Anesthesiology 2009; 110:766–73 Copyright © 2009, the American Society of Anesthesiologists, Inc. Lippincott Williams & Wilkins, Inc. Thalamic Microinfusion of Antibody to a Voltage-gated Potassium Channel Restores Consciousness during Anesthesia Michael T. Alkire, M.D.,* Christopher D. Asher, M.S.,† Amanda M. Franciscus, B.S.,‡ Emily L. Hahn, B.A.§

Background: The Drosophila Shaker mutant fruit-fly, with the nervous system, including voltage-gated (Kv), calci- its malfunctioning voltage-gated potassium channel, exhibits um-activated (KCa), inward rectifying (Kir), and 2-pore anesthetic requirements that are more than twice normal. 1 Shaker mutants with an abnormal Kv1.2 channel also demon- (K2P) domain background leak channels. Many authors have suggested that K channels are involved in produc-

strate significantly reduced sleep. Given the important role the Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/110/4/766/368020/0000542-200904000-00016.pdf by guest on 30 September 2021 1–6 thalamus plays in both sleep and arousal, the authors investi- ing the effects of anesthesia. Much recent focus has gated whether localized central medial thalamic (CMT) micro- been placed on distinguishing K2P channels as possible infusion of an antibody designed to block the pore of the Kv1.2 targets of anesthetic action,6,7 yet interactions with var- channel might awaken anesthetized rats. Methods: Male Sprague-Dawley rats were implanted with a ious other K channels like the Kv channels may also be 2,3,8,9 cannula aimed at the CMT or lateral thalamus. One week later, important. –unconsciousness was induced with either desflurane (3.6 ؎ Kv channels are further divided into 12 families (Kv1 Arousal Kv12) on the basis of sequence homology and similarity to .(51 ؍ or sevoflurane (1.2 ؎ 0.1%; n (55 ؍ n ;0.2% ␮ effects of a single 0.5- l infusion of Kv1.2 potassium channel Drosophila melanogaster (i.e., fruit-fly) genes.10 In Dro- blocking antibody (0.1–0.2 mg/ml) or a control infusion of Arc-protein antibody (0.2 mg/ml) were then determined. sophila, the Kv channel genes produce multiple versions of Results: The Kv1.2 antibody, but not the control antibody, a particular channel and are more commonly known by temporarily restored consciousness in 17% of all animals and historical nomenclature such as: Shaker (Kv1.1–Kv1.8), in 75% of those animals where infusions occurred within the Shab (Kv2.1–Kv2.2), Shaw (Kv3.1–Kv3.4), Shal (Kv4.1– CMT (P < 0.01 for each anesthetic). Lateral thalamic infusions Kv4.3), and ether-a-go-go (Kv10.1–Kv10.2). The eight (showed no effects. Consciousness returned on average (؎ SD –s after infusion and lasted a median time of 398 s members of the Shaker-related K channel family (Kv1.1 99 ؎ 170 (interquartile range: 279–510 s). Temporary seizures, without ap- Kv1.8) are involved with generating voltage-dependent parent consciousness, predominated in 33% of all animals. outward currents that regulate action potential threshold, Conclusions: These findings support the idea that the CMT as well as waveform and pacemaker activity in excitable plays a role in modulating levels of arousal during anesthesia tissue. Kv channels are generally composed of tetramers of and further suggest that voltage-gated potassium channels in the CMT may contribute to regulating arousal or may even be alpha subunits. When they are expressed as homomeric relevant targets of anesthetic action. 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 POTASSIUM (K) channels play a major role in regulating eight different subunits) can coassemble into channels with tissue excitability. There are at least four different types mixed heteromeric alpha subunit compositions. Further- of K channels that serve slightly different functions in more, Kv-beta subunits also exist and add on another layer of complex functional diversity in vivo.11 The subunit Supplemental digital content is available for this article. Direct composition of the Kv1 channels not only determines their
URL citations appear in the printed text and are available in gating and kinetic properties, it also dramatically affects both the HTML and PDF versions of this article. Links to the their expression and localization.11 digital files are provided in the HTML text of this article on the Journal’s Web site (www.anesthesiology.org). The idea that Kv channels might play a role in anes- thesia emerged from the discovery that Drosophila Shaker mutants shake their legs vigorously during ether 12,13 * Associate Professor in Residence, Department of Anesthesiology and Periop- anesthesia. The Shaker mutant lacks a normal func- erative Care and Fellow, the Center for the Neurobiology of Learning and tioning Kv1.x channel, suggesting that the suppression Memory, § Staff Research Associate, Department of Anesthesiology, † Medical Student, ‡ Undergraduate Student, University of California-Irvine. of neural activity under ether anesthesia depends to Received from the Department of Anesthesiology and Perioperative Care, and some extent on a properly functioning Kv1.x channel. the Center for the Neurobiology of Learning and Memory, University of California- Indeed, the amount of isoflurane needed to anesthetize a Irvine, Irvine, California. Submitted for publication September 14, 2008. Ac- cepted for publication December 30, 2008. Support was provided solely from Shaker mutant with a completely nonfunctioning Kv1.x institutional and/or departmental sources. Presented in part at the Annual Meet- channel is more than twice the dose needed to anesthe- ing of the American Society of Anesthesiologists, Orlando, Florida, October, 14 18–22, 2008, and at the Annual Meeting of the Society for Neurosciences, tize wild-type flies. Importantly, the changes in isoflu- Washington, D.C., November 15–19, 2008. rane doses needed to anesthetize various other Shaker Address correspondence to Dr. Alkire: UCIMC—Department of Anesthesiol- mutants parallels the expected reductions in ionic cur- ogy, 101 The City Dr. South, Bldg 53, Route 81-A, Orange, California 92868. [email protected]. Information on purchasing reprints may be found at rents mediated through the respective malfunctioning K www.anesthesiology.org or on the masthead page at the beginning of this issue. channels.14 In other words, the more defective the K ANESTHESIOLOGY’s articles are made freely accessible to all readers, for personal use only, 6 months from the cover date of the issue. channel (and the less current that passes through it), the

