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Administration of Epinephrine Does Not Increase Learning of Fear to Tone in Rats Anesthetized with Isoflurane or Desflurane

Sonner, James M. MD*; Xing, Yilei MD*; Zhang, Yi MD*; Maurer, Anya BS*; Fanselow, Michael S. PhD, MD; Dutton, Robert C. MD*; Eger, Edmond I. II MD*

doi: 10.1213/01.ANE.0000148619.77117.C7
Anesthetic Pharmacology: Research Report

Previous reports suggest that the administration of epinephrine increases learning during deep barbiturate-chloral hydrate anesthesia in rats but not during anesthesia with 0.4% isoflurane in rabbits. We revisited this issue, using fear conditioning to a tone in rats as our experimental model for learning and memory and isoflurane and desflurane as our anesthetics. Expressed as a fraction of the minimum alveolar anesthetic concentration (MAC) preventing movement in 50% of rats, the amnestic 50% effective dose (ED50) for fear to tone in control rats inhaling isoflurane and injected with saline intraperitoneally (i.p.) was 0.32 ± 0.03 MAC (mean ± se) compared with 0.37 ± 0.06 MAC in rats injected with 0.01 mg/kg of epinephrine i.p. and 0.38 ± 0.03 MAC in rats injected with 0.1 mg/kg of epinephrine i.p. For desflurane, the amnestic ED50 were 0.32 ± 0.05 MAC in control rats receiving a saline injection i.p. versus 0.36 ± 0.04 MAC in rats injected with 0.1 mg/kg of epinephrine i.p. We conclude that exogenous epinephrine does not decrease amnesia produced by inhaled isoflurane or desflurane, as assessed by fear conditioning to a tone in rats.

IMPLICATIONS: Administration of epinephrine does not affect fear conditioned learning in rats inhaling isoflurane or desflurane.

*Department of Anesthesia and Perioperative Care, University of California, San Francisco; and †Department of Psychology, University of California, Los Angeles

Supported, in part, by NIH grant 1PO1GM47818–07.

Dr. Eger is a paid consultant to Baxter Healthcare Corp. which donated the desflurane and isoflurane used in these studies.

Address correspondence and reprint requests to James M. Sonner, MD, Department of Anesthesia, S-455, University of California, San Francisco, CA 94143-0464. Address e-mail to sonnerj@anesthesia.ucsf.edu.

Weinberger et al. (1) found that the administration of epinephrine resulted in acquisition of conditional fear (i.e., learning) in rats deeply anesthetized with pentobarbital plus chloral hydrate. In the same laboratory, and using the same techniques, Gold et al. (2) duplicated this finding. However, using another learning paradigm in rabbits, El-Zahaby et al. (3) failed to replicate this finding. Weinberger and Gold (4) contended that this failure resulted from several possible causes, such as differences in species, depth of anesthesia, learning paradigm, and duration of training. In the present study, we revisited this issue, testing some of the explanations offered by Weinberger and Gold (4). Because epinephrine is used clinically in patients under anesthesia, it is critical to know for certain whether or not it will compromise any of the desirable effects of anesthesia. We examined fear conditioning in rats, using the clinical anesthetics isoflurane and desflurane, over a range of anesthetic depths from no anesthesia to those abolishing learning.

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Methods

The Committee on Animal Research of the University of California, San Francisco approved our study of male 8- to 10-wk old [Crl:CD(SD)BR] Sprague-Dawley rats, weighing 275–325 g, obtained from Charles River Laboratories (Wilmington, MA). Rats were housed 2 per cage in our animal care facility under 12 h cycles of light and dark for 1 wk before study and had continuous access to standard rat chow and tap water before the study.

We assessed the effect of intraperitoneal injection of epinephrine on fear conditioning to tone, in the absence and presence of various concentrations of desflurane or isoflurane (Tables 1 and 2). A given rat received one dose of epinephrine and one concentration of desflurane or isoflurane (i.e., was studied only once).

Table 1

Table 1

Table 2

Table 2

Training was accomplished in 4 identical rectangular-shaped chambers (25 cm × 20 cm × 17 cm) constructed of clear acrylic and located in a well-lit room, as previously described (5). The top of each training chamber contained an 8-cm diameter port sealed with a rubber cork. Inlet and outlet ports allowed continuous ventilation through the chambers. A circular flow through the 4 chambers was maintained by a fan producing a background noise of 70 dB (A-scale) (Sound Level Meter, Radio Shack, Ft. Worth, TX). A 5-L/min-oxygen flow through a variable-bypass isoflurane vaporizer or Tec-6 desflurane vaporizer (Datex-Ohmeda, Madison, WI) provided the fresh gas inflow. Carbon dioxide was removed with a soda lime canister, and gas concentrations were sampled from a port in the circle system. The floor of each training chamber consisted of 14 stainless steel rods (6-mm diameter), spaced 1.8 cm center to center wired to a shock scrambler (Gemini Avoidance System, San Diego Instruments, San Diego, CA). A speaker was mounted on the rear wall of each training chamber. Training chambers were cleaned with 2% ammonium hydroxide before and after each rat occupied it.

