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Anesthesiology:
doi: 10.1097/ALN.0b013e31819c461c
2008 Best Abstracts of the Meeting: Anesthesiology Editors' Picks

Thalamic Microinfusion of Antibody to a Voltage-gated Potassium Channel Restores Consciousness during Anesthesia

Alkire, Michael T. M.D.*; Asher, Christopher D. M.S.†; Franciscus, Amanda M. B.S.‡; Hahn, Emily L. B.A.§

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POTASSIUM (K) channels play a major role in regulating tissue excitability. There are at least four different types of K channels that serve slightly different functions in the nervous system, including voltage-gated (Kv), calcium-activated (KCa), inward rectifying (Kir), and 2-pore (K2P) domain background leak channels.1 Many authors have suggested that K channels are involved in producing the effects of anesthesia.1–6 Much recent focus has been placed on distinguishing K2P channels as possible targets of anesthetic action,6,7 yet interactions with various other K channels like the Kv channels may also be important.2,3,8,9
Kv channels are further divided into 12 families (Kv1–Kv12) on the basis of sequence homology and similarity to Drosophila melanogaster (i.e., fruit-fly) genes.10 In Drosophila, the Kv channel genes produce multiple versions of a particular channel and are more commonly known by historical nomenclature such as: Shaker (Kv1.1–Kv1.8), Shab (Kv2.1–Kv2.2), Shaw (Kv3.1–Kv3.4), Shal (Kv4.1–Kv4.3), and ether-a-go-go (Kv10.1–Kv10.2). The eight members of the Shaker-related K channel family (Kv1.1–Kv1.8) are involved with generating voltage-dependent outward currents that regulate action potential threshold, as well as waveform and pacemaker activity in excitable tissue. Kv channels are generally composed of tetramers of alpha subunits. When they are expressed as homomeric channels, most have “delayed rectifier” properties, and the others will exhibit fairly rapid inactivation.11 The different Kv1.x family members (where x is any one of the possible eight different subunits) can coassemble into channels with mixed heteromeric alpha subunit compositions. Furthermore, Kv-beta subunits also exist and add on another layer of complex functional diversity in vivo.11 The subunit composition of the Kv1 channels not only determines their gating and kinetic properties, it also dramatically affects their expression and localization.11
The idea that Kv channels might play a role in anesthesia emerged from the discovery that Drosophila Shaker mutants shake their legs vigorously during ether anesthesia.12,13 The Shaker mutant lacks a normal functioning Kv1.x channel, suggesting that the suppression of neural activity under ether anesthesia depends to some extent on a properly functioning Kv1.x channel. Indeed, the amount of isoflurane needed to anesthetize a Shaker mutant with a completely nonfunctioning Kv1.x channel is more than twice the dose needed to anesthetize wild-type flies.14 Importantly, the changes in isoflurane doses needed to anesthetize various other Shaker mutants parallels the expected reductions in ionic currents mediated through the respective malfunctioning K channels.14 In other words, the more defective the K channel (and the less current that passes through it), the greater the dose of isoflurane needed to anesthetize a particular Shaker mutant. This seems to suggest that anesthesia might work in part by hijacking the functioning of the Kv1.x channel.
Recently, the unconsciousness of sleep was also linked to a voltage-dependent K channel.15 Mutagenesis analysis was used to screen more than 9,000 Drosophila lines to identify those with a limited ability to sleep. Genetic analysis of these flies revealed a point mutation in a conserved domain of the Shaker gene that involves the voltage sensing portion of Kv1.2 channel.15,16 Thus, for Drosophila to sleep, it appears to need current to properly flow through its Kv1.2 channels. Taken together with the earlier anesthesia work, this suggests Kv1.2 channels might be involved with mediating the effects of volatile anesthetics on consciousness.
Concurrent with the developments in sleep neurophysiology, a possible role for the central medial thalamus (CMT) in contributing to the unconsciousness of anesthesia was recently identified.17 Anesthetic-induced unconsciousness can be reversed with a localized and site-specific microinfusion of nicotine into the CMT of rats.17 As the loss of consciousness associated with sleep and anesthesia may share overlapping neurobiological mechanisms,18–21 and nicotine is known to also block various K channels,22,23 including some Kv channels,24 the hypothesis is raised that anesthetic effects on consciousness might involve interactions with Kv1.2 channels located, in part, in the CMT. Herein, we open the investigation into this area of research by microinfusing a Kv1.2 channel blocking antibody directly into the CMT of rats placed in an anesthetic chamber exposed to a dose of inhalational agent that is just sufficient to render them unconscious. As in the case of Drosophila Shaker mutants, which have a chronic malfunction of Kv1.x channels, the acute conduction blockade of the Kv1.2 channels in the CMT should act to rapidly increase the anesthetic dose required to keep the animals unconscious. As the dose of anesthesia will be held constant after the localized antibody microinfusion, a positive result (indicating the possible contribution of Kv1.2 channels to inducing the unconsciousness of anesthesia) will be manifest as a behavioral arousal of the animals; that is, they should awaken in the chamber filled with anesthesia.
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Materials and Methods

