Effects of conspecific and predator odors on defensive behavior, analgesia, and spatial working memory.
The Effects of Previous Exposure to Inescapable Shock and the Odors of Shocked-Donor Conspecifics on Prod Burying and Freezing
To test the influence that exposure to inescapable shock and the presence of odors from shocked conspecifics might have on behavior, my students and I conducted a series of studies examining "shock-prod burying," rather than testing subjects on an escape-learning task in a shuttlebox. Pinel and his colleagues (e.g., Pinel & Treit, 1978, 1979, 1981; Terlecki, Pinel, & Treit, 1979) have demonstrated that rats show a reliable sequence of responses after they have experienced a single encounter of being shocked from an electric prod that protrudes from the wall of a chamber with movable bedding material on the floor. The first response occurring after the shock is for the rat to jump away from the prod and to freeze, motionless, in one of the corners that is opposite the wall on which the prod is mounted. Following a varying period of freezing, the rat then engages in repetitive approach-withdrawal reactions to and from the shock prod, and finally the rat begins to bury the prod with the bedding material using vigorous forepaw movements until the prod is typically covered (see Pinel & Wilkie, 1983, and Fanselow, Sigmundi, & Williams, 1987, for more complete descriptions and interpretations of the significance of this type of burying behavior). Of particular relevance to my stress research is the fact that this paradigm produces a reliable sequence of shock-elicited responses and the act of burying the prod requires that the rat has direct and prolonged contact with possible sources of odors which the experimenter can add to the bedding material. Therefore, I was interested in examining if previous experience with a series of inescapable shocks and/or the presence of urine and feces of shocked-donor rats in the bedding of the shock-prod test chamber resulted in altering the freezing and burying responses that have been typically observed after rats have been given a prod shock.
In order to study this problem, I conducted an experiment in which 32 adult male albino rats were first given four 30-min habituation sessions per day in an observational test chamber with clean corn-cob bedding (Bed-O-Cobs) on the floor (Williams, 1987). The chambers were illuminated with a dim light, and white noise was used to mask most extraneous sources of noise that might be distracting. Following the habituation sessions, one group of 16 rats was randomly assigned to a preshock (PS) condition and given 80 trials of 5-s presentations of inescapable shock (1 mA) via tail electrodes while restrained in a plastic tube for 90 min. Another group of 16 rats was given no shocks (NS) while being restrained in similar tubes for the same duration. Twenty-four hours later, both of these groups were randomly assigned to two subgroups, of 8 rats each, and tested in the observation chambers for prod burying and freezing following a single 100-ms presentation of a 6.5-mA shock delivered via a copper coil that was wrapped around the wooden wall prod. The duration of these observational-test sessions was 20 min after each subject made contact with the prod. One-half hour prior to this test session, stress odors (SO) were obtained by collecting a 1-liter sample of soiled bedding from male donor rats that had received five 5-s presentations of inescapable shock to their paws from the floor of a typical operant chamber. A similar size sample of bedding was collected from the bedding of nonshocked donor rats (NSO). These two types of samples were added to the bedding of two pairs of subgroups of rats immediately before the observation session in the test chamber. Thus, this experiment involved four groups of rats that comprised a 2 (PS/NS) pretreatment x 2 (SO/NSO) test-odor factorial design.
From behavior observed during the shock-prod test session, the total duration of freezing (i.e., crouched, immobile posture) and of prod burying (i.e., forepaw pushing of bedding in the direction of the prod) were recorded. The maximum height of the pile of bedding within 5 cm of the prod was also measured at the end of the session. All of the nonstressed rats in Group NS/NSO, and some of the subjects in the other groups, showed freezing, approach-avoidance responses, and prod burying after receiving the prod shock. Figure 1 presents the mean duration of prod burying and the heights of the piles for the four groups. Statistical analyses of these data, involving a factorial-design analysis of variance followed by post hoc comparison tests, indicated significant main effects of both the shock treatment and the test odors as well as a significant interaction between these variables. Specifically, the effects of preshock (PS) and the odors from shocked donors (SO) decreased the mean duration of burying and the heights of the bedding piles made by the subjects. Furthermore, the combination of having received the preshock condition and being tested in the presence of the odors of shocked-donor rats (i.e., Group PS/SO) resulted in very low levels of both of these measures of burying.
