation in locus coeruleus impulse activity remains intact ...

Does the inhibition of LC firing frequency caused by the orexin-1 receptor antagonist SB-334867-A during the active period, previously observed under halothane-induced hypnosis,29 also occur when the animal is under isoflurane-induced hypnosis? To answer this, we used combined microinjection-electrophysiological recording electrodes in a total of 6 animals (fig. 3). To ensure that any negative data would not be the result of inter-animal differences in either the active-period neuronal firing rate or responses to SB-334867-A, two animals were recorded under isoflurane anesthesia first and subsequently switched to halothane, while four animals were recorded under halothane anesthesia first, followed by isoflurane. Consistent with our previously published results,29 100μM SB-334867-A reduced LC impulse activity during the active period but not the rest period while the animals were under halothane anesthesia (2.66 ± 0.45 Hz preinjection, 1.58 ± 0.24 Hz SB-334867-A; paired two-tail t-test: n=8, p=0.008, t = 3.59, df = 7). Specifically, neurons with firing frequencies faster than the median frequency (2.76 Hz) were inhibited by SB-334867-A (3.59 ± 0.41 Hz preinjection vs. 1.94 ± 0.26 Hz SB-334867-A; paired two-tail t-test: n=4, p=0.001, t = 10.24, df = 3) whereas neurons firing slower than the median frequency were not (1.73 ± 0.45 Hz pre-injection vs. 1.21 ± 0.32 Hz SB-334867-A; paired two-tail t-test: n=4, p=0.32, t = 1.20, df = 3). In contrast, SB-334867-A did not attenuate active-period LC impulse activity during isoflurane-induced hypnosis (1.88 ± 0.17 Hz preinjection, 2.0 ± 0.19 Hz SB-334867-A; paired two-tail t-test: n=19, p=0.3, t = 1.07, df = 18). Neurons firing faster than the median frequency during isoflurane anesthesia (1.89 Hz) were not significantly inhibited in the presence of SB-334867-A (2.49 ± 0.11 Hz preinjection, 2.50 ± 0.25 Hz SB-334867-A; n=10, paired two-tail t-test: p=0.96, t = 0.10, df = 9) nor were those firing more slowly (1.19 ± 0.12 Hz preinjection, 1.44 ± 0.17 Hz SB-334867-A; n=9, paired two-tail t-test: p=0.01, t = 3.5, df = 8). SB-334867-A did not attenuate rest-period LC impulse activity during isoflurane-induced hypnosis (1.38 ± 0.17 Hz preinjection, 1.5 ± 0.18 Hz SB-334867-A; paired two-tail t-test: n=16, p=0.63, t = 2.3, df = 15). Altogether, the electrophysiological results suggest that while orexinergic neurons can maintain the potential to excite downstream targets under halothane, orexinergic activity is blunted under isoflurane.

Effects of SB-334867-A on locus coeruleus impulse activity. A) During halothane anesthesia faster-firing neurons exhibit greater impulse activity before SB-334867-A exposure. B) During isoflurane anesthesia, SB-334867-A fails to decrease the already low ...

Orexin deficient mice appear normal during induction and emergence from halothane anesthesia

Having demonstrated that halothane-induced hypnosis arises despite preservation of activity in two wake-active groups, we hypothesized that orexin/ataxin-3 mice would show delayed emergence from halothane general anesthesia with no change in sensitivity to induction of anesthesia as was the case for isoflurane and sevoflurane.15 As expected (fig. 4A), induction sensitivity was indistinguishable (F2,70 = 0.21) for orexin/ataxin-3 mice (halothane EC50= 0.87%, 95% confidence interval = 0.84%—0.90%, Hill slope = 13.2, 95% confidence interval = 6.5—19.8) and wild type sibling controls (halothane EC50 = 0.86%, 95% confidence interval = 0.83% — 0.89%, Hill slope = 15.3, 95% confidence interval = 7.6—23.1). However, following exposure to halothane, emergence times for halothane exposed orexin/ataxin-3 mice (20.0 ± 1.6 minutes) and wild type sibling controls (18.8 ± 1.0 min) were also indistinguishable (p = 0.52, by unpaired t-test) (fig. 4B).