All research activities were conducted with full approval of the Institutional Animal Care and Use Committee of the University of California, Irvine.
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Animals
A total of 106 Sprague-Dawley rats (250–280 g or approximately 9 weeks old on arrival) were obtained from Charles River Laboratories, Inc. (Wilmington, MA). They were housed individually in a temperature-controlled (22°C) colony room, with food and water available ad libitum. Animals were maintained on a 12-h light, 12-h dark cycle (0700–1900 lights on).
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Surgery
Rats were anesthetized with sodium pentobarbital (50 mg/kg, intraperitoneal) and placed into a stereotaxic frame (Benchmark Digital Stereotaxic, Saint Louis, MO). A guide cannula (23-gauge) was placed, aimed at the central medial thalamus (coordinates: anteroposterior –3.0 mm; mediolateral +1.7 mm, with 13-degree tilt; dorsoventral –4.5 mm; incisor bar, –3.3 mm). The guide cannula was 2 mm shorter in length than needed to reach the central medial thalamus. Indeed, the end of the guide cannula did not reach into the thalamus proper. The thalamus was entered only at the time of the experiments when the microinfusion was delivered through a microinfusion needle inserted into the guide cannula that was 2 mm longer than the guide cannula itself. For the animals given desflurane anesthesia (n = 55), all cannulae targeted the CMT. For the animals given sevoflurane (n = 51), most targeted the CMT, but ten animals were used as location controls; five targeted the ventral lateral thalamic nucleus (coordinates: anteroposterior –3.0 mm; mediolateral +1.7 mm; dorsoventral –4.5 mm), and five targeted the posterior thalamic nucleus (coordinates: anteroposterior –3.0 mm; mediolateral +1.7 mm; dorsoventral –3.5 mm). Dental acrylic and skull screws secured each cannula. Animals were allowed 6–7 days to recover before experiments.
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Drugs
The Kv1.2 antibody was a gift from Chiara Cirelli, M.D., Ph.D. (Associate Professor, Department of Psychiatry, University of Wisconsin, Madison, Wisconsin) and Giulio Tononi, M.D., Ph.D. (Professor, Department of Psychiatry, University of Wisconsin, Madison, Wisconsin). Kv1.2 rabbit polyclonal antibodies were made and affinity-purified through a contracted manufacturer (Genemed Synthesis Inc, San Francisco, CA), during performance of a grant with the Defense Advanced Research Projects Agency. The antibody was manufactured by following the specifications of Zhou et al.25 Zhou et al. generated specific antipeptide antibodies to epitopes in the external vestibule of the Kv1.2 delayed-rectifier potassium channel. Their antibody was found to block 70% of the whole-cell Kv1.2 currents in transfected cells in a concentration and time-dependent manner.25 Specificity was established by showing that the antibody did not block currents to Kv1.3 or Kv3.1 channels, and binding was mutually exclusive with α-dendrotoxin,25 a channel blocker that also binds to the external vestibule of the Kv1.2 channel.26 In the current work, the antibody was diluted in normal saline immediately before infusion into the thalamus of anesthetized rats. Initial infusions were performed with a concentration of 0.2 mg/ml antibody in 0.5-μl infusion volume given over 1 min. A large proportion of seizure responses prompted the lowering of the dose used to 0.1 mg/ml antibody in the same 0.5-μl infusion volume.
As antibodies are relatively large molecules (approximately 150 kDa), the in vivo use of an antibody infusion given directly into a discrete region of the brain might cause some type of nonspecific dysfunction to occur. Whereas many examples of using antibodies as in vivo probes for specific receptor-targets now exist,27–31 we nevertheless controlled for the possibility that a nonspecific arousal effect might occur due to the infusion of an antibody itself. To evaluate this possibility, we infused nine animals under sevoflurane anesthesia with an antibody directed against an intracellular nonreceptor target, the activity regulated cytoskeletal (Arc) protein. This Arc rabbit polyclonal antibody was purchased from a commercial vendor (BioVision Research Products, Mountain View, CA). We injected 0.2 mg/ml Arc antibody in phosphate-buffered saline given in a single 0.5-μl microinfusion.
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Consciousness Suppression with Anesthesia
Fig. 1
Fig. 1
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After recovery from cannula implantation (after 6–7 days), animals were anesthetized in a clear chamber as previously described.17 Briefly, animals were placed in a rectangular 8-l clear Plexiglas anesthetizing chamber and exposed to anesthesia in air at 2 l · min−1 until they lost their righting reflex (fig. 1). Anesthetic chamber agent concentrations were monitored continuously during the experiments using a Datex-Ohmeda Ultima Capnomac (Helsinki, Finland) and verified with gas chromatography (Model 80123B; SRI Instruments, Redondo Beach, CA). The chamber had a small door on one side, through which the animal was initially placed. The chamber also had small ports that served as the anesthetic gas inlet, the microinfusion tubing port inlet, two gas monitor sampling ports, and one gas chromatograph sampling port. Once each rat was well anesthetized, the door was partially opened, and a 25-gauge microinfusion needle was quickly inserted through the guide cannula, and the rat was placed onto its back in the center of the chamber. The needle was attached by a polyethylene tube through the wall of the anesthetizing chamber to a 10-μl syringe (Hamilton, Reno, NV), which was driven by a minipump (Harvard Apparatus, Holliston, MA). The chamber anesthesia concentration was then lowered to 3.6% for desflurane or to 1.2% for sevoflurane. The concentration was held stable for 20 min before a microinfusion was delivered. However, if a rat showed any spontaneous movement during this stabilization period, the chamber concentration was increased by 0.1% increments until each rat remained motionless for at least 20 min. Thus, the chamber concentration varied slightly depending on a specific animal’s behavior. Rats were thus anesthetized, in separate experiments, with either desflurane (3.6 ± 0.2%: n = 55) or sevoflurane (1.2 ± 0.1%: n = 51).
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Arousal Response Determinations
Responses to a single infusion of antibody per rat were graded as one of four levels: 1 = no effect-no visible movements; 2 = partial arousal – signs of arousal including eye opening and movements of extremities; 3 = full arousal-the complete turning of the animal onto its stomach, while exhibiting purposeful movements; 4 = seizures-focal or generalized tonic-clonic seizures.
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Histology
Brains were sliced into 40-μm sections and stained with thionin. Microinfusions were localized blinded to behavioral data. Data were incomplete in 7 rats that expired during surgery or were euthanized due to clogged or missing cannula. Infusion sites were projected onto the –3-mm coronal brain section from the atlas of Paxinos and Watson.32 However, a few infusions were located within ± 1.0 mm in the anteroposterior dimension.
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Statistics
The hypothesis that the CMT is involved with mediating the resumption of consciousness after antibody infusion was examined separately in both the desflurane- and the sevoflurane-exposed animals using Fisher exact test. We compared the histology of those animals showing a resumption of consciousness with those animals that failed to show an effect from the infusion. P < 0.05 was considered significant.
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Results