Figure 2 shows the mean duration of freezing for each of the four groups of subjects during the prod-burying observation session. Freezing behavior was found to occur very shortly after the subjects received the prod shock. The main effects of preshock and the odors from shocked conspecifics were both found to be a significant increase in the levels of freezing, and there was a significant interaction effect, with Group PS/SO clearly showing the greatest amount of freezing.
Exposure to shock and later testing with the stress odors from shocked conspecifics therefore resulted in a decrease in prod-burying behavior and an increase in freezing. Both of these changes were potentiated for rats that received the combination of preshock experience and testing with stress odors. Minor and Lolordo (1984) have reported that neutral odors associated with shock were effective mediators of fear reactions during subsequent tests that were conducted in a different environment. At the present time, it has not been determined whether the enhancement of changes in burying and freezing to the odors of stressed conspecifics, following shock exposure, is the result of fear conditioning and/or sensitization. However, the findings of another experiment by Williams (1987) have clearly demonstrated that this synergistic effect is found only if rats have received inescapable shock, as opposed to escapable shock, prior to testing with the odors of shocked conspecifics. This finding is consistent with the major assumption of Learned Helplessness Theory (Maier & Seligman, 1976), which claims that only exposure to uncontrollable stressors has the potential of producing alterations in subsequent responses.
The Effects of Exposure to Defeat by a Dominant Alpha Conspecific and the Odors of the Alpha Colony on Prod Burying and Freezing
Much of the research in the area of stress and coping with animals as subjects has involved the use of aversive or nociceptive stimuli. For example, electric shock is a good stressor because it can be easily and precisely controlled. However, it almost goes without saying that shock is not a very ethologically relevant source of stress for a wild rat in its natural environment. For this reason, the research that my students and I have been conducting during the past decade has focused on the effects of various types of social and predatory stressors that wild rats are likely to encounter. For example, Williams and Lierle (1988) found that when laboratory rats were defeated by a dominant (i.e., alpha) colony rat for repeated sessions, they demonstrated learning deficits and other physiological changes that paralleled the effects found in learned helplessness studies using inescapable shock as a stressor.
Based on our previous research involving stress exposure and testing with the odors of conspecifics, David Scott and I were interested in determining if a single session of defeat experience would result in rats showing a subsequent suppression in prod burying and an increase in freezing, particularly when subjects were tested with the odors associated with the alpha rat (Williams & Scott, 1989). Thirty-two male albino rats, which were used as subjects in this experiment, were first given four habituation sessions in the shock-prod chambers that were previously described. Then, they were randomly assigned to either a defeated (D) or nondefeated (ND) condition by being placed, as an intruder, in colony tub cages containing two 1-yr-old male resident rats after a female resident was removed from each cage. The subjects in the defeated condition were individually tested, as intruders, with residents that had been previously found to be consistently aggressive. After the intruder was placed inside a colony tub cage, the dominant or alpha male would typically sniff the anogenital area of the intruder, show lateral or broadside attack movements, pin the intruder on its back, and end a fighting bout by biting the intruder on its back. There were typically 3-5 aggressive bouts during a 15-min resident-intruder session (see Flannelly, Flannelly, & Blanchard, 1984; Williams, 1982; Williams & Lierle, 1988; for more details about the procedures used to establish aggressive colony males). The subjects that were assigned to the nondefeated condition were tested as intruders in colonies that consisted of two male rats, residing with a female, that were found previously to be nonaggressive during resident-intruder test sessions.
