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

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.1Many authors have suggested that K channels are involved in producing the effects of anesthesia.1–6Much recent focus has been placed on distinguishing K2P channels as possible targets of anesthetic action,6,7yet interactions with various other K channels like the Kv channels may also be important.2,3,8,9Kv 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.10In 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.11The 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 .11The subunit composition of the Kv1 channels not only determines their gating and kinetic properties, it also dramatically affects their expression and localization.11The 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.12,13The 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.14Importantly, 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.14In 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.15Mutagenesis 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.15,16Thus, 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.17Anesthetic-induced unconsciousness can be reversed with a localized and site-specific microinfusion of nicotine into the CMT of rats.17As the loss of consciousness associated with sleep and anesthesia may share overlapping neurobiological mechanisms,18–21and nicotine is known to also block various K channels,22,23including some Kv channels,24the 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.All research activities were conducted with full approval of the Institutional Animal Care and Use Committee of the University of California, Irvine.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).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.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. 25Zhou 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.25Specificity 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,25a channel blocker that also binds to the external vestibule of the Kv1.2 channel.26In 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,27–31we 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.After recovery from cannula implantation (after 6–7 days), animals were anesthetized in a clear chamber as previously described.17Briefly, animals were placed in a rectangular 8-l clear Plexiglas anesthetizing chamber and exposed to anesthesia in air at 2 l · min−1until 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).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.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.32However, a few infusions were located within ± 1.0 mm in the anteroposterior dimension.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.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.17Overall 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 1and can be seen online (see video, Supplemental Digital Content 1, which demonstrates the arousal response illustrated in fig. 1, https://links.lww.com/A823). Seizures were seen in of the the arousal to the antibody some type of we also the of arousal to animals under sevoflurane we arousal responses to a The animals did their and in response to this and four were to onto their during the with two onto their the of this type of arousal was different from the antibody lasted only as as the was and the animals did not to be with the antibody the animals to some of they in the chamber in a and they to and They did not to be in as they did not to focus on any particular part of their They were in their which might be from a was to the effects of the anesthesia on their or histology for desflurane are shown in figure and the histology for sevoflurane are shown in figure Infusion for the no-effect the for desflurane and sevoflurane are shown in and revealed that the resumption of consciousness was to infusions the CMT for both as also shown in and When the infusion was located in the CMT, of those animals from the rats also had infusions directly into the CMT, as shown in figure The animals that did not through an of more their movements were generally those of This was as a effect such that antibody delivered directly into the CMT of a generalized for a particular rats were allowed to recover from the infusion experiments, and including those that had to awaken days and before they all normal rat and were to and an antibody to block the external vestibule of Kv1.2 voltage-gated potassium channels into the CMT of anesthetized rats a of animals to a resumption of consciousness with in a chamber filled with inhalational histology of the it was found that an arousal response occurred in of those animals the infusion was located within the CMT. Taken these the CMT as an brain involved with regulating of arousal during anesthesia and further serve to suggest that the for this localized site-specific arousal effect involves anesthetic interactions with voltage-gated potassium by which consciousness is during anesthesia The by and in that anesthetic with the suppression of activity the for the of anesthesia from to have various channels are by anesthetic it which targets are the most for the effects of is that anesthetic on channels, such as type channels, or 2-pore domain background potassium channels, are the targets most directly to producing the effects of anesthetics on consciousness and a role for voltage-gated K channels in mediating the effects of anesthetics on consciousness has also been channels are located within the thalamus and the suggest that they are not as found within the CMT as one might have from the current This the of the CMT to be the focus of the current The CMT is part of the nonspecific thalamic arousal with areas mediating