Of the nine animals given a control antibody microinfusion, none showed any behavioral effects, regardless of cannula placement in the CMT (n = 4) or other thalamic areas (n = 5), data not shown. Previous control microinfusions of saline alone into the CMT were also found to be without effect.17
Table 1
Table 1
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Overall behavioral responses to the Kv1.2 antibody are summarized in table 1. The no-effect response was seen in 30.0% of the overall proportion of rats studied; partial arousal was seen in 13.4% of the rats; full return to consciousness was seen in 16.5% of the rats. Righting occurred on average (± SD) 170 ± 99 s after the infusion and lasted a median time of 398 s (interquartile range: 279–510 s). A representative example of the resumption of consciousness is shown in figure 1 and can be seen online (see video, Supplemental Digital Content 1, which demonstrates the arousal response illustrated in fig. 1, http://links.lww.com/A823). Seizures were seen in 33% of the rats.
To assess whether the arousal reactions to the antibody represented some type of internal pain response, we also qualitatively evaluated the appearance of arousal to pain. In seven pilot animals under sevoflurane anesthesia, we tested arousal responses to a 1-mA 60-s tail-shock stimulation. The animals did move their tails and feet in response to this stimulation, and four were able to slowly curl up onto their sides during the stimulation, with two flopping onto their stomach. Yet, the qualitative nature of this type of arousal was much different from the antibody effect. It lasted only as long as the stimulus was applied, and the animals did not seem to be focally conscious, with alert looking around. With the antibody infusion, the animals appeared to regain some level of higher consciousness; they could move around in the chamber in a crawling fashion, and they responded to environmental sights and sounds. They did not appear to be in pain, as they did not seem to focus on any particular part of their body. They were somewhat uncoordinated in their movements, which might be expected; from a systems perspective, essentially nothing was done to reduce the effects of the anesthesia on their cerebellum or spinal cord areas.
Fig. 2
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Fig. 3
Fig. 3
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Fig. 4
Fig. 4
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The histology results for desflurane are shown in figure 2, and the histology results for sevoflurane are shown in figure 3. Infusion needle-tip locations for the no-effect group versus the consciousness-restored groups for desflurane and sevoflurane are shown in figures 2A and 3A, respectively. Statistical analyses revealed that the resumption of consciousness was significantly related to infusions hitting the CMT for both agents, as also shown in figures 2B and 3B, respectively. When the infusion needle-tip was located in the CMT, 75% of those animals awoke from the anesthesia. Notably, rats having seizures often also had infusions directly into the CMT, as shown in figure 4. The animals that seized did not pass through an apparent state of progressively more arousal; rather their first movements were generally those of seizure-like activity. This was qualitatively interpreted as a dose-related effect such that too much antibody delivered directly into the CMT caused too much of a generalized excitation phenomenon for a particular rat.
The rats were allowed to recover from the infusion experiments, and all, including those that had seized, appeared to awaken normally. In subsequent days and before histologic examination, they all exhibited normal rat behaviors and were able to feed, drink, and groom normally.
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Discussion