Anesthesiology, V 110, No 4, Apr 2009 766 THALAMIC ANTIBODY REVERSAL OF ANESTHESIA 767 greater the dose of isoflurane needed to anesthetize a from Charles River Laboratories, Inc. (Wilmington, MA). particular Shaker mutant. This seems to suggest that They were housed individually in a temperature-con- anesthesia might work in part by hijacking the function- trolled (22°C) colony room, with food and water avail- ing of the Kv1.x channel. able ad libitum. Animals were maintained on a 12-h Recently, the unconsciousness of sleep was also linked light, 12-h dark cycle (0700–1900 lights on). to a voltage-dependent K channel.15 Mutagenesis analy- sis was used to screen more than 9,000 Drosophila lines Surgery to identify those with a limited ability to sleep. Genetic Rats were anesthetized with sodium pentobarbital (50 analysis of these flies revealed a point mutation in a mg/kg, intraperitoneal) and placed into a stereotaxic conserved domain of the Shaker gene that involves the frame (Benchmark Digital Stereotaxic, Saint Louis, MO). 15,16

voltage sensing portion of Kv1.2 channel. Thus, for A guide cannula (23-gauge) was placed, aimed at the Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/110/4/766/368020/0000542-200904000-00016.pdf by guest on 30 September 2021 Drosophila to sleep, it appears to need current to prop- central medial thalamus (coordinates: anteroposterior erly flow through its Kv1.2 channels. Taken together –3.0 mm; mediolateral ϩ1.7 mm, with 13-degree tilt; with the earlier anesthesia work, this suggests Kv1.2 dorsoventral –4.5 mm; incisor bar, –3.3 mm). The guide channels might be involved with mediating the effects of cannula was 2 mm shorter in length than needed to volatile anesthetics on consciousness. reach the central medial thalamus. Indeed, the end of the Concurrent with the developments in sleep neuro- guide cannula did not reach into the thalamus proper. physiology, a possible role for the central medial thala- The thalamus was entered only at the time of the exper- mus (CMT) in contributing to the unconsciousness of iments when the microinfusion was delivered through a anesthesia was recently identified.17 Anesthetic-induced microinfusion needle inserted into the guide cannula unconsciousness can be reversed with a localized and that was 2 mm longer than the guide cannula itself. For site-specific microinfusion of nicotine into the CMT of the animals given desflurane anesthesia (n ϭ 55), all rats.17 As the loss of consciousness associated with sleep cannulae targeted the CMT. For the animals given and anesthesia may share overlapping neurobiological sevoflurane (n ϭ 51), most targeted the CMT, but ten mechanisms,18–21 and nicotine is known to also block animals were used as location controls; five targeted the various K channels,22,23 including some Kv channels,24 ventral lateral thalamic nucleus (coordinates: anteropos- the hypothesis is raised that anesthetic effects on con- terior –3.0 mm; mediolateral ϩ1.7 mm; dorsoventral sciousness might involve interactions with Kv1.2 chan- –4.5 mm), and five targeted the posterior thalamic nu- nels located, in part, in the CMT. Herein, we open the cleus (coordinates: anteroposterior –3.0 mm; mediolat- investigation into this area of research by microinfusing eral ϩ1.7 mm; dorsoventral –3.5 mm). Dental acrylic and a Kv1.2 channel blocking antibody directly into the CMT skull screws secured each cannula. Animals were al- of rats placed in an anesthetic chamber exposed to a lowed 6–7 days to recover before experiments. dose of inhalational agent that is just sufficient to render them unconscious. As in the case of Drosophila Shaker Drugs mutants, which have a chronic malfunction of Kv1.x The Kv1.2 antibody was a gift from Chiara Cirelli, channels, the acute conduction blockade of the Kv1.2 M.D., Ph.D. (Associate Professor, Department of Psychi- channels in the CMT should act to rapidly increase the atry, University of Wisconsin, Madison, Wisconsin) and anesthetic dose required to keep the animals uncon- Giulio Tononi, M.D., Ph.D. (Professor, Department of scious. As the dose of anesthesia will be held constant Psychiatry, University of Wisconsin, Madison, Wiscon- after the localized antibody microinfusion, a positive sin). Kv1.2 rabbit polyclonal antibodies were made and result (indicating the possible contribution of Kv1.2 affinity-purified through a contracted manufacturer channels to inducing the unconsciousness of anesthesia) (Genemed Synthesis Inc, San Francisco, CA), during per- will be manifest as a behavioral arousal of the animals; formance of a grant with the Defense Advanced Re- that is, they should awaken in the chamber filled with search Projects Agency. The antibody was manufactured anesthesia. 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 Materials and Methods potassium channel. Their antibody was found to block 70% of the whole-cell Kv1.2 currents in transfected cells All research activities were conducted with full ap- in a concentration and time-dependent manner.25 Spec- proval of the Institutional Animal Care and Use Commit- ificity was established by showing that the antibody did tee of the University of California, Irvine. not block currents to Kv1.3 or Kv3.1 channels, and binding was mutually exclusive with ␣-dendrotoxin,25 a Animals channel blocker that also binds to the external vestibule A total of 106 Sprague-Dawley rats (250–280 g or of the Kv1.2 channel.26 In the current work, the anti- approximately 9 weeks old on arrival) were obtained body was diluted in normal saline immediately before