Behavioral studies show no change in sensitivity to induction of halothane anesthesia and no difference in emergence from halothane anesthesia in narcoleptic orexin/ataxin-3 mice as compared to their wild type control siblings. A) Loss of righting reflex ...


Our understanding of the mechanisms responsible for anesthetic-induced unconsciousness is at best rudimentary. The search for structures controlling arousal state whose function is reversibly modulated by anesthetic drugs remains essential for unraveling anesthetic action. Conscious perception requires an awake brain, though wakefulness by itself is not sufficient for consciousness.30 Recently, extensive insights have been made in the neurobiology of sleep-wake control (reviewed in31,32). The promotion, maintenance, and stabilization of wakefulness are functions of the ascending reticular activating system—a distributed neuroanatomic network with distinct structures in the brainstem, diencephalon, and forebrain.

Available evidence suggests that one way in which general anesthetics transiently and reversibly impair consciousness is via inhibition of wake promoting and sustaining arousal centers.3,4,3336 In this manuscript we demonstrate that inhibition of all such arousal centers is not necessary for anesthetic-induced unconsciousness. For instance, noradrenergic neurons in the LC and orexinergic neurons in the hypothalamus form part of the distributed neuroanatomic wake-promoting network.37 During active wakefulness, orexinergic neurons in the dorsomedial and perifornical hypothalamus are known to fire rapidly; whereas during both nonrapid eye movement and rapid eye movement sleep, orexinergic neurons become nearly quiescent.38,39 For orexinergic neurons, c-Fos protein expression reliably tracks antecedent electrophysiological activity.20 In awake animals, c-Fos is expressed in the nuclei of dorsomedial and perifornical hypothalamic orexinergic neurons whereas in anesthetic-induced hypnosis produced by isoflurane or sevoflurane, c-Fos expression is significantly reduced by 30 or 50%, respectively to levels found during nonrapid eye movement sleep.15,20 Here we show that for the prototype volatile alkane, halothane, anesthetic-induced hypnosis occurs despite ongoing wake-active levels of c-Fos expression and presumably of electrophysiological activity in the orexinergic population. Wake-active LC neurons also fire more rapidly during the active period (ZT 12 – 24) than the rest period (ZT 0 – 12) in halothane anesthetized rats.40 This is mediated at least in part by orexinergic inputs to the LC.29 Ongoing depolarization of orexinergic neurons with subsequent release of orexin-A at its efferent projections during halothane anesthesia is sufficient to explain preserved activity in the locus coeruleus which is heavily innervated by orexinergic neurons,16 depolarized by orexin-A,18 and is slowed during halothane anesthesia by local microinjection of the orexin antagonist SB-334867-A. This pattern of activity in orexinergic and LC neurons during halothane contrasts with that seen during comparable doses of isoflurane. We demonstrate that local microinjection of SB-334867-A during an isoflurane anesthetic had no effect upon the LC, confirming the absence of orexinergic tone. In stark contrast to halothane's effects, equipotent doses of isoflurane do not appear to preserve orexinergic activity as determined immunohistochemically and do not permit the expression of a diurnal rhythm of LC impulse activity.

Methodologic considerations and limitations

Several methodologic issues must be considered when examining our results. First, despite its two decades of use as a neuronal activity tracer, immunohistochemical detection of c-Fos protein may not accurately reflect electrophysiological activity in all cell types.41 For example, in populations that do not express c-Fos or in populations such as the LC, where basal c-Fos expression is extremely low during active wakefulness42,43 at a period in time when LC neurons fire maximally, it may not be possible to detect an anesthetic-induced decrease in c-Fos.