Microinfusing an antibody designed specifically to block the external vestibule of Kv1.2 voltage-gated potassium channels into the CMT of anesthetized rats caused a number of animals to display a temporary resumption of consciousness with restored mobility in a chamber filled with inhalational anesthesia. With histology examination of the animals’ brains, it was found that an arousal response occurred in 75% of those animals where the infusion needle-tip was located within the CMT. Taken together, these findings strongly implicate the CMT as an important brain site involved with regulating levels of arousal during anesthesia and further serve to suggest that the underlying mechanism for this localized site-specific arousal effect involves anesthetic interactions with voltage-gated potassium channels.
The mechanism by which consciousness is suppressed during anesthesia remains unknown. The seminal observation by Franks and Leib in 1984 that anesthetic potency correlates with the suppression of firefly luciferase protein activity shifted the search for the molecular mechanisms of anesthesia from lipids to proteins.33 Many studies have since detailed how various protein ion channels are affected by numerous anesthetic substances.2,7,34–36 Yet, it remains unclear which molecular targets are the most relevant for causing the clinical effects of anesthesia.2,7,37 The prevailing view is that anesthetic actions on ligand-gated ion channels, such as γ-aminobutyric acid type A, glycine, neuronal nicotinic channels, N-methyl-d-aspartate, or 2-pore domain background potassium channels, are the molecular targets most directly related to producing the clinical effects of anesthetics on consciousness and immobility.7,36,37 Yet, a role for voltage-gated K channels in mediating the effects of anesthetics on consciousness has also been proposed.2,8,14
Kv1.2 channels are densely located within the thalamus and cortex,38 but the early published reports suggest that they are not as densely found within the CMT as one might have anticipated from the current results. This raises the question of why exactly the CMT appeared to be the focus of the current effect. The CMT is part of the nonspecific intralaminar thalamic arousal system.39 It connects with brainstem areas mediating arousal and projects to wide expanses of cortical and basal ganglia areas.40,41 It receives afferents from hypothalamic areas involved with controlling sleep and arousal.18,20 Given its wide projection pattern onto cortex, it is possible that the effects found localized to the CMT area represent an influence on the projections to or from this area (or fibers of passage), rather than on the CMT cell bodies themselves. The large number of seizures found with injections around the midline thalamic area support the idea that this region is involved in regulating overall levels of cortical excitability.42 The findings reported here suggest that voltage-gated potassium channels in the CMT may contribute more to regulating arousal through localized network interactions than previously thought. Small effects on these channels can have large system-wide effects within neural networks and brain systems for which spike timing is a critical element of the transmission of information.2,21,43–45
However, it is important to clarify a number of issues related to these findings. First, the findings are primarily significant for adding further support to the idea that the CMT is an important node in an arousal network that may directly or indirectly interact with anesthesia. The amount of infusion volume used was only 0.5 μl. This is a much smaller volume than many in vivo studies use,27 and it suggests that the effects found are localized to a very small area immediately around the infusion sites that encompasses a size of less than approximately 0.25–0.5 mm. This small infusion volume was used to minimize spread from the needle-tip and help provide localization of the effects. A number of the infusions that hit the CMT or were near it did not cause an arousal reaction. This is likely due to the delivery of an insufficient dose with a particular infusion. The CMT interacts with both ascending (from brainstem),46,47 and descending (from cortex) arousal pathways.48 From an anesthesia perspective, the CMT receives input from the hypothalamus,40 a connection that helps modulate the sedation effects of anesthetics through GABAergic or orexinergic effects.18,49 It also interacts with the mesopontine tegmental anesthesia area,47 a brainstem region that induces an apparent anesthetic-like state when microinfused with barbiturate.50 Pharmacologic manipulations of the CMT can both directly enhance arousal and produce sedation effects.42,51 Microinfusion of nicotine into the CMT of anesthetized rats restores behavioral arousal despite continued anesthetic exposure.17 Microinfusion of a γ-aminobutyric acid agonist muscimol is reported to cause a sedation response.52 Taking these facts together with the current findings strongly suggests that the CMT is intimately involved with regulating levels of arousal during anesthesia.
Second, it should not be assumed without much further work that the arousal effect associated with the infusion of the Kv1.2 channel blocking antibody into the CMT is directly related to antagonism of a specific mechanism of anesthesia. This is certainly one possibility, but other evidence suggests this is an unlikely possibility. In vitro studies examining the effects of anesthetics on voltage-gated potassium channels show that these channels are affected by anesthetics,9 but generally only at doses much greater than those that are clinically relevant.53 One exception to this generalization is evident for Shaw2 mutant Kv channels, which appear to be highly sensitive to certain anesthetics.8 Yet, the effects on these and most other Kv channels is generally one of current blockade. Thus, in the present work, if anesthesia is acting to block currents through the Kv channels and the Kv1.2 antibody is also acting to block currents, then it seems more likely that the antibody should have enhanced sedation, rather than causing an arousal reaction. Nevertheless, the answer to this apparent contradiction may lie in the extreme biologic diversity of Kv channels, where it could be speculated that some heteromeric subunit combinations may exist that can produce anesthetic-sensitive channels that do open in response to anesthetic exposure. Another speculation further illustrates the potentially indirect nature of these findings. Assuming that the antibody did block the Kv1.2 channels in the CMT and functionally eliminated them, this would act to acutely change the firing patterns of the CMT neurons involved, but it would not stop the actions of the same anesthetic from affecting any other anesthetic- sensitive channels, such as GABAeric, glycinergic, cholinergic or K2P channels. The ultimate effect on thalamic neuronal firing patterns is likely the result of the combined contributions of multiple influences on the cell membrane potential7,54; therefore, the blocking of the Kv1.2 channel can be seen as just one additional influence that changes the cell’s membrane potential and hence its likelihood of entering a particular pattern of action potential firing.
