Acute and chronic morphine alters formalin pain in neonatal rats.

The present study tested the hypothesis that morphine exposure during the human developmental equivalent of the third trimester would alter inflammatory pain. This study examined whether acute or continuous opioid exposure in the neonatal rat alters formalin-induced nociception after 4 days of abstinence. Rats were exposed to a single acute administration of morphine on postnatal day 7 or 72 h of opioid infusion from postnatal days 5-7 via osmotic pump. When challenged with intraplantar formalin on postnatal day 11, rats exposed to acute or chronic morphine had increased phase II pain-associated behaviors. These findings suggest that neonatal morphine exposure may have unintended consequences on inflammatory pain.

Ethanol withdrawal-associated allodynia and hyperalgesia: age-dependent regulation by protein kinase C epsilon and gamma isoenzymes.

UNLABELLED: Ethanol (EtOH) withdrawal increases sensitivity to painful stimuli in adult rats. In this study, withdrawal from a single, acute administration of EtOH dose-dependently produced mechanical allodynia and thermal hyperalgesia in postnatal day 7 (P7) rats. In contrast, P21 rats exhibited earlier and more prolonged mechanical allodynia but not thermal hyperalgesia. For both P7 and P21 rats, blood and spinal cord EtOH levels peaked at 30 minutes after administration, with P7 rats achieving overall higher spinal cord concentrations. Protein kinase C (PKC) has been implicated in mediating pain responses. Inhibitory PKC- and gamma-specific peptides attenuated mechanical allodynia and thermal hyperalgesia in P7 rats, whereas only the PKCgamma inhibitor prevented mechanical allodynia in P21 rats. Immunoreactive PKC in dorsal root ganglion and PKCgamma in lumbar spinal cord increased at 6 hours after EtOH administration in P7 rats. In P21 rats, the density of PKC immunoreactivity remained unchanged, whereas the density of PKCgamma immunoreactivity increased and translocation occurred. These studies demonstrate developmental differences in neonatal nociceptive responses after withdrawal from acute EtOH and implicate a role for specific PKC isozymes in EtOH withdrawal-associated allodynia and hyperalgesia. PERSPECTIVE: This study examines age-specific nociceptive responses after ethanol exposure by using 2 different ages of rats. The results suggest that ethanol age-dependently alters sensitivity to mechanical and thermal stimuli via specific protein kinase C isozymes. These results begin to ascertain the mechanisms that produce abnormal pain after alcohol exposure.

Hyperresponsiveness on washout of volatile anesthetics from isolated spinal cord compared to withdrawal from ethanol.

We performed experiments in spinal cords isolated from neonatal rats to probe the mechanisms responsible for hyperresponsiveness of the population excitatory evoked potential (pEPSP) observed on washout of the volatile anesthetics halothane and isoflurane (1 minimal alveolar anesthetic concentration equivalent, MAC) compared with that observed after an anesthetic concentration of ethanol. After 30 min exposure to each anesthetic and washout, pEPSP area increased to levels significantly more than control (P < 0.01-0.001). Exposure to a very small (0.025 MAC) concentration of isoflurane over the same period itself produced a similarly exaggerated pEPSP (P < 0.05) in the continued presence of the drug, suggesting that the phenomenon is a direct excitatory effect of the small concentrations of anesthetic on washout, unlike the true withdrawal observed with ethanol. Isoflurane, but not halothane, significantly increased the amount of potassium-stimulated release of the excitatory neurotransmitters glutamate, aspartate, and substance P, suggesting the hyperresponsiveness for that drug is the result of a presynaptically mediated increase in transmitter release. A broad spectrum specific protein kinase C inhibitor, GF109203X, blocked ethanol withdrawal hyperresponsiveness but not hyperresponsiveness after halothane. If the behavioral symptoms of emergence from anesthesia are based on excitatory actions similar to those observed in the spinal cord, the results show that they represent direct excitatory actions rather than withdrawal and are attributable to direct actions on ion channels or receptors, rather than indirect effects mediated by protein kinase C.

Protein kinase Cgamma mediates ethanol withdrawal hyper-responsiveness of NMDA receptor currents in spinal cord motor neurons.

