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The Differential Effects of the Nonimmobilizer 1,2-Dichlorohexafluorocyclobutane (F6, 2N) and Isoflurane on Extrasynaptic Gamma-Aminobutyric AcidA Receptors

Perouansky, Misha MD; Banks, Mathew I. PhD; Pearce, Robert A. MD, PhD

doi: 10.1213/01.ANE.0000150942.68328.66
Anesthetic Pharmacology: Research Report

The nonimmobilizer 1,2-dichlorohexafluorocyclobutane (F6; also known as 2N) causes amnesia and seizures at concentrations less than and more than, respectively, than that predicted to cause immobility (MACpred). The molecular and cellular basis of these effects is not known. We reported previously that F6 has no effect on synaptic gamma aminobutyric acid (GABA)A receptors located on the somata of hippocampal pyramidal cells. However, in hippocampal neurons, GABAA receptors that are located subsynaptically have different pharmacologic properties from those at extrasynaptic sites, and these classes of receptors may serve different physiologic functions. Therefore, we investigated the effects of F6 and isoflurane on currents mediated predominantly by extrasynaptic GABAA receptors harvested from hippocampal neurons by exposing nucleated excised patches to brief, high-concentration pulses of GABA. We found that extrasynaptic GABAA receptors in the majority of neurons located in the pyramidal cell layer are insensitive to F6 at concentrations up to 110 μM, although receptors harvested from one putative interneuron were potently inhibited by 43 μM of F6. By contrast, isoflurane consistently reduced the peak amplitude and slowed deactivation of currents mediated by extrasynaptic receptors, similar to its effect on synaptic receptors. These results demonstrate the selective sensitivity of extrasynaptic GABAA receptors on pyramidal neurons to isoflurane but not F6.

IMPLICATIONS: Gamma aminobutyric acidA receptors are located either synaptically or extrasynaptically, and both can regulate the excitability of neurons. We have found that extrasynaptic receptors on hippocampal pyramidal neurons are insensitive to F6 but are blocked by isoflurane. Therefore, neither extrasynaptic nor synaptic receptors on pyramidal cells underlie the amnestic and proconvulsant effects of F6.

Department of Anesthesiology, University of Wisconsin, Madison

Presented, in part, at the American Society of Anesthesiologists annual meetings in New Orleans, LA, October 2001, Orlando, FL, October 2002, at the Society for Neuroscience annual meetings in New Orleans, LA, October 1997 and November 2000, San Diego, CA, November 2001, and at the 12th Neuropharmacology Conference, Orlando, FL, November 2002.

Supported, in part, by grant GM 55719 (to RAP).

Accepted for publication November 1, 2004.

Address correspondence and reprint requests to M. Perouansky, room 43, Bardeen labs, 1300 University Ave., Madison, WI 53792-3272. Address e-mail to mperouansky@wiscmail.wisc.edu.

A number of ligand-gated neuronal ion channels are affected to some degree by anesthetics. Of these, the gamma amino-butyric acid (GABA)A receptor is thought to play a pivotal role in general anesthetic action. In particular, GABAA receptor modulation may contribute to the amnesic and the proconvulsant properties of some volatile anesthetics (1,2). Separating relevant effects from irrelevant ones and linking effects on the receptor level to desirable and undesirable manifestations in vivo are fundamental aims of anesthesia-related research.

One approach to these aims is to compare the effects of anesthetics with those of drugs that have anesthetic-like physicochemical properties but do not produce the full spectrum of anesthetic actions in vivo. These compounds were initially termed “nonanesthetics” (3); but they have been referred to as nonimmobilizers after it was discovered that some drugs cause amnesia but do not prevent movement in response to noxious stimuli (4). The volatile compound, 1,2-dichlorohexafluorocyclobutane (designated F6 or 2N in the literature), is an extensively studied prototype nonimmobilizer. The minimum alveolar anesthetic concentration (MAC) at which it would be predicted to cause immobility (MACpred) is 0.042 atm (3), which is equivalent to an aqueous concentration of 16 μM at room temperature. Like anesthetics, it produces amnesia at a concentration of approximately one-third MACpred (3,4). At concentrations larger than MACpred it induces convulsions (3,5). We previously reported (6) that synaptic GABAA receptors on hippocampal pyramidal cells are insensitive to block by F6 and are therefore unlikely to contribute significantly to its in vivo effects. A growing body of literature, however, indicates that a substantial number of GABAA receptors are localized to extrasynaptic sites at the soma as well as the dendrites of neurons in various areas in the brain. The biophysical and pharmacologic properties of these receptors seem to differ from those of synaptic receptors (7–10), and they may have distinct physiologic functions. In particular, it has been suggested that tonic inhibition mediated by extrasynaptic receptors may play a role in the prevention of seizure activity (11) and may be a clinically important target of anesthetic and sedative drugs (12). We therefore investigated the effect of F6 on currents mediated largely by extrasynaptic receptors and compared it with the effects of isoflurane.