The chambers for tone testing provided a different environment from that provided by the training chambers. The clear acrylic testing chambers had an A-frame roof. Each had a 25 cm × 28 cm base and 21 cm × 28 cm sides and had a smooth floor, and all were in a different room from the training chambers. The test room was lit with a 25-W red light bulb, whereas the training room was lit by conventional white-light fluorescent ceiling lamps. The test cages were cleaned with a pine-scented solution, and there was no background noise. A speaker was mounted on the rear wall of each test chamber.

On the training day, rats (2 per cage) were brought to the training area, weighed, and their tails were marked with a permanent felt-tipped pen for identification. They were placed in an equilibration cage covered with a clear plastic plate surmounted by a chimney broad enough to allow us to easily reach into the cage and handle or extract a rat. The clear plastic plate was pierced to allow delivery of a >2-L/min inflow of oxygen with or without isoflurane or desflurane and to allow measurement of either anesthetic as monitored with an infrared analyzer (Capnomac II; Datex, Helsinki, Finland). The anesthetic concentration was adjusted to a predetermined target concentration identical to that provided in the training chambers. After 27 min of equilibration, each rat was injected either with normal saline or normal saline plus epinephrine at a dose of 0.01 mg/kg or 0.1 mg/kg and then returned to the equilibration chamber for an additional 3 min. After 30 min of equilibration, each rat was rapidly (<10 s) transferred from the equilibration cage via the chimney to a training chamber via the 8-cm port in the chamber top. (We separately determined that the introduction of the rats did not significantly change the chamber concentrations of anesthetic.) Anesthetic concentrations were separately measured by gas chromatography. The gas chromatograph was calibrated with secondary standards from tanks.

Rats were allowed to explore the chamber for 3 min before training began. Each rat then received 3 tone-shock pairs consisting of a 30-s tone (90 dB, A-scale, and 2000 Hz) coterminating with a 2-s electric shock (11 Hz bipolar square waves; 2 mA for all groups); 60 s separated repeated tone-shock trials. Rats were returned to their home cages (free of anesthetic) within 60 s after the last shock.

The next day, we assessed freezing to tone. Each rat was placed in an A-frame test chamber in the different room (see above), and after 3 min of exploration, a tone (90 dB, A-scale, and 2000 Hz) was continuously sounded for 8 min; shocks were not administered. Four rats, one in each of the four chambers, were observed via a video camera that allowed observation of all rats simultaneously. No personnel were in the testing room during this period.

Fear conditioning was assessed by a trained observer blind to the treatment received by the rats. To score freezing 24 h after training, an observation of 1 of the 4 rats was made every 2 s (6). Therefore, each rat was scored once every 8 s. Behavior was judged as freezing if there were no visible movement except for breathing. Rats were video recorded during training and testing for off-line analysis if required.

For each group, the anesthetic concentration was calculated as the mean of the concentrations measured in the cylinders and in the training chambers before and after training of that group.

The percentage of time a rat froze during the 8 min observation periods 24 h after training was calculated as the number of observations judged to be freezing divided by the total number of observations in 8 min, i.e., 60 observations (6).

For each group score for conditioning to tone, the mean and se were calculated. Median and quartiles also were calculated. One-way analysis of variance allowed comparisons of test and control (no epinephrine) groups at comparable anesthetic concentrations. Nonlinear regression was performed to calculate a 50% effective dose (ED50) according to the following equation:

CV

CV

where A is the maximum value of the dose-response curve, n is the hill coefficient, and ED50 is the concentration of anesthetic producing an effect equal to 50% of the maximum effect. A value of P < 0.05 was regarded as significant.

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Results

At a given MAC (the minimum alveolar anesthetic concentration of an inhaled anesthetic that is required to eliminate movement in 50% of animals in response to a noxious stimulus) fraction, the freezing score of rats given epinephrine did not differ significantly from the score for rats injected with normal saline (Tables 1 and 2; Figs. 1 and 2). Combining results for a given MAC fraction also did not produce significance. The ED50 values for isoflurane were: control (no epinephrine), 0.32 ± 0.03 MAC (mean ± se); 0.01 mg/kg of epinephrine, 0.37 ± 0.06 MAC; and 0.1 mg/kg of epinephrine, 0.38 ± 0.03 MAC. None of these differed significantly from the other by an analysis of variance (P = 0.28). The ED50 values for desflurane were: control, 0.32 ± 0.05 MAC; and 0.1 mg/kg of epinephrine, 0.36 ± 0.04 MAC. These ED50 values did not differ significantly (t-test; P = 0.54). There was no detectable effect of epinephrine on the amnesia produced by these anesthetics.