Third, generalized nonspecific effects of an antibody infusion into the CMT are unlikely to be the source of the arousal responses, as the control antibody infusions did not cause any reactions. In addition, a nonspecific arousal effect due to some type of internal pain-like state is unlikely because the qualitative nature of an arousal response to a painful stimulation was much different from that seen with the antibody effect. However, nonspecific binding effects of the Kv1.2 antibody cannot be ruled out. Antibodies are affinity reagents. This means that even though they have tremendous affinity for their target antigen, some level of low-affinity crossreactivity to other closely related protein sequences is common and often contributes to the signal. Thus, it is likely that most of the Kv1.2 antibody bound to the Kv1.2 receptor, but it might also have bound to other similar receptors or even to other similar channels. It is often seen with immunohistochemistry that nonspecific binding can occur to an extent that is sufficient to overwhelm the intended signal of interest.
How specific is this polyclonal antibody for blocking only Kv1.2 ion channels? From the original Zhou et al. work, the specificity between Kv1.2 and Kv1.3 is quite good.25 Yet, the antibody was not tested directly against Kv1.1 or Kv1.6 channels. This may be important because Kv1.1, Kv1.2, and Kv1.6 channels all show similar affinity for the binding of α-dendrotoxin.55 On this basis alone, it would be reasonable to assume that some crossreactivity of the Kv1.2 channel blocking antibody with the Kv1.1 and Kv1.6 channels may have occurred. Only further work with more specific versions of various toxins44 or the development of monoclonal antibodies might help clarify to what extent some crossreactivity may have influenced these findings.30,55 Indeed, the development of monoclonal antibodies for use in the specific blocking of various channels in vivo is now almost routine,28,31,56 though the polyclonal approach remains an established technique.29
If one speculates that the unconsciousness of anesthesia occurs through the opening of thalamic Kv1.2 channels and that the antibody blocked this open pore to restore consciousness, then why did the suppression of the unconsciousness response not last indefinitely? It is conceivable that the antibody may have dissociated from the receptor in a relatively short period of time, but this seems unlikely given the nature of antibody binding. More likely would be the possibility that receptor trafficking played some role in the termination of the response.57 In addition, the arousal response itself may have been due to presynaptic actions serving to decrease the functioning of GABAergic neurons, causing a temporary inhibition of inhibition or a disinhibition reaction.58,59 Another possibility is that a nonspecific generalized arousal reaction centered on the midline thalamus may have contributed to the response, such has been seen when acute ibotenic acid infusions are given into the midline thalamus.60 Further testing with other excitatory substances such as glutamate and even potassium itself would seem warranted.
One approach to identify appropriate targets of anesthetic action is to genetically modify specific ion channels and then evaluate the behavioral effects of such mutations on the various end-points of anesthesia in the mutant animals.61 Another relatively new approach is to use antibody-based validation of relevant ion channel drug targets.30 This approach has a number of potential advantages for use in anesthesia research. (1) After antibody development, the target selectivity and specificity of binding is essentially unparalleled. (2) Normal wild-type animals can be studied. This eliminates the fear that some compensatory mechanisms might interact with the behavior of interest, as may be the case with mutant animals. (3) The behavioral effects of any particular antibody-binding response can be determined, and then the underlying regional and even neuronal site-specificity associated with the responses can be identified with immunohistochemical techniques. The antibody approach, however, is not without its limitations. Proper validation and development of target specificity is enormously expensive, time consuming, and generally beyond the means and experience of most investigators.30 Thus, antibodies are usually borrowed from colleagues who have already developed them (i.e., limited availability), or they are commissioned from an antibody reagent company. The quality control on such products can vary substantially, and the inadvertent use of a not-so-specific antibody can prove greatly misleading.62 Nevertheless, the antibody approach may represent one pathway out of the quagmire that currently complicates the mechanism(s) of anesthesia research field.
Finally, it is important to note that this is not the first demonstration to link a site-specific change in potassium channel functioning to the hypnotic component of anesthesia and show an ability to antagonize the consciousness-suppressing effect of an anesthetic. Infusions of various K channel blockers, dendrotoxin (Kv), charybdotoxin (KCa), and quinine (KCa and Kv) were found to reduce the hypnotic effect of the selective α2-adrenergic agonist dexmedetomidine when they were delivered discretely into the locus coeruleus of rats.63 Taken together with the current findings, the ability of interactions with voltage-gated potassium channels to antagonize the hypnotic component of at least three different anesthetic agents (i.e., sevoflurane, desflurane, and dexmedetomidine) in vivo would seem to identify these channels as prime targets in need of further study.
The authors thank Giulio Tononi, M.D., Ph.D., Professor, and Chiara Cirelli, M.D., Ph.D., Associate Professor (University of Wisconsin, Madison, Wisconsin), for the generous gift of Kv1.2 channel blocking antibody. The authors also thank James L. McGaugh, Ph.D. (Professor of Neurobiology and Behavior, University of California-Irvine, Irvine, California), for his continued support and Yasmin Khowaja, B.S. (Staff Research Associate, University of California-Irvine), for technical assistance.
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References