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Methods

All experiments were conducted according to the guidelines laid out in the Guide for the Care and Use of Laboratory Animals and were approved by the University of Wisconsin Animal Care and Use Committee.

Juvenile male Sprague-Dawley rats (11–17 days of age) were decapitated under isoflurane anesthesia, and the brain was quickly removed and immersed in cold (4°C) artificial cerebrospinal fluid (ACSF) saturated with 95% O2/5% CO2 (carbogen gas). A block of tissue containing the hippocampus was removed and glued to a tissue tray using cyanoacrylate glue. Tissue slices 400-μm thick were prepared using a vibrating microtome (Leica VT1000, Bannockburn, IL) incubated at 32°C for 1 h and then kept in carbogen-saturated ACSF at room temperature until use.

Cells in the stratum pyramidale of CA1 were visualized, as previously described (10). Whole-cell recordings were obtained at room temperature (22°C–24°C) using a Multiclamp 700A patch-clamp amplifier and pClamp software (Axon Instruments, Foster City, CA). Data were filtered at 5 kHz, sampled at 10 kHz (Digidata 1200, Axon Instruments), and stored on a Pentium-based computer. Tight-seal whole-cell recordings from the somata of cells located in the stratum pyramidale of hippocampal CA1 layer were obtained using standard techniques. Patch pipettes had open-tip resistances of 2–4 MΩ when filled with the recording solution of composition (mM): CsCl 140, NaCl 10, HEPES 10, BAPTA 10, MgATP 2, and lidocaine N-methyl bromide (QX-314) 5, with a pH value of 7.3.

Nucleated patches were obtained, as previously described (10), by slowly withdrawing the patch pipette from the cell while applying negative pressure. Nucleated patches differ from excised outside-out patches in that they contain the cell nucleus and a larger cell membrane area. After isolation of the nucleated patch, the recording pipette was positioned in front of the control barrel of the θ pipette (Fig. 1). The patch was exposed to test and control solutions using a rapid application system, also previously described (10). In brief, the system consisted of a two barrel θ application pipette (fashioned from Thin Theta; Sutter Instruments, Novato, CA) connected to a piezoelectric stacked translator (model P-245.50; Physik Instruments, Costa Mesa, CA).

Figure 1

Figure 1

Solutions exchange rates (10%–90%) were estimated at the beginning of each experiment by measuring open-tip junction currents with dilute perfusion solution and ranged from 500 to 700 μs. Access resistance and capacitance of nucleated patches were measured using the amplifier circuitry. Series resistance was compensated 70%–90%. GABA (1 mM) was applied for 5 ms. We (10) showed that approximately 60%–75% of the current response to exogenously applied GABA in nucleated patches is carried by extrasynaptic receptors.

Slices were perfused continuously with ACSF of composition (mM): NaCl 127, KH2PO4 1.21, KCl 1.87, NaHCO3 26, CaCl2 2.17, MgSO4 1.44, and glucose 10, saturated with carbogen gas, with a pH value of 7.4. Solutions applied to nucleated patches were based on HEPES-buffered saline (HBS) of composition (mM): NaCl 130, KCl 3.1, NaHEPES 11, CaCl2 2.17, and MgSO4 1.44 adjusted to a pH value of 7.3. F6-containing solutions were prepared in Chemware Teflon FEP gas sampling bags (North Safety Products, Cranston, RI). F6 solutions were prepared by filling Teflon bags partially with HBS and adding to the head space appropriate quantities of F6-saturated air to achieve the desired aqueous concentration of F6, as calculated using a saline/gas partition coefficient for F6 of 0.026 (13). Isoflurane-containing solutions were prepared similarly by diluting isoflurane-saturated HBS to the desired concentration. Concentrations of F6 and of isoflurane in HBS from the Teflon bags were measured by gas chromatography.