On the day after each resident-intruder session, 1-liter samples were obtained of the odors from the soiled bedding of the aggressive alpha colonies (AO) and the nonaggressive colonies (NAO). New samples of soiled bedding were evenly distributed on top of the bedding on the floor of the shock-prod testing chambers. The subjects were placed individually in the shock-prod chamber with soiled bedding, and they were given the previously described prod shock upon their initial contact with the prod. The durations of prod burying and freezing were recorded for each of the four groups of subjects: nondefeated and tested with nonalpha-colony odors (Group ND/NAO), nondefeated and tested with alpha-colony odors (Group ND/AO), defeated and tested with nonalpha-colony odors (Group D/NAO), and defeated and tested with alpha-colony odors (Group D/AO).
The mean duration of prod burying and heights of the bedding piles for each of the groups are shown in Figure 3. Although the presence of alpha-colony odors in the bedding material of the shock-prod chamber resulted in a nonsignificant reduction of prod burying, the rats that were defeated and later tested with the alpha-colony odors (i.e., Group D/AO) showed significantly less prod burying and made smaller piles of bedding than the rats in the other groups. Finally, as can be seen in Figure 4, the duration of freezing behavior was clearly the greatest for Group D/AO.
Therefore, the findings of this experiment revealed that a single session of being defeated by an aggressive dominant conspecific was effective in disrupting prod burying and increasing freezing only when such rats were later tested with odors of alpha colonies present. Other studies done in my laboratory have also replicated these results using the odors from alpha rats to which the intruders were not exposed and testing defeated rats with clean bedding, rather than the soiled bedding nonaggressive colonies. In order to account for the changes in burying and freezing seen as a function of the combination of defeat and later testing with the alpha-colony odors, it is believed these odors may have become conditioned fear stimuli during the resident-intruder session. Consistent with this conditioning interpretation are the results of Williams, Rogers, and Adler (1990) which demonstrated that, when previously defeated rats were given a long period of exposure to alpha-colony odors, they subsequently showed extinction of fear and thus buried the prod and did not freeze in the presence of these odors.
Effects of a Single Session of Being Defeated by a Dominant Alpha Conspecific and the Odors of the Alpha Colony on Analgesia and Freezing
Many investigators have shown that exposure to various types of stressors (e.g., footshock, forced-swimming in water, rotation) reduces the perception of pain (i.e., analgesia or hypoalgesia) in rodents (see Amir & Galina, 1986; Maier, 1986, 1989; Rodgers, 1989, for an extensive review of this literature). Researchers have also shown that environmental stimuli, paired with shock, result in conditioned analgesia that is believed to be opioid mediated because it can be reversed by administering an opiate antagonist (e.g., naloxone or naltrexone) either prior to conditioning or later testing (Bolles & Fanselow, 1980; Fanselow, 1986; Fanselow & Baackes, 1982; MacLennan, Jackson, & Maier, 1980). Based on these results and the findings just described by Williams and Scott (1989), Paul Worland, Mindy Smith, and I speculated that the feared odors of alpha conspecifics by resident intruders might elicit an opioid-mediated, conditioned analgesic response. Rather than using the traditional tail-flick test for measuring analgesia, we decided that a more sensitive test of analgesia would be to give intruders a small injection of formalin in one of their hindpaws and record the level of paw licking they later showed when tested with the odors of alpha-colony bedding being present in an observational test chamber. Fanselow (1984, 1986) has established that suppressions in this type of formalin-induced, recuperative paw licking can be reliably used as an index of analgesia.
Specifically, one of the experiments by Williams, Worland, and Smith (1990) involved randomly assigning 40 rats to groups that comprised a 2 x 2 factorial design which was similar to the one used by Williams and Scott (1989), except that formalin tests were given rather than shock-prod tests. Thus, after four daily habituation sessions in the observation chambers, separate groups of 10 male rats were given the following treatments: defeated by an aggressive colony resident as an intruder and later formalin tested with alpha-colony odors present (D/AO), defeated and tested with clean bedding containing no alpha-colony odors (D/NAO), not defeated by a nonaggressive colony and tested with alpha-colony odors (ND/AO), or not defeated and tested with no alpha-colony odors (ND/NAO).