Figure 1

Figure 1

Figure 2

Figure 2

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Discussion

We found no statistically significant effect of epinephrine injection on fear to tone at any anesthetic concentration. Anesthetic concentrations ranged from zero to those that eliminated learning. The results for isoflurane did not differ from those for desflurane (Tables 1 and 2; Figs. 1 and 2). Thus, our results extend those reported by El-Zahaby et al. (3) but differed from those of Weinberger et al. (1) and Gold et al. (2).

The difference between our results and those found by Weinberger et al. (1) and by Gold et al. (2) are not due to a difference in study animals (both we and they studied Sprague-Dawley rats); also like the animals of Weinberger et al. (1), our rats were trained briefly in a single session and were tested soon after training. The duration between the time of training and testing is important because Gold et al. (2) found that a 7- or 15-day delay decreased measures of learning. Like Weinberger et al. (1), we studied rats that were awake and rats in which anesthesia was deep enough in control rats to suppress all learning. In addition, we studied the effects of epinephrine in rats subjected to anesthetic concentrations that permitted some (but decreased) learning to occur. We selected a dose of epinephrine found by Weinberger et al. (1) to increase learning. We also studied smaller (0.01 mg/kg) doses of epinephrine in groups of 4 rats and found no trend to a difference in learning for epinephrine and saline-injected rats, again in contrast to Weinberger et al. (1) who found that, if anything, 0.01 mg/kg of epinephrine produced a larger effect than 0.1 mg/kg of epinephrine.

Our study does differ from that of Weinberger et al. (1) in several ways. One of these is subtle: we assessed conditioning by the proportion of time the rat froze (did not move), and Weinberger et al. (1) assessed conditioning by the suppression of drinking. They also used an anesthetic (pentobarbital plus chloral hydrate) that is not used for clinical anesthesia and that has a different mechanism of action. Pentobarbital probably works by enhancing the action of γ-aminobutyric acid (GABA)A (7), whereas inhaled anesthetics such as isoflurane or desflurane affect multiple receptors (8,9).

The lack of an effect of epinephrine on anesthesia-induced amnesia is consistent with its lack of effect on MAC (10). Any effect it has likely is caused by changes in peripheral receptors that indirectly might influence the central nervous system.

Because epinephrine does not cross the blood-brain barrier, its action on memory is mediated possibly by increasing glucose concentrations (11). However, the studies reporting such an effect used instrumental forms of learning in unanesthetized animals (11). This effect of glucose was not found when Pavlovian forms of learning were used (12). The difference in forms of learning may be important; we, Weinberger et al. (1), and Gold et al. (2) used Pavlovian conditioning.

Epinephrine also might influence the kinetics of anesthetics. The large dose of epinephrine used in both our study and the study of Weinberger et al. (1) should increase cardiac output and the distribution/redistribution of anesthetic. This would be a smaller factor in the case of an inhaled anesthetic, where the increase in metabolism consequent to epinephrine administration would be expected to affect both cardiac output and ventilation (i.e., there should be limited effects of the MAC because of the counterbalancing effects of increased cardiac output and increased ventilation) (13). Changes in uptake would be less relevant with a poorly soluble anesthetic, such as desflurane (14), than a more soluble anesthetic, such as isoflurane (15). Although the differences from control were not statistically significant, note that the ED50 value for isoflurane after 0.1 mg/kg of epinephrine was 20% larger than control, whereas the ED50 value for desflurane was 13% larger.

An injected anesthetic, such as pentobarbital, differs in a crucially relevant manner from an inhaled anesthetic. With an injected anesthetic, increased ventilation cannot compensate for the more rapid redistribution of anesthetic secondary to an increased cardiac output. Thus, epinephrine administration would be expected to decrease the blood and brain concentration of pentobarbital by hastening redistribution to depots such as muscle and fat. Perhaps this explains at least part of the recovery of learning capacity seen by Weinberger et al. (1). Note further that only a small change in pentobarbital concentration at the effect site for learning and memory may be required to produce a large change in learning and memory if the dose-response relationship is steep (Figs. 1 and 2) and if the applied dose places the subject near the steeper portion of the dose-response curve. We do not know whether the rats in the study by Weinberger et al. (1) had a dose of pentobarbital that placed them near the steeper portion of the curve, because only one dose was given (i.e., unlike the present study, only a single point on the dose-response curve was obtained).

We conclude that epinephrine injection does not significantly increase conditional fear to a tone in rats under anesthesia with isoflurane or desflurane. If learning is increased, the increase is small and is of no clinical consequence. If a small increase occurs, it may have a pharmacokinetic rather than a pharmacodynamic basis.

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