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*Department of Anesthesia and Perioperative Care, University of California, San Francisco; and Department of Psychology, University of California, Los Angeles
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{at}anesthesia.ucsf.edu.
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Methods |
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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).
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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 x 28 cm base and 21 cm x 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:
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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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Discussion |
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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 
-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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Dr. Eger is a paid consultant to Baxter Healthcare Corp. which donated the desflurane and isoflurane used in these studies.
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