The procedure used during the resident-intruder sessions was the same as that used by Williams and Scott (1989). One day after their session as an intruder in either an aggressive or a nonaggressive colony, all subjects were given a .05-ml injection of a 15% solution of formalin in their right rear paw. Then, all subjects were primed by being placed in an aggressive colony's cage for 1 min. During this brief time, none of the subjects was attacked by the alpha resident. Immediately after this priming, the rats were placed in an observation-test chamber, containing a l-liter sample of the soiled bedding from either an aggressive alpha colony or from a nonaggressive colony. Videotapes were used to record the number of times that each rat was licking its formalin-injected paw, involving an 8-s time-sampling procedure for a total duration of 8 min, which was the same procedure used by Fanselow (1984).
Figure 5 shows the mean amount of recuperative paw licking by each of the four groups that occurred during the test sessions following the formalin injections and priming. The results of an analysis of variance and post hoc tests indicated that having a session of being attacked and defeated, as a resident intruder, did not have a significant effect on the level of paw licking when rats were later formalin tested without the odors. Likewise, the odors of the alpha colony, during testing, did not produce a significant effect on paw licking for subjects that were not previously defeated. However, rats that experienced being defeated by a dominant conspecific and were later formalin tested with alpha-colony odors (i.e., Group D/AO) showed a significant suppression in paw licking. The mean amount of freezing that each of the groups showed during the same observational test session, after the formalin injection, is shown in Figure 6. As was seen for paw licking, only one group of rats differed from the other three groups. Specifically, subjects that were defeated and tested with the alpha-colony odors (i.e., Group D/AO) were the only ones to show a significant amount of freezing during the formalin test. It should be noted that this level of freezing was not so high as to preclude the possibility of the subjects in Group D/AO from engaging in paw-licking behavior during the 8-min formalin-test session. The data from this experiment parallel the findings of Williams and Scott (1989) and suggest that fear conditioning to the alpha-colony odors occurs during resident-intruder defeat sessions, and it is expressed in terms of suppressions in prod burying and a paw licking and an increment in defensive freezing. Further support for the conditioning interpretation of the paw-lick analgesic reaction in this study is supported by the results of a separate experiment done by these investigators which demonstrated that a prolonged exposure session to alpha-colony odors, following a defeat session, resulted in the extinction of the fear-mediated analgesia elicited by these odors (Williams, Worland et al., 1990).
In another one of our experiments (Williams, Worland et al., 1990), an assessment was made to determine if the previously reported conditioned analgesia elicited by the odors of an alpha colony, following a single session of defeat for an intruder subject, was mediated by an endogenous opioid process and thus could be reversed by an opiate antagonist. In this study, 50 male adult albino rats were randomly assigned to five groups of 10 subjects each and given two daily habituation sessions in the chambers that would later be used for formalin testing of analgesia. Next, all subjects were tested as intruders in 10 separate colonies, of 2 male rats and a female, for which I of the males was previously found to be aggressive. Each resident-intruder session lasted 15 min, and 24 hr later all subjects were primed for 1 min in an alpha colony, without being defeated, and then formalin tested using the identical experimental and recording procedures that were used in the previously reported experiment by Williams, Worland et al. (1990). The only procedural difference was the groups were injected (IP) either with 7 mg/kg of naltrexone (an opiate antagonist) or with saline 20 min before the resident-intruder session or before the formalin test session. Twenty-four hours later, four groups were formalin tested after a sample of bedding from the alpha colony was evenly distributed on the floor of the test chamber, whereas a saline-injected control group was formalin tested with no alpha-colony odors.