During an experiment, the patch was kept in the solution streaming from the HBS barrel and exposed to the drug-containing solution at regular intervals (either 30 or 60 s) to minimize the effect of cumulative desensitization. Control responses were obtained first. For most experiments (“preapplication” protocol), the patches were then held in a HBS solution containing F6 or isoflurane at the concentration to be tested and then intermittently exposed to 1 mM of GABA + F6. For some experiments (“coapplication” protocol), patches were held in HBS solution lacking F6 and then intermittently exposed to 1 mM GABA + F6. Wash responses were obtained after switching back to drug-free solutions.

Aqueous samples (2 mL) were collected from Teflon bags using a gas-tight glass syringe (Hamilton Co, Reno, NV) fitted with a Teflon stopcock (Hamilton Co). Samples were transferred to 3.7-mL glass vials capped with mininert valves (Alltech, Nicholasville, KY). Drug concentrations in the vial head space were determined by gas chromatography using gas phase calibration standards for F6 or isoflurane. Concentrations in aqueous samples (Caq, sample) were calculated based on saline/gas partition coefficients (λsaline/gas) and the relative volumes of aqueous and gas phases within the vial (Vgas vial and Vaq, vial), according to the equation:

CV

CV

Gas phase concentrations (Cgas, vial) were measured using a Varian 3700 gas chromatograph (Varian Inc, Walnut Creek, CA) with a flame ionization detector. Separation was achieved by on-column injection into a 1.83 m × 3.2 mm stainless steel column packed with 80/100 Poropak Q.

Data were analyzed on a Pentium-based PC using Clampfit (Axon Instruments), Origin (MicroCal, Northampton, MA), and Excel (Microsoft, Seattle, WA). Data were filtered off-line at 2 kHz. The decay kinetics were characterized by exponential curve fitting. The decays of currents in response to a 5-ms application of GABA were best described by two or three exponential components. To quantify the overall decay time, we computed the weighted time constant τw = (A1 + A2+ A3τ3)/(A1+A2+A3), where τ is the time constant of decay and A is the amplitude. Results are expressed as mean ± sd, unless otherwise noted.

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Results

The data on F6 are based on recordings from 10 nucleated patches obtained from cells located in the stratum pyramidale of CA1. Two patches were each exposed to two different F6 concentrations. In one patch, the response to GABA showed a significant rundown over time and, despite being consistent with the other results, was not included in the summary graphs. In two experiments, the patches were lost before a wash could be obtained. Most of these cells are likely to be pyramidal cells (14). However, as we did not conduct histological analysis, an inhibitory cell may have also been included. This notion may explain the dichotomy in the observed effects of F6, as discussed in more detail below. The isoflurane data were obtained from 11 nucleated patches, 4 of which were exposed to 2 different concentrations.

Responses obtained under control and wash conditions did not differ significantly (P = 0.27, 0.15, and 0.59 for rise times [RT]10–90, amplitude, and τw, respectively; paired two-tailed t-test) and were therefore averaged. These values were used as controls for comparison with F6. Exposure of nucleated patches to a 5-ms pulse of 1 mM of GABA generated inward currents with 10%–90% RT10–90 of 2.06 ± 0.56 ms (range, 1.36–3.41 ms; n = 11) and a mean amplitude of −10.347 ± 2.762 nA (range, −7.4 to −16.9 nA; n = 11). The weighted time constant of decay (τw) was 73 ± 26 ms (range, 32–112 ms; n = 11).

We tested F6 at concentrations ranging from 24 to 110 μM (1.5–6.9 times MACpred). The illustrative example in Figure 2 features the cell exposed to the largest F6 concentration. Figure 2A shows the amplitudes and RT of the individual responses, evoked every 30 s throughout the course of the experiment (including the responses during solution exchanges). It is evident that responses to GABA obtained with concentrations <1 mM (Fig. 2A; during solution exchange) showed dramatically reduced amplitudes and slowed RT10–90. By contrast, exposure to F6 at both 110 and 55 μM had no effect on either the amplitude (Figs. 2, A and Bi), RT10–90 (Figs. 2, A and Bii), or kinetics of deactivation (Figs. 2Biii). Combined results from eight of the nine patches exposed to F6 at concentrations ranging from 24 to 110 μM (1.5–6.9 MAC) demonstrated that F6 had no significant effect on the amplitudes of currents mediated by GABAA receptors in the majority of cells (Fig. 3).