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Fig. 1. Thalamic antibody infusion awakens anesthetized rat. Sequential frames from a video showing a typical return to conscious- ness response after antibody infusion (approximately 1 s apart). (A) The animal is seen unconscious on its back in a chamber filled with 3.6–3.7% desflurane (as shown on the gas monitor, the green screen in the background). (B) At 2 min and 51 s after the infusion, the animal starts to arouse (the infusion pump is on top of the gas monitor in the background). (C) The animal rights itself. (D and E) The animal appears to look around before moving across the chamber. The anesthetic gas concentration is seen as 3.7% desflurane on the gas monitor in the background. Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/110/4/766/368020/0000542-200904000-00016.pdf by guest on 30 September 2021 infusion into the thalamus of anesthetized rats. Initial ber. The needle was attached by a polyethylene tube infusions were performed with a concentration of 0.2 through the wall of the anesthetizing chamber to a 10-␮l mg/ml antibody in 0.5-␮l infusion volume given over 1 syringe (Hamilton, Reno, NV), which was driven by a min. A large proportion of seizure responses prompted minipump (Harvard Apparatus, Holliston, MA). The the lowering of the dose used to 0.1 mg/ml antibody in chamber anesthesia concentration was then lowered to the same 0.5-␮l infusion volume. 3.6% for desflurane or to 1.2% for sevoflurane. The con- As antibodies are relatively large molecules (approxi- centration was held stable for 20 min before a microin- mately 150 kDa), the in vivo use of an antibody infusion fusion was delivered. However, if a rat showed any given directly into a discrete region of the brain might spontaneous movement during this stabilization period, cause some type of nonspecific dysfunction to occur. the chamber concentration was increased by 0.1% incre- Whereas many examples of using antibodies as in vivo ments until each rat remained motionless for at least 20 probes for specific receptor-targets now exist,27–31 we min. Thus, the chamber concentration varied slightly nevertheless controlled for the possibility that a nonspe- depending on a specific animal’s behavior. Rats were cific arousal effect might occur due to the infusion of an thus anesthetized, in separate experiments, with either antibody itself. To evaluate this possibility, we infused desflurane (3.6 Ϯ 0.2%: n ϭ 55) or sevoflurane (1.2 Ϯ nine animals under sevoflurane anesthesia with an anti- 0.1%: n ϭ 51). body directed against an intracellular nonreceptor tar- get, the activity regulated cytoskeletal (Arc) protein. This Arousal Response Determinations Arc rabbit polyclonal antibody was purchased from a Responses to a single infusion of antibody per rat were commercial vendor (BioVision Research Products, graded as one of four levels: 1 ϭ no effect-no visible Mountain View, CA). We injected 0.2 mg/ml Arc anti- movements; 2 ϭ partial arousal – signs of arousal includ- body in phosphate-buffered saline given in a single 0.5-␮l ing eye opening and movements of extremities; 3 ϭ full microinfusion. arousal-the complete turning of the animal onto its stom- ach, while exhibiting purposeful movements; 4 ϭ sei- Consciousness Suppression with Anesthesia zures-focal or generalized tonic-clonic seizures. After recovery from cannula implantation (after 6–7 days), animals were anesthetized in a clear chamber as Histology previously described.17 Briefly, animals were placed in a Brains were sliced into 40-␮m sections and stained rectangular 8-l clear Plexiglas anesthetizing chamber and with thionin. Microinfusions were localized blinded to exposed to anesthesia in air at 2 l · minϪ1 until they lost behavioral data. Data were incomplete in 7 rats that their righting reflex (fig. 1). Anesthetic chamber agent expired during surgery or were euthanized due to concentrations were monitored continuously during the clogged or missing cannula. Infusion sites were pro- experiments using a Datex-Ohmeda Ultima Capnomac jected onto the –3-mm coronal brain section from the (Helsinki, Finland) and verified with gas chromatography atlas of Paxinos and Watson.32 However, a few infusions (Model 80123B; SRI Instruments, Redondo Beach, CA). were located within Ϯ 1.0 mm in the anteroposterior The chamber had a small door on one side, through dimension. which the animal was initially placed. The chamber also had small ports that served as the anesthetic gas inlet, Statistics the microinfusion tubing port inlet, two gas monitor The hypothesis that the CMT is involved with mediat- sampling ports, and one gas chromatograph sampling ing the resumption of consciousness after antibody infu- port. Once each rat was well anesthetized, the door was sion was examined separately in both the desflurane- and partially opened, and a 25-gauge microinfusion needle the sevoflurane-exposed animals using Fisher exact test. was quickly inserted through the guide cannula, and the We compared the histology of those animals showing a rat was placed onto its back in the center of the cham- resumption of consciousness with those animals that

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Table 1. Summary of Behavioral Responses to Intrathalamic Kv1.2 Antibody Infusion

Desflurane Sevoflurane Behavioral Response (Number of Animals) (Number of Animals)

1, no effect 17 12 2, partial arousal 8 5 3, consciousness restored 8 8 4, seizure 19 13 failed to show an effect from the infusion. P Ͻ 0.05 was considered significant. Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/110/4/766/368020/0000542-200904000-00016.pdf by guest on 30 September 2021

Results

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

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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 obser- vation by Franks and Leib in 1984 that anesthetic po- tency 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 sub- 2,7,34–36