1. Yost CS: Potassium channels: Basic aspects, functional roles, and medical significance. Anesthesiology 1999; 90:1186–203

2. Arhem P, Klement G, Nilsson J: Mechanisms of anesthesia: Towards integrating network, cellular, and molecular level modeling. Neuropsychopharmacology 2003; 28(Suppl 1):S40–7

3. Yamakura T, Lewohl JM, Harris RA: Differential effects of general anesthetics on G protein-coupled inwardly rectifying and other potassium channels. Anesthesiology 2001; 95:144–53

4. Franks NP, Honore E: The TREK K2P channels and their role in general anaesthesia and neuroprotection. Trends Pharmacol Sci 2004; 25:601–8

5. Patel AJ, Honore E: Anesthetic-sensitive 2P domain K+ channels. Anesthesiology 2001; 95:1013–21

6. Patel AJ, Honore E: 2P domain K+ channels: Novel pharmacological targets for volatile general anesthetics. Adv Exp Med Biol 2003; 536:9–23

7. Franks NP: General anaesthesia: From molecular targets to neuronal pathways of sleep and arousal. Nat Rev Neurosci 2008; 9:370–86

8. Covarrubias M, Rubin E: Ethanol selectively blocks a noninactivating K+ current expressed in Xenopus oocytes. Proc Natl Acad Sci U S A 1993; 90:6957–60

9. Zorn L, Kulkarni R, Anantharam V, Bayley H, Treistman SN: Halothane acts on many potassium channels, including a minimal potassium channel. Neurosci Lett 1993; 161:81–4

10. Gutman GA, Chandy KG, Adelman JP, Aiyar J, Bayliss DA, Clapham DE, Covarriubias M, Desir GV, Furuichi K, Ganetzky B, Garcia ML, Grissmer S, Jan LY, Karschin A, Kim D, Kuperschmidt S, Kurachi Y, Lazdunski M, Lesage F, Lester HA, McKinnon D, Nichols CG, O’Kelly I, Robbins J, Robertson GA, Rudy B, Sanguinetti M, Seino S, Stuehmer W, Tamkun MM, Vandenberg CA, Wei A, Wulff H, Wymore RS: International Union of Pharmacology.XLI. Compendium of voltage-gated ion channels: Potassium channels. Pharmacol Rev 2003; 55:583–6

11. Trimmer JS, Rhodes KJ: Localization of voltage-gated ion channels in mammalian brain. Annu Rev Physiol 2004; 66:477–519

12. Trout WE, Kaplan WD: A relation between longevity, metabolic rate, and activity in shaker mutants of Drosophila melanogaster. Exp Gerontol 1970; 5:83–92