However, in the orexinergic neurons c-Fos expression in the two hours preceding sacrifice has been validated as a surrogate of antecedent neuronal activity. Second, for all experiments that compare the effects among volatile anesthetics, equipotent doses must be defined. For our in vivo extracellular recordings of LC neurons of rats acutely anesthetized with isoflurane or halothane, we used a dose of 1.3 times the ED50 for loss of righting in rats based upon published studies of uninstrumented animals.21,22 While the actual ratio of isoflurane's ED50 to halothane's ED50 is numerically different than that of work by Imas and colleagues,44 inspection of the 95% confidence limits in the original work by Kissin and colleagues is not inconsistent with more recent work. Nonetheless, we acknowledge that our chosen ratio relying upon the increased potency of halothane relative to isoflurane may not be perfect. Third, compounding the question of anesthetic potency is the development of mild hypothermia in our rats, which developed despite several attempts to maintain euthermia including warming blankets above and below all rats. As core temperatures did not differ between rats exposed to isoflurane or halothane, the major impact would be an increase in volatile anesthetic potency relative to our measured concentrations. Finally, with respect to studies in orexin/ataxin-3 transgenic mice, we acknowledge the potential for compensatory changes due to postnatal destruction of all orexinergic neurons. These animals and their sibling controls were studied at 10 weeks of age shortly after onset of narcolepsy and cataplexy.45 Therefore, any potential compensatory adaptations should be minimized in these animals that undergo embryogenesis and development with an intact orexin signaling system relative to studies in mice with a constitutive knockout of the preproorexin gene or its receptors.

General anesthesia can occur without inactivation of orexinergic or noradrenergic LC neurons

Studies with barbiturates, propofol, chloral hydrate, and ketamine have previously demonstrated that inactivation of the LC is not required for induction of general anesthesia even though it is considered fundamental for dexmedetomidine.3,4,34,42 Both indirect as well as direct assessments indicated that halogenated ether anesthetics such as isoflurane as well as propofol and etomidate do inhibit orexinergic neurons of the hypothalamus.15,42,46,47 Meanwhile, halothane, similar to dexmedetomidine,34 fails to depress activity of orexinergic neurons.

A relative preservation of orexinergic signaling during halothane exposure might be anticipated to bias other cortically projecting arousal systems in addition to the LC such as the basal forebrain46, intralaminar, and midline thalamic nuclei48 and may begin to mechanistically explain how hypnotic doses of halothane produce less cortical electroencephalographic depression than comparable doses of isoflurane.79 Such a finding is also consistent with the demonstration that intracerebroventricular addition of exogenous orexin-A during 1.2% isoflurane, when endogenous levels should be very low, changed the cortical electroencephalographic signature from a state of burst suppression, indicative of deep anesthesia, to a more aroused pattern.47

Recently, supra-hypnotic doses of sevoflurane have been shown to directly depolarize LC neurons recorded from a brainstem slice via gap junction channels. Significantly reduced inward depolarizing currents were also elicited from LC neurons in the slice by hypnotic and subhypnotic levels of sevoflurane as well as sub- to suprahypnotic doses of isoflurane, but not by halothane or propofol.49 Our results with isoflurane and halothane suggest that the direct depolarizing contributions of volatile anesthetics (sevoflurane >> isoflurane >> halothane) at gap junctions measured in the chemically deafferented LC could be outweighed by orexinergic and other presently undefined afferent inputs to the LC in the intact animal. However, it should be noted that we did not measure the firing rates of LC neurons in intact rats exposed to equipotent hypnotic doses of sevoflurane.