stances. Yet, it remains unclear which molecular Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/110/4/766/368020/0000542-200904000-00016.pdf by guest on 30 September 2021 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 ␥-aminobutyric acid type A, glycine, neuronal nicotinic channels, N-methyl-D-aspartate, or 2-pore domain back- ground 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 Fig. 3. The return to consciousness effect found with antibody effects of anesthetics on consciousness has also been infusion during sevoflurane anesthesia is also site-specific. (A) proposed.2,8,14 The central medial thalamus target is again shown on the rat Kv1.2 channels are densely located within the thala- atlas figure (arrowed), located to the left. Expanded insets show 38 infusion sites (black dots) for the infusions that had no-effect mus and cortex, but the early published reports sug- versus those that restored consciousness. (B) The return to gest that they are not as densely found within the CMT consciousness response is significantly associated with infu- as one might have anticipated from the current results. sions that hit the central medial thalamus (gray highlighted area in A) versus those that missed. This raises the question of why exactly the CMT ap- peared to be the focus of the current effect. The CMT is tassium channels into the CMT of anesthetized rats part of the nonspecific intralaminar thalamic arousal 39 caused a number of animals to display a temporary re- system. It connects with brainstem areas mediating sumption of consciousness with restored mobility in a arousal and projects to wide expanses of cortical and 40,41 chamber filled with inhalational anesthesia. With histol- basal ganglia areas. It receives afferents from hypo- thalamic areas involved with controlling sleep and aro- ogy examination of the animals’ brains, it was found that 18,20 an arousal response occurred in 75% of those animals usal. Given its wide projection pattern onto cortex, where the infusion needle-tip was located within the it is possible that the effects found localized to the CMT area represent an influence on the projections to or from CMT. Taken together, these findings strongly implicate this area (or fibers of passage), rather than on the CMT the CMT as an important brain site involved with regu- cell bodies themselves. The large number of seizures lating levels of arousal during anesthesia and further found with injections around the midline thalamic area serve to suggest that the underlying mechanism for this support the idea that this region is involved in regulating overall levels of cortical excitability.42 The findings re- ported here suggest that voltage-gated potassium chan- nels in the CMT may contribute more to regulating arousal through localized network interactions than pre- viously 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 Fig. 4. Infusion sites associated with seizure responses for both significant for adding further support to the idea that the desflurane and sevoflurane. The central medial thalamus target is again shown on the rat atlas figure (arrowed), located to the CMT is an important node in an arousal network that left. Expanded insets show infusion sites (black dots) for the may directly or indirectly interact with anesthesia. The infusions causing seizures. Qualitatively, the infusions causing amount of infusion volume used was only 0.5 ␮l. This is seizures appear to cluster in the intermedial dorsal thalamic 27 nucleus, located just above the central medial thalamus, or on a much smaller volume than many in vivo studies use, the border between the two. and it suggests that the effects found are localized to a

Anesthesiology, V 110, No 4, Apr 2009 THALAMIC ANTIBODY REVERSAL OF ANESTHESIA 771 very small area immediately around the infusion sites of the same anesthetic from affecting any other anesthetic- that encompasses a size of less than approximately 0.25– sensitive channels, such as GABAeric, glycinergic, cho- 0.5 mm. This small infusion volume was used to mini- linergic or K2P channels. The ultimate effect on thalamic mize spread from the needle-tip and help provide local- neuronal firing patterns is likely the result of the com- ization of the effects. A number of the infusions that hit bined contributions of multiple influences on the cell the CMT or were near it did not cause an arousal reac- membrane potential7,54; therefore, the blocking of the tion. This is likely due to the delivery of an insufficient Kv1.2 channel can be seen as just one additional influ- dose with a particular infusion. The CMT interacts with ence that changes the cell’s membrane potential and both ascending (from brainstem),46,47 and descending hence its likelihood of entering a particular pattern of (from cortex) arousal pathways.48 From an anesthesia action potential firing.