13. Timpe LC, Schwarz TL, Tempel BL, Papazian DM, Jan YN, Jan LY: Expression of functional potassium channels from Shaker cDNA in Xenopus oocytes. Nature 1988; 331:143–5

14. Tinklenberg JA, Segal IS, Guo TZ, Maze M: Analysis of anesthetic action on the potassium channels of the Shaker mutant of Drosophila. Ann N Y Acad Sci 1991; 625:532–9

15. Cirelli C, Bushey D, Hill S, Huber R, Kreber R, Ganetzky B, Tononi G: Reduced sleep in Drosophila Shaker mutants. Nature 2005; 434:1087–92

16. Douglas CL, Vyazovskiy V, Southard T, Chiu SY, Messing A, Tononi G, Cirelli C: Sleep in Kcna2 knockout mice. BMC Biol 2007; 5:42

17. 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

18. 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

19. Lydic R, Baghdoyan HA: Sleep, anesthesiology, and the neurobiology of arousal state control. Anesthesiology 2005; 103:1268–95

20. 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

21. Alkire MT, Hudetz AG, Tononi G: Consciousness and anesthesia. Science 2008; 322:876–80

22. Wang H, Shi H, Zhang L, Pourrier M, Yang B, Nattel S, Wang Z: Nicotine is a potent blocker of the cardiac A-type K(+) channels. Effects on cloned Kv4.3 channels and native transient outward current. Circulation 2000; 102:1165–71

23. Wang H, Yang B, Zhang L, Xu D, Wang Z: Direct block of inward rectifier potassium channels by nicotine. Toxicol Appl Pharmacol 2000; 164:97–101

24. Fu XW, Nurse C, Cutz E: Characterization of slowly inactivating KV{alpha} current in rabbit pulmonary neuroepithelial bodies: Effects of hypoxia and nicotine. Am J Physiol Lung Cell Mol Physiol 2007; 293:L892–902

25. Zhou BY, Ma W, Huang XY: Specific antibodies to the external vestibule of voltage-gated potassium channels block current. J Gen Physiol 1998; 111:555–63

26. Hurst RS, Busch AE, Kavanaugh MP, Osborne PB, North RA, Adelman JP: Identification of amino acid residues involved in dendrotoxin block of rat voltage-dependent potassium channels. Mol Pharmacol 1991; 40:572–6

27. Faraguna U, Vyazovskiy VV, Nelson AB, Tononi G, Cirelli C: A causal role for brain-derived neurotrophic factor in the homeostatic regulation of sleep. J Neurosci 2008; 28:4088–95

28. Gomez-Varela D, Zwick-Wallasch E, Knotgen H, Sanchez A, Hettmann T, Ossipov D, Weseloh R, Contreras-Jurado C, Rothe M, Stuhmer W, Pardo LA: Monoclonal antibody blockade of the human Eag1 potassium channel function exerts antitumor activity. Cancer Res 2007; 67:7343–9

29. Liao YJ, Safa P, Chen YR, Sobel RA, Boyden ES, Tsien RW: Anti-Ca2+ channel antibody attenuates Ca2+ currents and mimics cerebellar ataxia in vivo. Proc Natl Acad Sci U S A 2008; 105:2705–10

30. Rhodes KJ, Trimmer JS: Antibody-based validation of CNS ion channel drug targets. J Gen Physiol 2008; 131:407–13

31. Sheehan KC, Lai KS, Dunn GP, Bruce AT, Diamond MS, Heutel JD, Dungo-Arthur C, Carrero JA, White JM, Hertzog PJ, Schreiber RD: Blocking monoclonal antibodies specific for mouse IFN-alpha/beta receptor subunit 1 (IFNAR-1) from mice immunized by in vivo hydrodynamic transfection. J Interferon Cytokine Res 2006; 26:804–19

32. Paxino G, Watson C: The Rat Brain in Stereotaxic Coordinates. 5th edition. Burlington, MA, Elsevier Academic Press, 2005

33. Franks NP, Lieb WR: Seeing the light: Protein theories of general anesthesia. Anesthesiology 2004; 101:235–7

34. Campagna JA, Miller KW, Forman SA: Mechanisms of actions of inhaled anesthetics. N Engl J Med 2003; 348:2110–24

35. Rudolph U, Antkowiak B: Molecular and neuronal substrates for general anaesthetics. Nat Rev Neurosci 2004; 5:709–20

36. Franks NP: Molecular targets underlying general anaesthesia. Br J Pharmacol 2006; 147(Suppl 1):S72–81

37. Sonner JM, Antognini JF, Dutton RC, Flood P, Gray AT, Harris RA, Homanics GE, Kendig J, Orser B, Raines DE, Rampil IJ, Trudell J, Vissel B, Eger EI 2nd: Inhaled anesthetics and immobility: Mechanisms, mysteries, and minimum alveolar anesthetic concentration. Anesth Analg 2003; 97:718–40