The present studies were designed to test the hypothesis that neuronal-specific protein kinase Cgamma (PKCgamma) plays a critical role in acute ethanol withdrawal hyper-responsiveness in spinal cord. Patch-clamp studies were carried out in motor neurons in neonatal rat spinal cord slices. Postsynaptic currents were evoked by brief pulses of 2 mM N-methyl-D-aspartic acid (NMDA) in the presence of bicuculline methiodide 10 microM; strychnine 5 microM and tetrodotoxin 0.5 microM. Both ethanol depression and withdrawal hyper-responsiveness of NMDA-evoked currents are dependent on increases in intracellular Ca(2+). Blocking intracellular increase in Ca(2+) by 30 mM 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA) not only decreased the ethanol-induced depression of NMDA-evoked currents (33+/-5% in control vs 20+/-3% in BAPTA, P<0.05) but also eliminated acute ethanol withdrawal hyper-responsiveness. Immunohistochemistry studies revealed that neonatal spinal cord motor neurons contain an abundance of nuclear PKCgamma. Exposure to ethanol (100 mM) induced PKCgamma translocation from the nucleus to cytoplasm in motor neurons. Pretreatment with the gamma-isozyme-specific peptide PKC inhibitor, gammaV5-3, blocked ethanol-induced translocation and also blocked withdrawal hyper-responsiveness. The results show that PKCgamma mediates ethanol withdrawal hyper-responsiveness in spinal motor neurons; the results may be relevant to some symptoms of ethanol withdrawal in vivo.

Acute and chronic ethanol exacerbates formalin pain in neonatal rats.

We have previously reported that withdrawal from acute ethanol (EtOH) exposure lowers mechanical thresholds in post-natal day 7 (P7) and post-natal day 21 (P21) rats. The present study tested the hypothesis that daily administration of 4 g/kg 15% EtOH for 5 days in rats during the human developmental equivalent of the third trimester, but not at a later time in development, would alter mechanical thresholds and formalin-induced pain behaviors. A transient decrease in mechanical thresholds (allodynia) was observed in P7 rats upon withdrawal from repeated EtOH between P3 and P7. When challenged with intraplantar formalin on P11, rats exposed to acute or chronic EtOH had enhanced phase II pain behaviors. In contrast to chronic EtOH administration to rats between P3 and P7, prolonged mechanical allodynia was observed in P21 rats upon withdrawal from chronic EtOH between P17 and P21. Formalin responses were unchanged in P25 rats exposed to acute or chronic EtOH. The affects of EtOH on somatosensory processing are dependent upon the age at which exposure occurs.

Acute and chronic ethanol does not affect incisional pain in neonatal rats.

We have previously found that in post-natal day 7 rats withdrawal from acute and chronic ethanol (EtOH) exposure lowers mechanical thresholds during withdrawal and exacerbates spontaneous pain responses to an inflammatory injury 4 days post-withdrawal. These findings suggested alterations in somatosensory pathways following EtOH exposure during the third trimester developmental equivalent. In this study we wanted to determine whether EtOH exposure during the third trimester equivalent exacerbates mechanical allodynia and thermal hyperalgesia produced by an incisional model of post-operative pain at post-natal day 21. The extent and duration of mechanical allodynia and thermal hyperalgesia following incision was measured and found to be unaffected by prior EtOH exposure.

Inhibition of spinal protein kinase C-epsilon or -gamma isozymes does not affect halothane minimum alveolar anesthetic concentration in rats.

Anesthetic effects on receptor or ion channel phosphorylation by enzymes such as protein kinase C (PKC) have been postulated to underlie some aspects of anesthesia. In vitro studies show that anesthetic effects on several receptors are mediated by PKC. To test the importance of PKC for the immobility produced by inhaled anesthetics, we measured the effect of intrathecal injections of PKC-epsilon and -gamma inhibitors on halothane minimum alveolar anesthetic concentration (MAC) in 7-day-old and 21-day-old Sprague-Dawley rats. The inhibitors were made as solutions of 100 pmol/5 microL and were given in a volume of 5 microL (7-day-old [P7] rats) or 10 microL (21-day-old [P21] rats). Controls were saline injections or injections of the peptide carrier at the same concentration and volumes; there were six animals in each group. In P7 rats, MAC values (in percentage of an atmosphere) were 1.63 +/- 0.0727 (mean +/- SEM) in saline controls, 1.55 +/- 0.141 in carrier controls, 1.54 +/- 0.0800 in rats given PKC-epsilon, and 1.69 +/- 0.0554 in rats given PKC-gamma. In P21 animals, the values were 1.20 +/- 0.0490, 1.31 +/- 0.0124, 1.27 +/- 0.0367, and 1.15 +/- 0.0483, respectively. Injection of the inhibitors did not change MAC in either age group. These results do not support an anesthetic effect on phosphorylation as a mechanism underlying the capacity of inhaled anesthetics to prevent movement in response to noxious stimulation, and they indirectly support a direct action on receptors or ion channels.