Figure 2

Figure 2

Figure 3

Figure 3

Although eight of nine patches showed no effect of F6 on current amplitude or kinetics of deactivation at concentrations up to 110 μM (Fig. 4), one patch exposed to an intermediate concentration of F6 (43 μM) did show a marked reduction in current amplitude (Fig. 4A). Deactivation was slowed in this same patch as well (Fig. 4B). These results suggested that we harvested patches from at least two neuronal populations (an insensitive majority and a sensitive minority) based on the susceptibility of their extrasynaptic GABAA receptors to block by F6. However, because this result was observed for only a single patch, we performed additional tests to ensure that the observation was not the result of a technical artifact, such as inadequate solution exchange. Figure 5A illustrates a time series of amplitudes and RT of the individual responses obtained in this cell. The illustration is analogous to Figure 2 (with the exception that agonists were applied every 60 s). When GABA + F6 were applied in the usual manner (i.e., after preexposure of the patch to the 43 μM of F6 in saline), the response was blocked by 53%. By contrast, when F6 was coapplied with GABA, the effect was greatly reduced (average 7% block). This result demonstrates that the solution flow rate and drug content of the test barrel were appropriate and that such an artifact cannot explain the reduced current amplitude. Examination of the original current traces (Fig. 5Bi) and the normalized rising and decaying phases (Fig. 5B, ii and iii) shows that the decay was slowed and the rising phase was accelerated with the preapplication protocol. The lack of slowing of RT10–90 values supports proper flow and GABA content; indeed, acceleration of RT10–90 as well as slowed deactivation are both consistent with classical open channel block (15). In this patch, we also tested the effect of F6 on prolonged exposures to 1 mM of GABA (2000 ms). These results were consistent with the block observed with brief exposures (not shown).

Figure 4

Figure 4

Figure 5

Figure 5

In a separate set of experiments, we analyzed the effect of isoflurane on responses of extrasynaptic GABAA receptors to 1 mM of GABA. An example of the effect of isoflurane 0.5 mM (1.6 MAC) is shown in Figure 6A. In addition to blocking the current, isoflurane slowed its deactivation (Fig. 6B) by markedly prolonging the slow component of the decay (Fig. 6B). In all experiments, isoflurane (0.15–0.6 mM) blocked the peak currents and slowed the decay (Fig. 6C). On average, 0.5 mM of isoflurane blocked the peak current by 69.9% ± 8.9% and slowed τw by 2.52 ± 0.5-fold (n = 8). These effects were concentration-dependent and are qualitatively similar to isoflurane’s modulation of synaptic GABAA receptors in the same neuronal population (2).

Figure 6

Figure 6

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Discussion

The main finding of our experiments is that extrasynaptic GABAA receptors of hippocampal pyramidal cells, similar to their synaptic counterparts, display differential sensitivity to anesthetics versus nonimmobilizers. When tested under our activation conditions (i.e., saturating GABA), these receptors are blocked by isoflurane but are insensitive to F6. This leads us to conclude that inhibition of GABAA receptors in pyramidal cells does not underlie the in vivo effects of F6. We found, in addition, that GABAA receptors that are susceptible to inhibition by F6 are present in some hippocampal neurons. Variable, subunit-specific block of GABAA receptors by anesthetics and anesthetic-like compounds, which we and others (16–18) have observed in expression systems, has an analog in vivo. A caveat to this conclusion is that we activated extrasynaptic receptors using conditions substantially different than those that exist in situ, where extrasynaptic receptors may be continuously exposed to approximately 0.2–2 μM of GABA (19) or to GABA transients generated by spillover from neighboring synapses (20).

The role played by extrasynaptic GABAA receptors in vivo is not precisely defined. It has been suggested that these receptors mediate a tonic form of inhibition that controls neuronal excitability. Extrasynaptic receptors are present in both pyramidal cells and inhibitory interneurons and, at least in the acute slice preparation, the tonic inhibitory current seems to be more prominent in interneurons (21). However, its contribution to the balance between excitation and inhibition remains to be defined. Seizure-inducing, seizure-suppressing, and amnestic drugs potently modulate the tonic current (9,11). In particular, midazolam and propofol enhance the tonic current more than synaptic currents (12), which seem to be mediated by α5 subunit-containing GABAA receptors (22). Assuming that extrasynaptic receptors underlie tonic inhibition of pyramidal cells, our finding that extrasynaptic receptors are insensitive to F6 implies that tonic inhibition is also not affected. We tested F6 in a range of concentrations reaching well above the threshold that induces seizures in vivo. Therefore, seizure generation by F6 seems to be due neither to reduce extrasynaptic (i.e., tonic) nor to block subsynaptic (i.e., phasic) GABAAergic inhibition of pyramidal cells. By contrast, isoflurane at clinical concentrations reliably blocked the peak currents generated by saturating agonist concentrations while also slowing its decay. This is reminiscent of its effect on intrinsically activated synaptic receptors (2) and is compatible with the dual effect on currents exogenously evoked by slow to intermediate GABA concentrations (23).