38. Kues WA, Wunder F: Heterogeneous expression patterns of mammalian potassium channel genes in developing and adult rat brain. Eur J Neurosci 1992; 4:1296–308

39. Bogen JE: Some neurophysiologic aspects of consciousness. Semin Neurol 1997; 17:95–103

40. Van der Werf YD, Witter MP, Groenewegen HJ: The intralaminar and midline nuclei of the thalamus. Anatomical and functional evidence for participation in processes of arousal and awareness. Brain Res Brain Res Rev 2002; 39:107–40

41. Krout KE, Belzer RE, Loewy AD: Brainstem projections to midline and intralaminar thalamic nuclei of the rat. J Comp Neurol 2002; 448:53–101

42. Miller JW, Hall CM, Holland KD, Ferrendelli JA: Identification of a median thalamic system regulating seizures and arousal. Epilepsia 1989; 30:493–500

43. Barnes-Davies M, Barker MC, Osmani F, Forsythe ID: Kv1 currents mediate a gradient of principal neuron excitability across the tonotopic axis in the rat lateral superior olive. Eur J Neurosci 2004; 19:325–33

44. Dodson PD, Barker MC, Forsythe ID: Two heteromeric Kv1 potassium channels differentially regulate action potential firing. J Neurosci 2002; 22:6953–61

45. Trussell LO: Cellular mechanisms for preservation of timing in central auditory pathways. Curr Opin Neurobiol 1997; 7:487–92

46. Dringenberg HC, Olmstead MC: Integrated contributions of basal forebrain and thalamus to neocortical activation elicited by pedunculopontine tegmental stimulation in urethane-anesthetized rats. Neuroscience 2003; 119:839–53

47. Sukhotinsky I, Zalkind V, Lu J, Hopkins DA, Saper CB, Devor M: Neural pathways associated with loss of consciousness caused by intracerebral microinjection of GABA A-active anesthetics. Eur J Neurosci 2007; 25:1417–36

48. French JD, Hernandez-Peon R, Livingston RB: Projections from cortex to cephalic brain stem (reticular formation) in monkey. J Neurophysiol 1955; 18:74–95

49. 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

50. Devor M, Zalkind V: Reversible analgesia, atonia, and loss of consciousness on bilateral intracerebral microinjection of pentobarbital. Pain 2001; 94:101–12

51. Miller JW, Ferrendelli JA: The central medial nucleus: Thalamic site of seizure regulation. Brain Res 1990; 508:297–300

52. Miller JW, Ferrendelli JA: Characterization of GABAergic seizure regulation in the midline thalamus. Neuropharmacology 1990; 29:649–55

53. Franks NP, Lieb WR: Stereospecific effects of inhalational general anesthetic optical isomers on nerve ion channels. Science 1991; 254:427–30

54. 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

55. Harvey AL, Robertson B: Dendrotoxins: Structure-activity relationships and effects on potassium ion channels. Curr Med Chem 2004; 11:3065–72

56. Kim SJ, Park Y, Hong HJ: Antibody engineering for the development of therapeutic antibodies. Mol Cells 2005; 20:17–29

57. Connors EC, Ballif BA, Morielli AD: Homeostatic regulation of Kv1.2 potassium channel trafficking by cyclic AMP. J Biol Chem 2008; 283:3445–53

58. Shimada H, Uta D, Nabekura J, Yoshimura M: Involvement of Kv channel subtypes on GABA release in mechanically dissociated neurons from the rat substantia nigra. Brain Res 2007; 1141:74–83

59. Finnegan TF, Chen SR, Pan HL: Mu opioid receptor activation inhibits GABAergic inputs to basolateral amygdala neurons through Kv1.1/1.2 channels. J Neurophysiol 2006; 95:2032–41

60. Stienen PJ, van Oostrom H, Hellebrekers LJ: Unexpected awakening from anaesthesia after hyperstimulation of the medial thalamus in the rat. Br J Anaesth 2008; 100:857–9

61. Yost CS, Oh I, Eger EI 2nd, Sonner JM: Knockout of the gene encoding the K(2P) channel KCNK7 does not alter volatile anesthetic sensitivity. Behav Brain Res 2008; 193:192–6

62. Rhodes KJ, Trimmer JS: Antibodies as valuable neuroscience research tools versus reagents of mass distraction. J Neurosci 2006; 26:8017–20

63. Nacif-Coelho C, Correa-Sales C, Chang LL, Maze M: Perturbation of ion channel conductance alters the hypnotic response to the alpha 2-adrenergic agonist dexmedetomidine in the locus coeruleus of the rat. Anesthesiology 1994; 81:1527–34

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