perspective, the CMT receives input from the hypothal- Third, generalized nonspecific effects of an antibody Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/110/4/766/368020/0000542-200904000-00016.pdf by guest on 30 September 2021 amus,40 a connection that helps modulate the sedation infusion into the CMT are unlikely to be the source of effects of anesthetics through GABAergic or orexinergic the arousal responses, as the control antibody infusions effects.18,49 It also interacts with the mesopontine teg- did not cause any reactions. In addition, a nonspecific mental anesthesia area,47 a brainstem region that induces arousal effect due to some type of internal pain-like state an apparent anesthetic-like state when microinfused is unlikely because the qualitative nature of an arousal with barbiturate.50 Pharmacologic manipulations of the response to a painful stimulation was much different CMT can both directly enhance arousal and produce from that seen with the antibody effect. However, non- sedation effects.42,51 Microinfusion of nicotine into the specific binding effects of the Kv1.2 antibody cannot be CMT of anesthetized rats restores behavioral arousal de- ruled out. Antibodies are affinity reagents. This means spite continued anesthetic exposure.17 Microinfusion of that even though they have tremendous affinity for their a ␥-aminobutyric acid agonist muscimol is reported to target antigen, some level of low-affinity crossreactivity cause a sedation response.52 Taking these facts together to other closely related protein sequences is common with the current findings strongly suggests that the CMT and often contributes to the signal. Thus, it is likely that is intimately involved with regulating levels of arousal most of the Kv1.2 antibody bound to the Kv1.2 receptor, during anesthesia. but it might also have bound to other similar receptors Second, it should not be assumed without much fur- or even to other similar channels. It is often seen with ther work that the arousal effect associated with the immunohistochemistry that nonspecific binding can oc- infusion of the Kv1.2 channel blocking antibody into the cur to an extent that is sufficient to overwhelm the CMT is directly related to antagonism of a specific mech- intended signal of interest. anism of anesthesia. This is certainly one possibility, but How specific is this polyclonal antibody for blocking other evidence suggests this is an unlikely possibility. In only Kv1.2 ion channels? From the original Zhou et al. vitro studies examining the effects of anesthetics on work, the specificity between Kv1.2 and Kv1.3 is quite voltage-gated potassium channels show that these chan- good.25 Yet, the antibody was not tested directly against nels are affected by anesthetics,9 but generally only at Kv1.1 or Kv1.6 channels. This may be important because doses much greater than those that are clinically rele- Kv1.1, Kv1.2, and Kv1.6 channels all show similar affin- vant.53 One exception to this generalization is evident ity for the binding of ␣-dendrotoxin.55 On this basis for Shaw2 mutant Kv channels, which appear to be alone, it would be reasonable to assume that some cross- highly sensitive to certain anesthetics.8 Yet, the effects reactivity of the Kv1.2 channel blocking antibody with on these and most other Kv channels is generally one of the Kv1.1 and Kv1.6 channels may have occurred. Only current blockade. Thus, in the present work, if anesthe- further work with more specific versions of various sia is acting to block currents through the Kv channels toxins44 or the development of monoclonal antibodies and the Kv1.2 antibody is also acting to block currents, might help