The mean levels of recuperative paw licking for all of these defeated groups are shown in Figure 7. As would be expected from our previously described research, there was significantly less paw licking (i.e., analgesia) for groups that had saline or naltrexone given prior to defeat and formalin tested with saline in the presence of alpha-colony odors (i.e., Groups S/S-AO and N/S-AO) as compared to the paw licking of the control group of subjects (i.e., Group S/S-NAO) that were given a saline injection prior to their defeat session and again prior to being tested with no alpha-colony odors. In contrast to this analgesic reaction observed with the combination of defeat experience and alpha-odor testing for saline-injected rats, administering naltrexone prior to the test session resulted in a significant increase in paw licking for Groups S/N-AO and N/N-AO. These results indicate that an endogenous opioid system was definitely involved in the regulation of the analgesia that defeated rats showed when tested with the alpha-colony odors. However, because this analgesic reaction was not completely reversed, it is believed that this type of conditioned analgesia may be regulated by a combination of both opioid and nonopioid processes. Another interesting finding is that even though the opioid processes were blocked for the rats in Group N/S-AO, at the time of defeat, these subjects showed analgesia when subsequently formalin tested with saline and with the alpha-colony odors being present. This suggests that the observed analgesia, during testing, was the result of the conditioning of a central state (e.g., fear) that did not require the actual activation, or expression, of opioid processes during the time of conditioning (i.e., the resident-intruder session). In contrast to the paw-licking data, the results of defensive freezing for these same groups of subjects indicated that naltrexone had no effect if it was given prior to the formalin test, but naltrexone did increase freezing when given prior to the resident-intruder session. This finding suggests that the intensity of the pain from being bitten by the alpha residents may have been greater for intruders that were given naltrexone prior to their defeat experience, thus resulting in stronger fear conditioning.
Conditioned Fear and Analgesia to Alpha-Colony Odors Following Defeat by a Dominant Conspecific: Involvement of GABA and Serotonin Receptors
When benzodiazepines bind to the GABA (gamma-aminobutyric acid) receptors, decreases in neuronal transmission are known to occur and fear can be significantly attenuated (Shephard, 1987). Injecting rats with a benzodiazepine, prior to exposing them to repeated trials of inescapable shock, has been found to prevent the occurrence of learned helplessness when the rats are later tested for escape learning in a shuttlebox (Drugan, Ryan, Minor, & Maier, 1984). It has also been shown that injecting rats with a benzodiazepine, such as diazepam, successfully blocks the conditioning of fear to contextual stimuli associated with electric shock (Fanselow & Helmstetter, 1988; Westbrook, Greeley, Nabke, & Swinbourne, 1991). Furthermore, it has been demonstrated that the activation of the serotonin autoreceptors by buspirone, a 5-H[T.sub.1A] agonist, results in preventing learned helplessness (Drugan, Crawley, Paul, & Skolnick, 1987) and can effectively relieve fear (Blanchard, D.C., Rodgers, Hendrie, & Hori, 1988; Costall, Kelly, Naylor, & Onaivi, 1988). Finally, it has been reported that the activation of 5-H[T.sub.1A] autoreceptors can be as effective in reducing fear as are benzodiazepines (Fernandez-Guasti & Hong, 1989; Treit & Fundytus, 1988).
Based on the previously mentioned psychopharmacological findings and our published research, which showed that alpha-colony odors can result in a suppression of shock-prod burying and an increase in freezing for defeated intruders, Greg Hotsenpiller and I were interested in determining if these fear-mediated changes might be prevented by injecting rats with either diazepam or buspirone prior to their being defeated by a dominant conspecific (Hotsenpiller & Williams, 1996). Following habituation sessions in the previously described shock-prod chambers, 8 rats were randomly assigned to one of six groups of a 2 x 3 factorial design: saline administered prior to a nondefeat session (SAL/ND), diazepam (2.5 mg/kg) administered prior to a nondefeat session (DZP/ND), buspirone (5 mg/kg) administered prior to a nondefeat session (BUS/ND), saline administered prior to a defeat session (SAL/D), diazepam administered prior to a defeat session (DZP/D), and buspirone administered prior to a defeat session (BUS/D). The injections were given 30 min before the defeat and nondefeat sessions to ensure that the drugs were absorbed. The subjects experiencing defeat received a 25-min session in a colony cage containing 2 male residents, with a female removed. Previous resident-intruder tests were conducted to establish the fact that 1 of the resident males in each colony was very aggressive. The precise procedure used during a defeat session was similar to that used in our previous research (e.g., Williams & Scott, 1989; Williams, Worland et al., 1990). The subjects in the groups not exposed to defeat were placed in a tub with the odors of aggressive male rats, but without the colony rats being present.