arousal and to of and from areas involved with sleep and its onto it is possible that the effects found localized to the CMT area an on the to or from this area of than on the CMT The large of found with the thalamic area the idea that this region is involved in regulating overall of suggest that voltage-gated potassium channels in the CMT may more to regulating arousal through localized interactions than previously effects on these channels can have large effects within neural and brain for which is a of the of it is to a of to these the are for further to the idea that the CMT is an in an arousal that may directly or with The amount of infusion volume used was only This is a volume than many in vivo it suggests that the effects found are localized to a small area immediately the infusion sites that a of less than approximately This small infusion volume was used to from the and of the A of the infusions that the CMT or were it did not cause an arousal This is due to the of an dose with a particular infusion. The CMT with both arousal an anesthesia the CMT from the that the effects of anesthetics through or also with the anesthesia region that an when with of the CMT can both directly arousal and produce of nicotine into the CMT of anesthetized rats behavioral arousal anesthetic of a is to cause a these together with the current suggests that the CMT is involved with regulating of arousal during it should not be without further work that the arousal effect associated with the infusion of the Kv1.2 channel blocking antibody into the CMT is directly to of a specific of This is one possibility, but other suggests this is an the effects of anesthetics on voltage-gated potassium channels show that these channels are by generally only at doses greater than those that are to this is for mutant Kv channels, which to be to the effects on these and most other Kv channels is generally one of current Thus, in the work, if anesthesia is to block currents through the Kv channels and the Kv1.2 antibody is also to block then it seems more that the antibody should have than an arousal the to this may in the diversity of Kv channels, it be that some heteromeric subunit may exist that can produce channels that open in response to anesthetic further the of these that the antibody did block the Kv1.2 channels in the CMT and this act to the of the CMT but it not the of the same anesthetic from any other channels, such as or K2P channels. The effect on thalamic is the result of the of multiple on the the blocking of the Kv1.2 channel can be seen as just one that changes the potential and its of a particular of action potential generalized nonspecific effects of an antibody infusion into the CMT are to be the of the arousal as the control antibody infusions did not cause any a nonspecific arousal effect due to some type of is the of an arousal response to a was different from that seen with the antibody However, nonspecific binding effects of the Kv1.2 antibody be are This that they have for their some of to other is and to the Thus, it is that most of the Kv1.2 antibody to the Kv1.2 but it might also have to other or to other channels. is seen with that nonspecific binding can occur to an extent that is sufficient to the of specific is this polyclonal antibody for blocking only Kv1.2 the Zhou et al. work, the Kv1.2 and Kv1.3 is the antibody was not directly against or channels. This may be and channels all show for the binding of this basis it be to that some of the Kv1.2 channel blocking antibody with the and channels may have further work with more specific versions of various the of antibodies might to extent some may have these the of antibodies for use in the specific blocking of various channels in vivo is now the polyclonal an established one that the unconsciousness of anesthesia through the opening of thalamic Kv1.2 channels and that the antibody this open to then did the suppression of the unconsciousness response not is that the antibody may have from the in a relatively of but this seems given the of antibody be the possibility that some role in the of the the arousal response may have been due to to the functioning of a of or a possibility is that a nonspecific generalized arousal on the thalamus may have to the such has been seen when acute infusions are given into the with other such as and potassium to identify targets of anesthetic action is to specific channels and then evaluate the behavioral effects of such on the various of anesthesia in the mutant relatively is to use of channel has a of potential for use in anesthesia antibody the and of binding is wild-type animals can be This the that some might with the of as may be the case with mutant The behavioral effects of any particular response can be and then the and associated with the responses can be with The antibody is not without its and of is time and generally the and of most antibodies are from have them (i.e. , limited or they are from an antibody The control on such can and the use of a antibody can the antibody may one of the that the of anesthesia research it is to that this is not the to a site-specific in potassium channel functioning to the of anesthesia and show an ability to the effect of an of various K channel (Kv), (KCa), and and were found to the effect of the when they were delivered into the of together with the current the ability of interactions with voltage-gated potassium channels to the of at least different anesthetic (i.e. , and in vivo to identify these channels as targets in need of further authors Giulio Tononi, M.D., Professor, and Chiara Cirelli, M.D., of Wisconsin, Madison, for the gift of Kv1.2 channel blocking The authors also Ph.D. of and University of for and Research University of for