clarify to what extent some crossreactivity then it seems more likely that the antibody should have may have influenced these findings.30,55 Indeed, the de- enhanced sedation, rather than causing an arousal reac- velopment of monoclonal antibodies for use in the spe- tion. Nevertheless, the answer to this apparent contra- cific blocking of various channels in vivo is now almost diction may lie in the extreme biologic diversity of Kv routine,28,31,56 though the polyclonal approach remains channels, where it could be speculated that some het- an established technique.29 eromeric subunit combinations may exist that can pro- If one speculates that the unconsciousness of anesthe- duce anesthetic-sensitive channels that do open in sia occurs through the opening of thalamic Kv1.2 chan- response to anesthetic exposure. Another speculation nels and that the antibody blocked this open pore to further illustrates the potentially indirect nature of these restore consciousness, then why did the suppression of findings. Assuming that the antibody did block the Kv1.2 the unconsciousness response not last indefinitely? It is channels in the CMT and functionally eliminated them, conceivable that the antibody may have dissociated from this would act to acutely change the firing patterns of the the receptor in a relatively short period of time, but this CMT neurons involved, but it would not stop the actions seems unlikely given the nature of antibody binding.

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More likely would be the possibility that receptor traf- notic component of at least three different anesthetic ficking played some role in the termination of the re- agents (i.e., sevoflurane, desflurane, and dexmedetomi- sponse.57 In addition, the arousal response itself may dine) in vivo would seem to identify these channels as have been due to presynaptic actions serving to decrease prime targets in need of further study. the functioning of GABAergic neurons, causing a tempo- rary inhibition of inhibition or a disinhibition reac- The authors thank Giulio Tononi, M.D., Ph.D., Professor, and Chiara Cirelli, 58,59 M.D., Ph.D., Associate Professor (University of Wisconsin, Madison, Wisconsin), tion. Another possibility is that a nonspecific gener- for the generous gift of Kv1.2 channel blocking antibody. The authors also thank alized arousal reaction centered on the midline thalamus James L. McGaugh, Ph.D. (Professor of Neurobiology and Behavior, University of California-Irvine, Irvine, California), for his continued support and Yasmin may have contributed to the response, such has been Khowaja, B.S. (Staff Research Associate, University of California-Irvine), for tech- seen when acute ibotenic acid infusions are given into nical assistance. 60

the midline thalamus. Further testing with other exci- Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/110/4/766/368020/0000542-200904000-00016.pdf by guest on 30 September 2021 tatory substances such as glutamate and even potassium itself would seem warranted. References One approach to identify appropriate targets of anes- thetic action is to genetically modify specific ion chan- 1. Yost CS: Potassium channels: Basic aspects, functional roles, and medical significance. ANESTHESIOLOGY 1999; 90:1186–203 nels and then evaluate the behavioral effects of such 2. Arhem P, Klement G, Nilsson J: Mechanisms of anesthesia: Towards inte- grating network, cellular, and molecular level modeling. Neuropsychopharma- mutations on the various end-points of anesthesia in the cology 2003; 28(Suppl 1):S40–7 61 mutant animals. Another relatively new approach is to 3. 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