Mechanistic inferences from studies of halothane-induced hypnosis in orexin/ataxin-3 mice

We also demonstrate that mice lacking orexinergic neurons emerge from halothane anesthesia in a manner indistinguishable from their sibling controls. In contrast, such mice have delayed emergence from isoflurane and sevoflurane.15 The neuronal substrates governing anesthetic emergence remain largely unknown. However, distinct patterns of emergence in narcoleptic-cataplexic mice following exposure to two different halogenated ethers as compared to the halogenated alkane, halothane, highlight an opportunity to exploit agent-specific diversity, leading ultimately to novel hypothesis testing. Although our results show that hypnotic doses of halothane do not depress firing of orexinergic neurons nor of locus coeruleus neurons, this cannot account for the difference in recovery from anesthesia compared to isoflurane/sevoflurane because orexinergic neurons have been completely destroyed in adult orexin/ataxin-3 mice.45

Redundancy in the neural control of arousal state may have evolved as a survival advantage to protect wakefulness. Both lesion and genetic knockout studies suggest the functional integration of arousal promoting networks is sufficient to overcome permanent destruction at one or more ascending reticular activating nodes.50,51 However, transient anesthetic-induced inactivation at wake-promoting reticular activating loci occurring either in parallel or at critical sites appears sufficient to induce anesthetic hypnosis.33,52,53 Presumably, reactivation of wake-promoting reticular activating loci is essential for anesthetic emergence.15,35,54 Hence, to explain the normal emergence of orexin/ataxin-3 mice from halothane, we predict the existence of an afferent input to orexinergic neurons with the following features: it should show circadian changes in neuronal activity, should modulate activity of orexinergic neurons while directly or indirectly modulating LC activity, and should be inhibited by isoflurane/sevoflurane while remaining unaffected by equipotent hypnotic doses of halothane. This hypothetical wake active locus may also reinforce activity of LC neurons in addition to the indirect effects such a nucleus would promote by activating the orexinergic neurons.18 Of the many potential candidates for such a group, basal forebrain cholinergic neurons are known to display state dependent activity, send efferent projections to orexinergic neurons, and depolarize orexinergic neurons via release of acetylcholine.55,56 Manipulation of central cholinergic signaling modulates the behavioral expression of sleep, wake, and anesthetized states.14,57,58 Pharmacologic enhancement of cholinergic neurotransmission using physostigmine, an acetylcholinesterase inhibitor that crosses the blood brain barrier, has been demonstrated to speed emergence from both intravenous anesthetics5962 and volatile anesthetics including isoflurane, sevoflurane, and halothane.6365 Although no study describes a direct comparison between the efficacy of physostigmine as a partial antagonist of halothane- versus isoflurane- or sevoflurane-induced hypnosis, equipotent doses of halothane and isoflurane have been shown to significantly, but differentially alter presynaptic cholinergic signaling in a rat synaptosome preparation.66 Halothane antagonizes the inhibition of pharmacologically induced release of the inhibitory neurotransmitter γ-aminobutyric acid mediated by muscarinic receptors in striatum whereas isoflurane does not. Whether such an effect persists elsewhere in the brain and whether it occurs with physiological depolarization remains unknown, but this finding highlights the potential mechanistic differences between equipotent doses of halothane and isoflurane upon efficacy at cholinergic sites.66

Together, these facts make cholinergic basal forebrain neurons an attractive subject for current46,67,68 and future investigations.


The authors wish to thank Dr. Andrew Ochroch, M.D., M.S.C.E. (Associate Professor, Department of Anesthesiology and Critical Care, University of Pennsylvania) for his expert statistical input.

Support: This work was supported by National Institute of Health grants K08 GM077357, NS24698, and DA06214, by the Foundation for Anesthesia Education and Research, Rochester, Minnesota, and by the Department of Anesthesiology and Critical Care, University of Pennsylvania, Philadelphia, Pennsylvania


Attribution: Department of Anesthesiology and Critical Care, University of Pennsylvania, Philadelphia, Pennsylvania

Summary Statement: Diversity in neuronal mechanisms of anesthetic action is demonstrated by showing that halothane-induced hypnosis does not depend upon inhibition of hypothalamic orexinergic neurons nor of pontine noradrenergic neurons in the locus coeruleus. accessed April 27, 2009