Mechanical allodynia and thermal hyperalgesia upon acute opioid withdrawal in the neonatal rat.

Upon withdrawal from opioids many patients experience a heightened sensitivity to stimuli and an exaggerated pain response. We present evidence that neonatal rats exhibit allodynia and hyperalgesia on acute opiate withdrawal. Postnatal 7 and 21 day rats were used to approximately model a full term human infant and a human child, respectively. The opiate antagonist naloxone was used to precipitate withdrawal at 30 or 120 min after a single acute administration of morphine. Alternatively, rats were allowed to undergo spontaneous withdrawal. Behavioral manifestations of withdrawal syndrome were not observed when naloxone was administered at 30 min post-morphine, but were present when withdrawal was precipitated at 120 min. Spontaneous and precipitated withdrawal from a single acute administration of morphine produced mechanical allodynia and thermal hyperalgesia in postnatal day 7 rats and mechanical allodynia in postnatal day 21 rats. A higher dose of morphine was required to produce mechanical allodynia in postnatal day 21 versus 7 rats but this increase was independent of the analgesic efficacy of morphine at these two ages. The present work illustrates the need to examine the phenomenon of hypersensitivity upon opioid withdrawal in the human pediatric population.

Exaggerated nociceptive responses on morphine withdrawal: roles of protein kinase C epsilon and gamma.

On withdrawal from opioids many patients experience a heightened sensitivity to stimuli and an exaggerated pain response. The phenomenon has been little studied in infants. We present evidence that in postnatal day 7 rats an exaggerated nociceptive ventral root response of spinal cords in vitro and withdrawal-associated thermal hyperalgesia in vivo are dependent on protein kinase C (PKC), and we document the roles of PKC and gamma isozymes. In vitro, the slow ventral root potential (sVRP) is a nociceptive-related response in spinal cord that is depressed by morphine and recovers to levels significantly above control on administration of naloxone. A broad-spectrum PKC antagonist, GF109213X, blocked withdrawal hyperresponsiveness of the sVRP whereas an antagonist specific to Ca(++)-dependent isozymes, Go69076, did not. Consistent with this finding, a specific peptide inhibitor of calcium-independent PKC, but not an inhibitor of calcium-dependent PKC gamma, blocked withdrawal hyperresponsiveness of the sVRP. Similarly, in vivo in 7-day-old rat pups, inhibition of PKC, but not PKC gamma, prevented thermal hyperalgesia precipitated by naloxone at 30 min post-morphine. In contrast, thermal hyperalgesia during spontaneous withdrawal was inhibited by both PKC and gamma inhibitors. The consistency between the in vivo and in vitro findings with respect to naloxone-precipitated withdrawal provides further evidence that the sVRP reflects nociceptive neurotransmission. In addition the difference between naloxone-precipitated and spontaneous withdrawal in vivo suggests that in postnatal day 7 rats, morphine exposure produces an early phase of primary afferent sensitization dependent upon PKC translocation, followed by a later phase involving spinal sensitization mediated by PKC gamma.

Protein kinase C epsilon and gamma: involvement in formalin-induced nociception in neonatal rats.

