Certain volatile drugs called nonimmobilizers do not cause anesthesia despite having lipid solubilities that the Meyer-Overton hypothesis predicts would make them anesthetics (1–4). Nonimmobilizers do not cause immobilization at their predicted MAC (the minimum alveolar concentration of anesthetic required to suppress movement in response to noxious stimulus), at large concentrations modestly increase the MAC of simultaneously administered anesthetics, and can cause convulsions.
Even so, the nonimmobilizers 1,2-dichlorohexafluoro-cyclobutane (2N, also termed F6) and perfluoropentane, like the anesthetic desflurane, suppress learning in a classical conditioning paradigm, the fear-potentiated startle (5,6). The mechanism that mediates 2N suppression of this learning is unknown. The mechanism seems to be different from that mediating immobility, because movement response to noxious stimulation is not suppressed whereas fear-potentiated startle is.
Multiple brain systems are responsible for learning and memory. To examine some of these systems, we previously studied the effect of isoflurane on two other forms of learning: fear conditioning to tone and fear conditioning to context (7), learning paradigms mediated by different, albeit partially overlapping, neural substrates (8,9). In these forms of classical conditioning, a tone, or the surrounding environment (termed, “context”), is paired with a noxious electric shock (8,9). If a rodent learns an association between either of these previously neutral cues and the shock, upon reexposure to the cue, it will take a characteristic motionless posture termed “freezing”(10,11). We found that the concentration of isoflurane required to suppress fear conditioning to tone was twice that for fear conditioning to context (7). Because the hippocampus plus amygdala mediate freezing to context, whereas the amygdala alone mediates freezing to tone, our results suggested that the hippocampal-dependent aspects of learning are more vulnerable to the depressant effects of anesthetics (7). These differences prompted the present study of the effects of 2N. We anticipated that 2N would, like isoflurane, differentially influence fear conditioning to context and tone tested 24 h after training. Additionally, because 2N does not cause sedation at concentrations expected to suppress learning (12), we could study the short-term memory processes occurring during the conditioning procedure and compare these results with the results for more conventional long-term memory studied 24 h after the training (7–9,11,13). We hypothesized that short-term memory might be less vulnerable to disruption because it does not require the additional processing necessary for the retention of long-term memory.
The Committee on Animal Research of the University of California, San Francisco, approved our study of 140 male specific-pathogen-free Sprague-Dawley rats weighing 275 to 325 g (Charles River Laboratories, Hollister, CA). Animals were housed in our animal care facility for 1 wk before study under 12-h cycles of light and dark, 2 per cage, and had continuous access to standard rat chow and tap water before the study. The dose-response effect of 2N on fear conditioning was measured in 4 groups trained to context (n = 8 per group) and 6 groups trained to tone (n = 8 per group for 5 groups and n = 4 for 1 group). Untrained control groups receiving 2N (n = 8 per group) were included for each of the 2 conditioning preparations. An additional 3 groups (n = 8 per group) were studied to determine the duration of short-term memory for training to context and 3 groups (n = 8) for training to tone.
Cylinders and Chambers
Exposure to 2N before training was accomplished in 4 identical clear acrylic cylinders (7-cm diameter and 26-cm length) sealed with rubber corks perforated with inlet and outlet ports that permitted delivery of gas purged of carbon dioxide. Fresh gas inflow was provided by a 1 L/min oxygen flow through an enflurane vaporizer containing 2N only. Gases were recirculated through a soda lime canister, and gas concentrations were sampled from a port in the circle system.
Training was accomplished in 4 identical rectangular-shaped chambers (25 × 20 × 17 cm) constructed of clear acrylic and located in a well-lit room. 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). Fresh gas inflow was provided by a 5 L/min oxygen flow through an enflurane vaporizer filled with 2N. 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 animal occupied it.