1. Alkire MT, Haier RJ, Fallon JH. Toward a unified theory of narcosis: Brain imaging evidence for a thalamocortical switch as the neurophysiologic basis of anesthetic-induced unconsciousness. Conscious Cogn. 2000;9:370–86. [PubMed]
2. Alkire MT, McReynolds JR, Hahn EL, Trivedi AN. Thalamic microinjection of nicotine reverses sevoflurane-induced loss of righting reflex in the rat. Anesthesiology. 2007;107:264–72. [PubMed]
3. Nelson LE, Guo TZ, Lu J, Saper CB, Franks NP, Maze M. The sedative component of anesthesia is mediated by GABA(A) receptors in an endogenous sleep pathway. Nat Neurosci. 2002;5:979–84. [PubMed]
4. Nelson LE, Lu J, Guo T, Saper CB, Franks NP, Maze M. The alpha2-adrenoceptor agonist dexmedetomidine converges on an endogenous sleep-promoting pathway to exert its sedative effects. Anesthesiology. 2003;98:428–36. [PubMed]
5. Franks NP. General anaesthesia: From molecular targets to neuronal pathways of sleep and arousal. Nat Rev Neurosci. 2008;9:370–86. [PubMed]
6. Lydic R, Baghdoyan HA. Sleep, anesthesiology, and the neurobiology of arousal state control. Anesthesiology. 2005;103:1268–95. [PubMed]
7. Antunes LM, Golledge HD, Roughan JV, Flecknell PA. Comparison of electroencephalogram activity and auditory evoked responses during isoflurane and halothane anaesthesia in the rat. Vet Anaesth Analg. 2003;30:15–23. [PubMed]
8. Hudetz AG. Effect of volatile anesthetics on interhemispheric EEG cross-approximate entropy in the rat. Brain Res. 2002;954:123–31. [PubMed]
9. Orth M, Bravo E, Barter L, Carstens E, Antognini JF. The differential effects of halothane and isoflurane on electroencephalographic responses to electrical microstimulation of the reticular formation. Anesth Analg. 2006;102:1709–14. [PubMed]
10. Eckenhoff MF, Eckenhoff RG. Quantitative autoradiography of halothane binding in rat brain. J Pharmacol Exp Ther. 1998;285:371–6. [PubMed]
11. Correa AM. Gating kinetics of Shaker K+ channels are differentially modified by general anesthetics. Am J Physiol. 1998;275:C1009–21. [PubMed]
12. Peterlin Z, Ishizawa Y, Araneda R, Eckenhoff R, Firestein S. Selective activation of G-protein coupled receptors by volatile anesthetics. Mol Cell Neurosci. 2005;30:506–12. [PubMed]
13. Nakayama T, Penheiter AR, Penheiter SG, Chini EN, Thompson M, Warner DO, Jones KA. Differential effects of volatile anesthetics on M3 muscarinic receptor coupling to the Gα heterotrimeric G protein. Anesthesiology. 2006;105:313–24. [PubMed]
14. Keifer JC, Baghdoyan HA, Lydic R. Pontine cholinergic mechanisms modulate the cortical electroencephalographic spindles of halothane anesthesia. Anesthesiology. 1996;84:945–54. [PubMed]
15. Kelz MB, Sun Y, Chen J, Cheng Meng Q, Moore JT, Veasey SC, Dixon S, Thornton M, Funato H, Yanagisawa M. An essential role for orexins in emergence from general anesthesia. Proc Natl Acad Sci U S A. 2008;105:1309–14. [PubMed]
16. Peyron C, Tighe DK, van den Pol AN, de Lecea L, Heller HC, Sutcliffe JG, Kilduff TS. Neurons containing hypocretin (orexin) project to multiple neuronal systems. J Neurosci. 1998;18:9996–10015. [PubMed]
17. Trivedi P, Yu H, MacNeil DJ, Van der Ploeg LH, Guan XM. Distribution of orexin receptor mRNA in the rat brain. FEBS Lett. 1998;438:71–5. [PubMed]