We harvested patches from visualized neurons located in the pyramidal cell layer of the hippocampal CA1 area. Light microscopy in acute slices without specialized staining techniques (e.g., in vivo labeling in transgenic animals) or electrophysiological characterization does not allow differentiation of whether any particular neuron is a pyramidal cell or an interneuron located within the pyramidal layer, e.g., of the basket or calretinin-immunoreactive cell types (24,25). Interneurons account for approximately 6% of neurons in the CA1 area of the hippocampus but for an even smaller percentage of neurons in stratum pyramidale, where the majority of pyramidal cells are localized (26). Therefore, whereas it may not be surprising that we recorded by chance from one interneuron (of 10 cells in this series), it also follows that the chances of successfully harvesting a patch from another interneuron of the same type are low, even in a large sample of randomly selected neurons. For that reason, instead of attempting to buttress our observation with additional data, we scrutinized this experiment with special emphasis on artifact detection. Future interneuron-directed experiments could use transgenic animals with green fluorescent protein-labeled interneurons (27) to substantiate our observation.

Although we cannot exclude technical causes with absolute certainty, we are reasonably confident that the observations in this aberrant patch (that, we assume, was obtained from an interneuron) are indeed real. The following lines of argument support our conclusion. First, the RT10–90 of the responses to 1 mM of GABA in the presence of F6 were not slower than those of control responses. Slower RT10–90 would be expected if there were flow irregularities. Second, coapplication of F6 and GABA yielded currents with amplitudes that were similar to the control currents, confirming proper flow and GABA content of the test barrel. This also shows that F6 is a relatively slow blocker compared with the rate of channel activation. Third, the results of experiments in which we have exposed this patch to GABA for 2000 ms support the observations made with brief GABA exposures (not shown). Fourth, the amplitude of the response in this patch was more than three sd larger than the responses in all other patches, indicating heterogeneity in the sampled population. Finally, the effect of F6 on the RT and the decay time constant in this cell (shortening and slowing, respectively, as opposed to no effect in the other cells) is also compatible with the presence of a blocking action (15).

The subunit composition of GABAA receptors influences their susceptibility to modulation by a variety of drugs, including anesthetics. Some of the best-characterized interactions involve benzodiazepines: recombinant GABAA receptors must contain a γ2-subunit and must not include either α4 or α6 subunits (28) to be modulated by these drugs. The blocking effect of large concentrations of isoflurane (18) and F6 (16) on expressed GABAA receptors is also mitigated by the presence of the γ2-subunit. Therefore, if some neurons in the pyramidal cell layer do not express the γ2-subunit, their GABAA receptors may be sensitive to block by F6.

Most GABAA receptors are heteropentamers consisting of 2α, 2β, and either 1γ or 1δ subunit, with α1, β2, and γ2 subunits being the most abundant in the brain. However, 50% of receptors containing the α4 subunit do not contain either a γ or a δ subunit (whether δ-containing receptors are sensitive to F6 is unknown). Because α4-containing receptors constitute approximately 13% of GABAA receptors in the hippocampus, it is likely that non-γ-containing receptors are indeed present (29). However, the cellular and subcellular localization of such receptors is unknown.

In summary, we have shown that, unlike isoflurane, F6 does not alter responses of excised extrasynaptic receptors to GABA. We have also serendipitously discovered heterogeneity in their susceptibility to the amnestic, convulsant nonimmobilizer F6. We believe that the most parsimonious explanation of this result is that somatic, extrasynaptic receptors on hippocampal CA1 pyramidal cells are, like their synaptic counterparts, sensitive to isoflurane and insensitive to F6, whereas some neurons located in the pyramidal cell layer express extrasynaptic receptors that are sensitive to inhibition by F6.

We thank Drs. Michael Laster and Edmund Eger 2nd (UCSF) for providing gas phase calibration standards for isoflurane and F6, and Sarah Smith (UW) for technical support.

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