Tone testing was performed in chambers providing a different environment from that provided by the training chambers. The clear acrylic testing chambers had an A-frame roof. They measured 25 × 28 cm base and 21 × 28 cm sides, had a smooth floor, and 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 testing cages were cleaned with a pine-scented solution, and there was no background noise. A speaker was mounted on the rear wall of each testing chamber.
On the training day, animals were brought to the training area and their tails marked with a permanent felt-tipped pen for identification. After at least 1 h of habituation in their home cages, animals were placed in the acrylic cylinders where 2N was delivered for 30 min at the target concentration. Each animal was then rapidly (<10 s) transferred to a training chamber via the 8-cm port in the chamber top. (We separately determined that the introduction of the rats did not materially change the chamber concentrations of 2N.) Animals were allowed to explore the chamber for 3 min before training began. 2N concentrations were measured by gas chromatography. The gas chromatograph was calibrated with secondary standards from tanks.
For tone conditioning, animals received three tone-shock pairs consisting of a 30-s tone (90 dB, A-scale, 2000 Hz) co-terminating with a 2-s electric shock (11 Hz bi-polar square waves, 2 mA for all groups); shock pairs were 90 s apart. Animals were returned to their home cages within 60 s after the last shock. For context conditioning, the identical procedure was used except that no tone was delivered. For Control groups, the identical tone and context procedures were used except that no shock was delivered, i.e., Unshocked Control groups.
The next day we assessed freezing to context and tone. For context testing, each animal that had received context training was returned to the chamber in which it was trained and allowed to explore the chamber for 8 min; neither tone nor shock were administered. Four animals were observed simultaneously, one in each of the four chambers. For tone testing, each animal that had received tone training was placed in an A-frame testing chamber in the different room (see above) and, after 3 min of exploration, a tone (90 dB, A-scale, 2000 Hz) was continuously sounded for 8 min; shocks were not administered. Again, four animals were observed simultaneously, one in each of the four chambers. Additionally, after a 4-h period after the tone test, each tone-trained animal was tested for context by an 8-min observation in the identical chamber in which it was trained and then tested for tone-context by applying the tone for an additional 4 min in that chamber.
Six additional groups were tested for duration of short-term memory. After training, the animals were returned to their home cages and moved out of the training room. The Context groups tested 3 or 20 min after training were either returned to the identical chambers in which they had been trained (Same Context groups) or placed in the A-frame chambers in the different room (Different Context group). The Tone groups tested 3 or 20 min after training were placed in the A-frame chambers at 3 or 17 min, allowed to explore for an additional 3 min, and then exposed to the tone for 8 min. Figure 1 shows a diagram of the four training and testing procedures.
Animal observations were via a video camera that allowed observation of all rats simultaneously. No personnel were in the training or testing rooms during these periods. Fear conditioning measured 3 min, 20 min, or 24 h after training was assessed by 2 trained observers. To score freezing, an observation of 1 of the 4 animals was made every 2 s (11). Therefore, each animal was scored once every 8 s. Behavior was judged as freezing if there was no visible movement except for breathing. The observation periods were also videorecorded for scoring by a blinded observer.
Freezing during training was assessed by viewing videotapes recorded during the conditioning procedures. To score freezing during training, an observation of each animal was made once every 2 s. Freezing during training was scored every 2 s rather than every 8 s because the training shocks and tones caused relatively rapid changes in freezing behavior.
For each group, the 2N concentration was calculated as the mean and standard deviation (sd) of the concentrations measured in the cylinders and in the training chambers before and after training of that group.
The percentage of time an animal froze during the 8-min observation periods for 3 min, 20 min, and 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 (11).
For the Context groups, the percentage of time an animal froze during the training procedure was calculated for the 60 s (30 observations) before the third (final) shock (or the comparable period for the Control [Unshocked] group). For the Tone groups, freezing scores during training were calculated for the 20 s (10 observations) before the third shock (i.e., 8 s after the tone onset). Additionally, so that freezing during training scores could be consecutively graphed, scores were calculated for each 10-s interval, that is, the number of observations in 10 s divided by 5.