18. Hagan JJ, Leslie RA, Patel S, Evans ML, Wattam TA, Holmes S, Benham CD, Taylor SG, Routledge C, Hemmati P, Munton RP, Ashmeade TE, Shah AS, Hatcher JP, Hatcher PD, Jones DN, Smith MI, Piper DC, Hunter AJ, Porter RA, Upton N. Orexin A activates locus coeruleus cell firing and increases arousal in the rat. Proc Natl Acad Sci U S A. 1999;96:10911–6. [PubMed]
19. Sun Y, Chen J, Pruckmayr G, Baumgardner JE, Eckmann DM, Eckenhoff RG, Kelz MB. High throughput modular chambers for rapid evaluation of anesthetic sensitivity. BMC Anesthesiology. 2006;6:13. [PMC free article] [PubMed]
20. Estabrooke IV, McCarthy MT, Ko E, Chou TC, Chemelli RM, Yanagisawa M, Saper CB, Scammell TE. Fos expression in orexin neurons varies with behavioral state. J Neurosci. 2001;21:1656–62. [PubMed]
21. Kissin I, Morgan PL, Smith LR. Anesthetic potencies of isoflurane, halothane, and diethyl ether for various end points of anesthesia. Anesthesiology. 1983;58:88–92. [PubMed]
22. Kissin I, Morgan PL, Smith LR. Comparison of isoflurane and halothane safety margins in rats. Anesthesiology. 1983;58:556–61. [PubMed]
23. Jodo E, Aston-Jones G. Activation of locus coeruleus by prefrontal cortex is mediated by excitatory amino acid inputs. Brain Res. 1997;768:327–32. [PubMed]
24. Shiekhattar R, Aston-Jones G. NMDA-receptor-mediated sensory responses of brain noradrenergic neurons are suppressed by in vivo concentrations of extracellular magnesium. Synapse. 1992;10:103–9. [PubMed]
25. Hirata H, Aston-Jones G. A novel long-latency response of locus coeruleus neurons to noxious stimuli: Mediation by peripheral C-fibers. J Neurophysiol. 1994;71:1752–61. [PubMed]
26. Akaoka H, Aston-Jones G. Opiate withdrawal-induced hyperactivity of locus coeruleus neurons is substantially mediated by augmented excitatory amino acid input. J Neurosci. 1991;11:3830–9. [PubMed]
27. Shiekhattar R, Aston-Jones G, Chiang C. Local infusion of calcium-free solutions in vivo activates locus coeruleus neurons. Brain Res Bull. 1991;27:5–12. [PubMed]
28. Yamuy J, Fung SJ, Xi M, Chase MH. Hypocretinergic control of spinal cord motoneurons. J Neurosci. 2004;24:5336–45. [PubMed]
29. Gompf HS, Aston-Jones G. Role of orexin input in the diurnal rhythm of locus coeruleus impulse activity. Brain Res. 2008;1224:43–52. [PMC free article] [PubMed]
30. Young GB. Coma. Ann N Y Acad Sci. 2009;1157:32–47. [PubMed]
31. Datta S, Maclean RR. Neurobiological mechanisms for the regulation of mammalian sleep-wake behavior: Reinterpretation of historical evidence and inclusion of contemporary cellular and molecular evidence. Neurosci Biobehav Rev. 2007;31:775–824. [PMC free article] [PubMed]
32. Jones BE. From waking to sleeping: Neuronal and chemical substrates. Trends Pharmacol Sci. 2005;26:578–86. [PubMed]
33. Vanini G, Watson CJ, Lydic R, Baghdoyan HA. γ-aminobutyric acid-mediated neurotransmission in the pontine reticular formation modulates hypnosis, immobility, and breathing during isoflurane anesthesia. Anesthesiology. 2008;109:978–88. [PMC free article] [PubMed]