The central nervous system undergoes dynamic changes as it matures. However, until recently, very little was known about the impact of these changes on pain and analgesia. This study tested the hypothesis that the epsilon and gamma isozymes of protein kinase C (PKC) contribute to formalin-induced nociception in an age-dependent manner. Expression of epsilon and gamma PKC and the contributions of these isozymes in formalin-induced nociception was examined in postnatal day 7, 15, and 21 rats. epsilonPKC expression in dorsal root ganglion neurons and gammaPKC expression in lamina II of the spinal cord increased from the first to the third postnatal week. Coupling immunohistochemical and Western analysis, translocation of epsilonPKC followed intraplantar formalin in all ages. In contrast, formalin-induced gammaPKC translocation was observed only in postnatal day 21 rats. Behaviorally, intrathecal administration of the epsilonPKC-specific inhibitor (epsilonV1-2) attenuated phase 1 and phase 2 formalin behaviors at all ages. In contrast, intrathecal administration of the gammaPKC-specific inhibitor (gammaV5-3) attenuated only phase 2 responses in postnatal day 15 and 21 rats. Functionally, inhibition of epsilonPKC decreased capsaicin-stimulated release of glutamate and calcitonin gene-related peptide in spinal cords isolated from postnatal day 7 rats. These results suggest that epsilonPKC age independently mediates inflammatory pain produced by intraplantar formalin. In contrast, gammaPKC contributes to formalin-induced nociception in an age-dependent manner. Identifying the molecular mechanisms responsible for age-specific patterns of nociception is necessary for the rational development of novel therapeutic strategies for treating pediatric pain.

Ethanol withdrawal hyper-responsiveness mediated by NMDA receptors in spinal cord motor neurons.

1. Following ethanol (EtOH) exposure, population excitatory postsynaptic potentials (pEPSPs) in isolated spinal cord increase to a level above control (withdrawal hyper-responsiveness). The present studies were designed to characterize this phenomenon and in particular to test the hypothesis that protein kinases mediate withdrawal. 2. Patch-clamp studies were carried out in motor neurons in rat spinal cord slices. Currents were evoked by brief pulses of glutamate, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) or N-methyl-D-aspartic acid (NMDA). 3. Of 15 EtOH-sensitive neurons in which currents were evoked by glutamate, four (27%) displayed withdrawal hyper-responsiveness in the washout period. Mean current area after washout was 129.6+/-5% of control. 4. When currents were evoked by AMPA, two of 10 neurons (20%) displayed withdrawal hyper-responsiveness, with a mean current area 122+/-8% of control on washout. 5. Of a group of 11 neurons in which currents were evoked by NMDA, nine (82%) displayed withdrawal hyper-responsiveness. Mean increase in current area at the end of the washout period was to 133+/-6% of control (n=9, P<0.001). When NMDA applications were stopped during the period of EtOH exposure, mean area of NMDA-evoked responses on washout was only 98.0+/-5% of control (n=6, P>0.05). 6. The tyrosine kinase inhibitor genistein (10-20 microM) blocked withdrawal hyper-responsiveness. Of six EtOH-sensitive neurons, the mean NMDA-evoked current area after washout was 89+/-6% of control, P>0.05. 7 The protein kinase A (PKA) inhibitor Rp-cAMP (20-500 microM) did not block withdrawal hyper-responsiveness. On washout, the mean NMDA-evoked current area was 124+/-6% of control (n=5, P<0.05). 8 Two broad-spectrum specific protein kinase C (PKC) inhibitors, GF-109203X (0.3 microM) and chelerythrine chloride (0.5-2 nM), blocked withdrawal hyper-responsiveness. Responses on washout were 108+/-7%, n=5 and 88+/-4%, n=4 of control, respectively, P>0.05. 9 NMDA activation during EtOH exposure is necessary for withdrawal hyper-responsiveness. Both tyrosine kinase and PKC, but not PKA, appear to be essential for EtOH withdrawal hyper-responsiveness mediated by postsynaptic NMDA receptors in spinal cord motor neurons.

Ethanol tachyphylaxis in spinal cord motorneurons: role of metabotropic glutamate receptors.