In addition to the 8-min scores calculated for the observations made 3 min, 20 min, and 24 h after training, we calculated scores for these groups by using intervals comparable to the intervals used to calculate scores for freezing during training. To do so, for the Context groups, these freezing scores were calculated for the 60 s (30 observations) beginning 30 s after placing the animals in the testing chambers. For the Tone groups, these freezing scores were calculated for the 60 s (30 observations) before and 20 s (10 observations) after the tone onset (allowing an 8-s latency after the tone onset).
For each group score, the mean and standard error of the mean (sem) were calculated. Single-factor analysis of variance StatView (Abacus Concepts, Inc., Berkeley, CA) was used for overall comparison of the differences among the Context-Conditioned groups and the Tone-Conditioned groups. Student-Newman-Keuls testing provided pairwise comparisons among the groups. Freezing scores were defined as impaired when the group score differed significantly from the corresponding 0% group. Freezing was defined as abolished when the score did not differ significantly from the corresponding Control (Unshocked) 3% 2N group. We use the term “suppressed” to collectively refer to freezing that was impaired and/or abolished. Two-factor analysis of variance was used for overall comparison of the difference between Context versus Tone groups at identical concentrations. A two-tailed t-test test was used to compare groups tested 3 and 20 min after training. A P value < 0.05 was regarded as significant for all comparisons.
Additionally, the 8-min freezing score of each animal at 24 h was dichotomously classified as either freezing or nonfreezing, defining the thresholds for classification according to the scores in the Control (Unshocked) groups (7). For the context paradigm, the classification threshold was chosen as the highest freezing score of any animal in the Context Control (Unshocked) group; the threshold for the tone paradigm was similarly chosen from the scores in the Tone Control (Unshocked) group. The thresholds for the freezing scores during training were similarly chosen. Logistic regression analysis was then applied to provide the 50% effective doses and SEMs for suppression of learned context-fear and tone-fear associations (BMDP Statistical Software; University of California Press, Berkeley, CA). The statistical significance of the difference between the 50% effective doses was calculated using a two-tailed t-test.
We calculated the correlations between the on-line freezing scores of the two observers, and between the on-line and blinded scores of one observer, measured for individual animals.
The average measured concentration of 2N was within 5% of the target concentration for all groups.
Two of the 4 animals in the 3.7% group convulsed when the tone was applied. Because 2 previous animals in pilot studies had similarly convulsed at 3.7%, we did not study additional animals in this group. Data for 2 animals in the 3-min tone groups were deleted because of technical problems.
Table 1 and Figure 2 show the percent freezing at each concentration step for animals trained to context with unsignaled shocks (i.e., without tones). The freezing scores are for testing 24 h after training using the 8-min observation scores. As shown, memory for context was neither impaired nor abolished by 1% 2N and was both impaired and abolished by 2% and 3% 2N. The alternative 60-s freezing scores yielded the same conclusions.
Rats trained with shocks signaled by a tone preceding the shock showed minimal freezing during the 1 min before the onset of the test tone; the highest baseline group score before the tone was 4% ± 3%. Table 1 and Figure 2 show freezing scores for these animals during the test tone. Memory for tone was neither impaired nor abolished by 2% 2N, and was impaired although not abolished by 3% and 3.5% 2N. (Although 3.7% 2N yielded group scores that were not statistically different compared with the 3% Control [Unshocked] group, the small number of animals in the 3.7% group (n = 2) argues against describing memory as being abolished.) The alternative 20-s freezing scores yielded the same conclusions. Two-factor analysis of variance yielded significant main effects of tone versus context, F(1, 56) = 36.2, P < 0.001, and concentration, F(3, 56) = 10.5, P < 0.001, although the interaction between the two was not significant, F(3, 56) = 1.05, P = 0.38. Thus, for long-term memory, context training was more vulnerable to the effects of 2N than tone training.