34. Zecharia AY, Nelson LE, Gent TC, Schumacher M, Jurd R, Rudolph U, Brickley SG, Maze M, Franks NP. The involvement of hypothalamic sleep pathways in general anesthesia: Testing the hypothesis using the GABAA receptor beta3N265M knock-in mouse. J Neurosci. 2009;29:2177–87. [PubMed]
35. Hirota K, Kushikata T. Central noradrenergic neurones and the mechanism of general anaesthesia. Br J Anaesth. 2001;87:811–3. [PubMed]
36. 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. [PubMed]
37. Jones BE. Arousal systems. Front Biosci. 2003;8:s438–51. [PubMed]
38. Lee MG, Hassani OK, Jones BE. Discharge of identified orexin/hypocretin neurons across the sleep-waking cycle. J Neurosci. 2005;25:6716–20. [PubMed]
39. Mileykovskiy BY, Kiyashchenko LI, Siegel JM. Behavioral correlates of activity in identified hypocretin/orexin neurons. Neuron. 2005;46:787–98. [PubMed]
40. Aston-Jones G, Chen S, Zhu Y, Oshinsky ML. A neural circuit for circadian regulation of arousal. Nat Neurosci. 2001;4:732–8. [PubMed]
41. Dragunow M, Faull R. The use of c-fos as a metabolic marker in neuronal pathway tracing. J Neurosci Methods. 1989;29:261–5. [PubMed]
42. Lu J, Nelson LE, Franks N, Maze M, Chamberlin NL, Saper CB. Role of endogenous sleep-wake and analgesic systems in anesthesia. J Comp Neurol. 2008;508:648–62. [PMC free article] [PubMed]
43. Zhu Y, Fenik P, Zhan G, Mazza E, Kelz M, Aston-Jones G, Veasey SC. Selective loss of catecholaminergic wake active neurons in a murine sleep apnea model. J Neurosci. 2007;27:10060–71. [PubMed]
44. Imas OA, Ropella KM, Ward BD, Wood JD, Hudetz AG. Volatile anesthetics enhance flash-induced γ oscillations in rat visual cortex. Anesthesiology. 2005;102:937–47. [PubMed]
45. Hara J, Beuckmann CT, Nambu T, Willie JT, Chemelli RM, Sinton CM, Sugiyama F, Yagami K, Goto K, Yanagisawa M, Sakurai T. Genetic ablation of orexin neurons in mice results in narcolepsy, hypophagia, and obesity. Neuron. 2001;30:345–54. [PubMed]
46. 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. [PubMed]
47. Yasuda Y, Takeda A, Fukuda S, Suzuki H, Ishimoto M, Mori Y, Eguchi H, Saitoh R, Fujihara H, Honda K, Higuchi T. Orexin a elicits arousal electroencephalography without sympathetic cardiovascular activation in isoflurane-anesthetized rats. Anesth Analg. 2003;97:1663–6. [PubMed]
48. Bayer L, Eggermann E, Saint-Mleux B, Machard D, Jones BE, Muhlethaler M, Serafin M. Selective action of orexin (hypocretin) on nonspecific thalamocortical projection neurons. J Neurosci. 2002;22:7835–9. [PubMed]
49. Yasui Y, Masaki E, Kato F. Sevoflurane directly excites locus coeruleus neurons of rats. Anesthesiology. 2007;107:992–1002. [PubMed]
50. Blanco-Centurion C, Gerashchenko D, Shiromani PJ. Effects of saporin-induced lesions of three arousal populations on daily levels of sleep and wake. J Neurosci. 2007;27:14041–8. [PMC free article] [PubMed]
51. Szentirmai E, Kapas L, Sun Y, Smith RG, Krueger JM. Spontaneous sleep and homeostatic sleep regulation in ghrelin knockout mice. Am J Physiol Regul Integr Comp Physiol. 2007;293:R510–7. [PubMed]
52. Devor M, Zalkind V. Reversible analgesia, atonia, and loss of consciousness on bilateral intracerebral microinjection of pentobarbital. Pain. 2001;94:101–12. [PubMed]