1. Ethanol (EtOH) tachyphylaxis (acute tolerance), a time-dependent decrease in apparent potency, is known in vivo and in some neuronal preparations. The present studies characterize EtOH tachyphylaxis in spinal motorneurons and test the hypothesis that metabotropic glutamate receptors (mGluRs) play a role. 2. Patch clamp studies were carried out in motorneurons in rat spinal cord slices. Currents were evoked by pulses of glutamate, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) or N-methyl-D-aspartic acid (NMDA). 3. In nine of 15 cells, ethanol depression of glutamate-evoked currents was time-dependent. EtOH depressed current area 36.9+/-3% at 8-10 min, but only 16.8+/-3% at 20 min. Mean reduction in depression was 20.1+/-1%, N=9. Tachyphylaxis was less prominent in currents evoked by AMPA or NMDA, appearing in two of 10 AMPA and three of 11 NMDA currents. 4. The mGluR agonist trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid (ACPD) increased, the antagonist (+/-)-alpha-methyl-4-carboxyphenylglycine (MCPG) decreased the area of glutamate-evoked currents. ACPD also increased the area of NMDA- and AMPA-evoked currents. 5. ACPD increased the incidence of tachyphylaxis in glutamate-evoked currents to 100% (N=9); MCPG markedly reduced tachyphylaxis. ACPD also increased the incidence of tachyphylaxis in currents evoked by NMDA and AMPA to five of eight and four of seven neurons, respectively. 6. Block of G-protein pathways by intracellular GDP-beta-s abolished tachyphylaxis in glutamate-evoked currents (N=8); however, currents recovered only partially following EtOH washout. 7. Activation of mGluRs contributes to neuronal tachyphylaxis to EtOH in spinal cord motorneurons, probably via G-protein pathways.

Enflurane decreases glutamate neurotransmission to spinal cord motor neurons by both pre- and postsynaptic actions.

UNLABELLED: We have previously reported volatile anesthetic actions on glycinergic inhibitory transmission to spinal motor neurons. The present study is a comparable set of experiments on glutamatergic excitatory transmission. We tested the hypothesis that the balance between excitation and inhibition is shifted toward inhibition by larger depressant actions on excitation. Patch-clamp techniques were used to study spontaneous and evoked glutamate alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid currents in rat spinal cord slices. Enflurane (0.6 mM, 1 minimum alveolar anesthetic concentration) significantly decreased spontaneous miniature current frequencies either when sodium channels were blocked (miniature excitatory postsynaptic currents, mEPSCs), or when sodium channels were not blocked (spontaneous excitatory postsynaptic currents, sEPSCs). Enflurane did not affect mEPSC or sEPSC amplitude or kinetics. The effects on mEPSCs and sEPSCs did not differ. Enflurane significantly decreased both amplitude and area of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-evoked currents with no change in kinetics (P < 0.05 and 0.01, respectively). In contrast, enflurane increased miniature glycinergic current frequency when sodium channels were blocked, and prolonged glycinergic current duration. Enflurane actions on glutamatergic excitatory transmission are purely depressant both pre- and postsynaptically, whereas glycinergic inhibition is enhanced presynaptically under some conditions, and always prolonged postsynaptically. Thus, enflurane shifts the balance between synaptic excitation and inhibition in the direction of inhibition. IMPLICATIONS: Explanations proposed for anesthetic-induced central nervous system depression include enhancement of synaptic inhibition and depression of excitation. The results reported herein suggest that, in the case of enflurane, the mechanism is a shift in the balance toward inhibition. Excitation is uniformly depressed by multiple mechanisms, whereas some anesthetic actions tend to enhance inhibition.

Pre- and postsynaptic volatile anaesthetic actions on glycinergic transmission to spinal cord motor neurons.

1. A common anaesthetic endpoint, prevention of withdrawal from a noxious stimulus, is determined primarily in spinal cord, where glycine is an important inhibitory transmitter. To define pre- and postsynaptic anaesthetic actions at glycinergic synapses, the effects of volatile anaesthetic agents on spontaneous and evoked glycinergic currents in spinal cord motor neurons from 6 - 14-day old rats was investigated. 2. The volatile anaesthetic agents enflurane, isoflurane and halothane significantly increased the frequency of glycinergic mIPSCs, enflurane to 190.4% of control+/-22.0 (mean+/-s.e.m., n=7, P<0.01), isoflurane to 199.0%+/-28.8 (n=7, P<0.05) and halothane to 198.2%+/-19.5 (n=7, P<0.01). However without TTX, isoflurane and halothane had no significant effect and enflurane decreased sIPSC frequency to 42.5% of control+/-12.4 (n=6, P<0.01). All the anaesthetics prolonged the decay time constant (tau) of both spontaneous and glycine-evoked currents without increasing amplitude. With TTX total charge transfer was increased; without TTX charge transfer was unchanged (isoflurane and halothane) or decreased (enflurane). 3. Enflurane-induced mIPSC frequency increases were not significantly affected by Cd(2+) (50 microM), thapsigargin (1 - 5 microM), or KB-R7943 (5 microM). KB-R7943 and thapsigargin together abolished the enflurane-induced increase in mIPSC frequency. 4. There are opposing facilitatory and inhibitory actions of volatile anaesthetics on glycine release dependent on calcium homeostatic mechanisms and sodium channels respectively. Under normal conditions (no TTX) the absolute amount of glycinergic inhibition does not increase. The contribution of glycinergic inhibition to anaesthesia may depend on its duration rather than its absolute magnitude.