The tone-trained animals were later retested, this time in their original training chambers. When tested to context, as expected (7) and shown in Table 1, the freezing was infrequent for all groups, and we do not draw any conclusions from these groups. When these animals were then tested to tone context, freezing again seemed to be impaired at 3%, whereas no concentration abolished learning. Thus, the results for tone context are in accord with the results to tone alone.
In preparation for logistic regression analysis, freezing scores were classified as either freezing or nonfreezing (7). For the freezing-to-context paradigm, the freezing scores of the animals in the Control (Unshocked) group ranged from 0% to 7%, and 7% was taken as the threshold: scores >7 were classified as freezing whereas scores ≤7 were classified as nonfreezing. Similarly, for the freezing-to-tone paradigm, the freezing scores of the animals in the Control (Unshocked) group ranged from 0% to 30%, and 30% was taken as the threshold. (By comparison, an alternative method for defining the threshold values might be the Control (Unshocked) group score mean plus 2 SDs, which for the context preparation would be 7% and for the tone preparation would be 31%.) Logistic regression analysis applied to the classified data for testing at 24 h yielded a 50% effective concentration (EC50) for the suppression of freezing to context of 2.00% ± 0.01% (value ± sem) and of freezing to tone of 3.45% ± 0.26%; the difference between EC50s was significant (P < 0.05).
Figure 3 shows the 10-s freezing scores during the training to context procedure. For the 0%, 1%, and 2% 2N groups, each shock caused a burst of activity (14) (i.e., decreased freezing) followed by markedly increased levels of freezing. In contrast, minimal freezing developed in the 3% 2N group. As expected, no increase occurred in the Control (Unshocked) group. Table 2 and Figure 4 show the freezing during training scores (60 s before the third shock) and Figure 4 additionally shows the scores of these same animals tested 24 h after training (8-min scores). Importantly, 2% 2N did not impair or abolish freezing to context during training but did impair and abolish freezing to context measured 24 h after training. Three percent 2N impaired and abolished all forms of contextual learning and memory. Thus, long-term memory for context training was more vulnerable than short-term memory to the effects of 2N. Consistent with this conclusion, the EC50 for suppression of freezing to context measured during training, 2.59% ± 21% (60-s score), was more than for that measured at 24 h, with either 8-min or 60-s scores, 2.00% ± 0.01% and 1.32% ± 0.22%, respectively; the differences between EC50s for suppression measured during training and at 24 h were significant (P < 0.05).
Figure 5 shows the freezing scores during training to tone. Like context training, each shock caused an activity burst. So did each tone. Table 2 and Figure 6 show the scores for freezing (20 s before the third shock) and Figure 6 additionally shows the scores of these animals tested 24 h after training (8-min scores). Three percent and 3.5% 2N did not impair freezing to tone during training but did impair freezing measured 24 h after training. An EC50 for freezing during training was not calculated because all animals continued to freeze even at 3.5 vol%, the largest concentration group we studied completely. In contrast, concentrations of 3.28% ± 0.22% (20-s scores) and 3.45% ± 0.26%, (8-min scores) suppressed freezing in 50% of animals measured at 24 h (i.e., the EC50s). Thus, long-term memory for tone training was more vulnerable to the effects of 2N than short-term memory.
Two-factor analysis of variance of freezing during training scores yielded significant main effects of tone versus context, F(1, 56) = 7.35, P < 0.01, and concentration, F (3, 56) = 10.3, P < 0.001, and significant interaction between the two, F(3, 56) = 8.46, P < 0.001. Thus, for short-term memory, context training was more vulnerable to the effects of 2N than tone training.
To ascertain that freezing observed during training represented context-shock associations and to evaluate the duration of short-term memory for the associations, groups were trained while breathing 2% 2N and tested 3 min and 20 min after training. Figure 7 shows the 60-s scores for the Control group trained to context and tested at 3 min in the different chamber (Different Context group) and the similarly trained groups tested at 3 and 20 min in the training chamber (Same Context groups). The results show that freezing to context during training represented context-shock associations and that short-term memory of the associations endured for at least 3 min.