53. Correa-Sales C, Rabin BC, Maze M. A hypnotic response to dexmedetomidine, an α-2 agonist, is mediated in the locus coeruleus in rats. Anesthesiology. 1992;76:948–52. [PubMed]
54. Alkire MT, Asher CD, Franciscus AM, Hahn EL. Thalamic microinfusion of antibody to a voltage-gated potassium channel restores consciousness during anesthesia. Anesthesiology. 2009;110:766–73. [PubMed]
55. Yoshida K, McCormack S, Espana RA, Crocker A, Scammell TE. Afferents to the orexin neurons of the rat brain. J Comp Neurol. 2005;494:845–61. [PMC free article] [PubMed]
56. Sakurai T, Nagata R, Yamanaka A, Kawamura H, Tsujino N, Muraki Y, Kageyama H, Kunita S, Takahashi S, Goto K, Koyama Y, Shioda S, Yanagisawa M. Input of orexin/hypocretin neurons revealed by a genetically encoded tracer in mice. Neuron. 2005;46:297–308. [PubMed]
57. Coleman CG, Lydic R, Baghdoyan HA. M2 muscarinic receptors in pontine reticular formation of C57BL/6J mouse contribute to rapid eye movement sleep generation. Neuroscience. 2004;126:821–30. [PubMed]
58. Douglas CL, Bowman GN, Baghdoyan HA, Lydic R. C57BL/6J and B6.VLEPOB mice differ in the cholinergic modulation of sleep and breathing. J Appl Physiol. 2005;98:918–29. [PubMed]
59. Bellur SN, Wojcik WJ, Radulovacki M. Effects of cholinomimetic agents on pentobarbital anesthesia in mice. Res Commun Chem Pathol Pharmacol. 1978;22:601–4. [PubMed]
60. Caldwell CB, Gross JB. Physostigmine reversal of midazolam-induced sedation. Anesthesiology. 1982;57:125–7. [PubMed]
61. Meuret P, Backman SB, Bonhomme V, Plourde G, Fiset P. Physostigmine reverses propofol-induced unconsciousness and attenuation of the auditory steady state response and bispectral index in human volunteers. Anesthesiology. 2000;93:708–17. [PubMed]
62. Toro-Matos A, Rendon-Platas AM, Avila-Valdez E, Villarreal-Guzman RA. Physostigmine antagonizes ketamine. Anesth Analg. 1980;59:764–7. [PubMed]
63. Artru AA, Hui GS. Physostigmine reversal of general anesthesia for intraoperative neurological testing: Associated EEG changes. Anesth Analg. 1986;65:1059–62. [PubMed]
64. Plourde G, Chartrand D, Fiset P, Font S, Backman SB. Antagonism of sevoflurane anaesthesia by physostigmine: Effects on the auditory steady-state response and bispectral index. Br J Anaesth. 2003;91:583–6. [PubMed]
65. Hudetz AG, Wood JD, Kampine JP. Cholinergic reversal of isoflurane anesthesia in rats as measured by cross-approximate entropy of the electroencephalogram. Anesthesiology. 2003;99:1125–31. [PubMed]
66. Salord F, Keita H, Lecharny JB, Henzel D, Desmonts JM, Mantz J. Halothane and isoflurane differentially affect the regulation of dopamine and gamma-aminobutyric acid release mediated by presynaptic acetylcholine receptors in the rat striatum. Anesthesiology. 1997;86:632–41. [PubMed]
67. Dong HL, Fukuda S, Murata E, Higuchi T. Excitatory and inhibitory actions of isoflurane on the cholinergic ascending arousal system of the rat. Anesthesiology. 2006;104:122–33. [PubMed]
68. Laalou FZ, de Vasconcelos AP, Oberling P, Jeltsch H, Cassel JC, Pain L. Involvement of the basal cholinergic forebrain in the mediation of general (propofol) anesthesia. Anesthesiology. 2008;108:888–96. [PubMed]