Acetylcholine receptors do not mediate isoflurane's actions on spinal cord in vitro.

UNLABELLED: Extensive studies on anesthetic mechanisms have focused on the nicotinic acetylcholine receptor, and to a lesser extent on the muscarinic receptor. We designed the present study to test the hypothesis that cholinergic receptors mediate some of the depressant actions of a volatile anesthetic in rat spinal cord. The cord was removed from 2- to 7-day-old rats and superfused in vitro; ventral root potentials were evoked by stimulating a lumbar dorsal root and recording from the corresponding ipsilateral ventral root. Both nicotine and muscarine depressed the nociceptive-related slow ventral root potential (sVRP). The nicotinic antagonists mecamylamine, methyllycaconitine, dihydro-beta-erythroidine, and the muscarinic antagonist atropine blocked the depressant effects of the respective agonists. Isoflurane 0.3 mini- mum alveolar anesthetic concentration depressed the sVRP area to approximately 40% of control. None of the antagonists changed the extent of isoflurane depression of the sVRP. The depressant actions of cholinergic agonists suggest that cholinergic receptors are important in spinal neurotransmission, but the lack of interaction between antagonists and isoflurane suggests that cholinergic receptors have little part in mediating the actions of this anesthetic in spinal cord. Because minimum alveolar anesthetic concentration is determined primarily in spinal cord, cholinergic receptors may be eliminated as molecular targets for this anesthetic end-point. IMPLICATIONS: Neither nicotinic nor muscarinic acetylcholine receptor antagonists altered spinal cord actions of isoflurane, suggesting that these receptors have little role in isoflurane actions in spinal cord. Cholinergic receptors thus may be eliminated as molecular targets in determining the anesthetic end-point of immobility in response to a noxious stimulus (minimum alveolar anesthetic concentration).

Acetylcholine receptors do not mediate the immobilization produced by inhaled anesthetics.

UNLABELLED: Acetylcholine receptors transmit excitatory impulses, are broadly distributed throughout the central nervous system, and are particularly sensitive to the depressant effects of inhaled anesthetics. Thus these receptors are potential mediators of the immobility produced by inhaled anesthetics. We tested this potential in rats by giving intraperitoneal atropine, scopolamine, and mecamylamine to block muscarinic (atropine and scopolamine) and neuronal nicotinic (mecamylamine) acetylcholine receptors. Block with scopolamine (up to 100 mg/kg), atropine (10 mg/kg), mecamylamine (up to 4 mg/kg), or atropine (10 mg/kg) plus mecamylamine (up to 4 mg/kg) did not significantly decrease the isoflurane concentration required to suppress movement to noxious stimulation (minimum alveolar anesthetic concentration). We also gave atropine intrathecally, finding that the infusions that did not cause permanent paralysis produced slight or no decreases in the minimum alveolar anesthetic concentration. We conclude that acetylcholine receptors do not seem to play a role as mediators of immobilization by inhaled anesthetics. IMPLICATIONS: Inhaled anesthetics produce two crucial effects: amnesia and immobility in the face of noxious stimulation. Block of muscarinic and neuronal nicotinic acetylcholine receptors in rats does not significantly decrease the isoflurane concentration required to suppress movement to stimulation. Thus, acetylcholine receptors do not seem to play a major role as mediators of the immobilization produced by inhaled anesthetics. Their capacity to mediate other effects of inhaled anesthetics (e.g., amnesia) remains to be tested.