The duration of short-term memory of the tone-shock associations was evaluated by comparing Tone-Shocked and Tone-Unshocked groups trained while breathing 3% 2N. Figure 8 shows the 60-s scores before, and the 20-s scores after, tone onset for the Control Tone-Unshocked group tested at 3 min and the Tone-Shocked groups tested at 3 and 20 min. The results show that freezing to tone during training represented tone-shock associations and that short-term memory of the associations endured for at least 20 min.
The independently obtained on-line scores of the two observers, and the on-line and blinded scores of one of the observers, correlated closely, r2 = 0.99 and 0.98, respectively. These three sets of observations provided nearly identical group freezing scores and yielded the same conclusions.
The administration of 2N during fear conditioning caused distinctive patterns of suppression of learning and memory. Long-term memory of tone-shock training was impaired. The concentration producing a 50% impairment (EC50) was 3.45% ± 0.26% 2N (mean ± sem). For context-shock training, the EC50 was 2.00% ± 0.01%. This 1.7 times larger concentration to impair fear conditioning to tone compared with context, is consistent with our previous finding that the isoflurane concentration suppressing fear condition to tone was 1.9 times larger than the concentration suppressing fear to context (7). Long-term memory of tone-shock training was not abolished by 3.5% 2N, whereas context-shock training was abolished by 2%. These findings support the concept that processes mediating long-term memory for fear conditioning to context are more vulnerable to the effects of inhaled anesthetics than those to tone. The findings are also consistent with similar mechanisms mediating suppression of long-term memory during 2N and isoflurane.
The effect of 2N on short-term memory could be studied because 2N, unlike isoflurane, does not cause sedation (12). This lack of sedative effect is shown in Figures 3 and 5 by the very low scores (i.e., nearly continuous mobility) before the onset of training shocks. Short-term memory for context-shock training was impaired with an EC50 of 2.59% ± 0.21% 2N, yet short-term memory for tone-shock training was not impaired with 3.5% [the largest concentration studied, 83% of the predicted MAC calculated according to the oil/gas partition coefficient of 2N (1)]. These results for short-term memory are consistent with those for long-term memory, that context-shock associations are more vulnerable to the effect of 2N than tone-shock associations.
Further, because long-term memory was suppressed at concentrations that did not suppress short-term memory, these results also show that short-term memory processes are more resistant to the depressant effects of 2N than long-term memory processes.
To ascertain that freezing measured during training was, in fact, evidence of short-term memory, we studied freezing measured 3 and 20 minutes after training. Animals trained with context shocks during 2% 2N and with tone shocks during 3%, concentrations that suppressed long-term memory, did not suppress freezing to context or tone measured 3 minutes nor freezing to tone measured 20 minutes after training, showing that short-term memory persisted for up to 3 to 20 minutes.
During training, animals displayed a burst of activity to both tone and shock (Fig. 5), showing that the stimuli were perceived by the central nervous system. They also developed increasing freezing to the second and third tones. This freezing shows that tone and shock signals were transmitted to the site where initial learning occurred. These observations are consistent with previous findings that the mechanism whereby 2N suppresses learning and memory does not include suppression of impulse transmission to the brain (15–17).
Whereas two animals in the 3.7% group convulsed at the time of tone presentation, smaller concentrations produced no motor manifestations of seizure activity, and our previous study did not find any electroencephalographic evidence of epileptic activity at such concentrations (12). Thus, the mechanism for suppressing memory at concentrations of 3.5%, or less, does not include convulsive activity.
Our results may be summarized in terms of the stages of information processing involved in learning and memory: acquisition (memory formation), retention (storage), and retrieval (expression) (18). For acquisition, information must be transmitted to the site where memory formation occurs. Then, information is encoded to form a new memory trace, a process that seems to involve enhanced synaptic efficiency (19). Retention of this trace requires transformation to a more enduring form, a process sometimes referred to as consolidation (20). A crucial step in this transformation is gene induction and the synthesis of new protein (21–23). This step provides an important threshold for defining short-term and long-term memories, a step that occurs within an hour or so of training (21–23). Thus, we defined short-term memories as those retrieved during or up to 20 minutes after training, and long-term memories as those retrieved 24 hours after acquisition.
Our findings suggest that 2N does not interfere with transmission of tone-shock signals, or of context-shock signals except perhaps during concentrations of at least 3%. 2N does not impair encoding of tone-shock associations, and impairs those for context-shock associations only at concentrations of at least 3%. Importantly, 2N disrupts the transformation of initial memory traces to more enduring forms of memory for both tone-shock and context-shock training.
The finding that fear conditioning to context is more vulnerable to the effect of 2N than fear conditioning to tone may be explained by the additional processing required for the contextual conditioning. The neural substrates underlying fear conditioning to tone and context differ. The amygdala mediates learning of freezing to tone whereas both amygdala and hippocampus are required for learning freezing to context. Disruption of hippocampal processing suppresses fear conditioning to context yet does not suppress fear conditioning to tone (8,9).
What mechanisms might explain the resistance of short-term relative to long-term memory? This pattern can be produced by disruptions of intracellular signal processing leading to protein synthesis. For example, mutants with alterations of the mitogenic-activated protein kinase cascade demonstrate normal fear conditioning to tone and context when measured 30 minutes after training but not after 24 hours (24). Disruptions of protein kinase A (22,23,25), cAMP-responsive element-binding protein (21), and protein synthesis (22,23) provide other examples.
Perhaps importantly, disruptions of certain metabotropic glutamate receptors (mGluRs) can also impair long-term memory more than short-term memory (26). For example, Frohardt et al. (27), infused a mGluR antagonist into the hippocampus of rats before fear conditioning. The animals froze to context during training yet freezing measured at 24 hours was suppressed, showing that short-term memory was intact yet long-term memory impaired (27). Further, Aiba et al. (28), reported that genetic disruption of mGluR activity impaired context-specific learning more than tone-specific learning, a result consistent with our finding that context conditioning was more vulnerable to the effects of 2N than tone conditioning.
This pattern of disruption with mGluRs is interesting because Minami et al. (29–31), reported that 2N suppressed mGluR1 and mGluR5 activity in oocytes at concentrations that suppressed learning in rodents (5,6). These authors proposed that suppression of mGluRs may explain 2N’s suppression of learning (30,32).
In contrast, at the concentrations we studied, 2N does not depress ligand-gated ion channels associated with synaptic activity, notably γ-aminobutyric acid type A, N-methyl-d-aspartate, and α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (32–35). These results are consistent with our findings that the acquisition of tone-shock associations remained intact and those for context shock were only minimally impaired by 2N. We do not know why 3% 2N impaired acquisition of training to context.
Our findings confirm our previous reports that 2N suppresses or abolishes fear conditioning in rats tested using fear-potentiated startle (5,6). The present study differs in methodology and in the capacity to dissect components of the learning process (5,6,36). Differences in study method can produce quantitative differences in results. Consider our finding that 0.25 MAC isoflurane suppressed freezing to context and our previous finding that 0.25 MAC desflurane suppressed fear-potentiated startle (5,6). In contrast, 2% 2N suppressed freezing to context in the present study, a larger concentration than the 1% required to suppress fear-potentiated startle. Although these 2N concentrations differ, possibly because of the differences in methods, the results show that 2N suppresses fear conditioning over the wide range of conditions used for the freezing and the startle paradigms.
In summary, we find that the administration of 2N during fear conditioning causes less suppression of short-term memory than of long-term memory, and less suppression of tone-shock associations than of context-shock associations. Such results suggest that both anesthetics and nonimmobilizers affect memory and learning at different sites and that the effect at each site may be mediated by different mechanisms. They also suggest that amnesia for remembrances of one form of sensory input may not indicate amnesia for another form.
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