On the day after the defeat or nondefeat sessions, all of the subjects were placed in the shock-prod chambers with the respective bedding odors of their prior experience. Again, the specific details of the shock-prod test session and the definitions of the measures of prod-burying and freezing behaviors were the same as those used in our previously described research (e.g., Williams & Scott, 1989; Williams, Worland et al., 1990). The mean durations of prod burying for each of the groups, which were all tested with alpha-colony odors being present, are depicted in Figure 8. The results of an analysis of variance and planned-comparison tests indicated that exposure to a session of conspecific defeat produced a lack of responding in terms of prod burying when alpha-colony odors were present during testing, thus confirming the previous results of Williams and Scott (1989). Furthermore the defeated groups that received diazepam or buspirone (i.e., Groups DZP/D and BUS/D) buried significantly longer than the saline control group, and these two experimental groups did not differ in terms of their burying from the three nondefeated groups. Therefore, the administration of diazepam or buspirone was sufficient to prevent the lack of prod burying that was observed as a conditioned reaction to the alpha-colony odors for the defeated groups that had been injected with saline. Hence the contingency between a state of fear at the time of defeat and the odors of the alpha colony was never established in the subjects that were given anxiolytics just before the defeat session. The mean heights of the bedding piles made by these six groups of subjects were also examined, and the pattern of group differences was found to be exactly the same as that for duration of prod-burying data, shown in Figure 8. Finally, both of these measures of prod burying were found to be highly correlated within each of the groups.
The mean durations of defensive freezing, which typically was observed immediately following the prod shock, appear in Figure 9. The results of the statistical analyses indicated that there was significantly more freezing by the resident-intruder defeated group that was given a saline injection (i.e., Group SAL/D) than by all of the other groups. These findings are also consistent with the interpretation that the conditioning of fear to the odors of the colonies during the defeat sessions can be effectively blocked by the administration of either diazepam or buspirone. Finally, it is highly unlikely that these results can be attributed to the disrupting side effects of these drugs because such disruptions would have caused a decrease, rather than an increase, in prod-burying behavior.
The simultaneous display of fear and analgesia suggests that both of these reactions can be conditioned to the same stimuli during exposure to a stressor. The concurrent conditioning of analgesia and fear was seen in our previous research involving defeat sessions and formalin testing for analgesia. In these studies, it was consistently found that a single session of defeat resulted in the intruder rat showing less licking of its formalin-injected paw and greater levels of freezing when testing was done in the presence of conditioned alpha-colony odors, as opposed to their absence (Williams, Just, & Worland, 1994; Williams, Worland et al., 1990).
Based on these findings and the results of our previously described experiment on the effects of anxiolytics during defeat on subsequent shock-prod burying, Greg Hotsenpiller and I were also interested in determining if benzodiazepine and a 5-H[T.sub.1A] autoreceptor agonist could block the conditioning of analgesia that occurs when an intruder is defeated by an aggressive conspecific (Hotsenpiller & Williams, 1996). In order to examine this hypothesis, we basically replicated our initial experiment by using six groups of rats in the same type of factorial design, with separate groups of subjects receiving a saline injection, a 2.5-mg/kg injection of diazepam, or a 5-mg/kg injection of buspirone, 30 min prior to either a defeat or a nondefeat session. Twenty-four hours later, rather than being tested with the shock-prod paradigm, each subject had its right rear paw injected with a dilute solution of formalin, was then given a priming session for 1 min with an aggressive colony, and was finally observed for a 16-min test session in a tub cage with the bedding from alpha-colony residents on the floor. During the formalin tests, the number of time-sampling instances of paw-licking and freezing responses were recorded (see Williams, Worland et al., 1990, for a complete description of the rationale and specific details concerning this testing procedure).
Figure 10 displays the means for paw licking for the six groups during the formalin tests. These data and the statistical analyses indicated that the control group of rats given a saline injection prior to a defeat session (i.e., Group SAL/D) showed a low level of paw licking, which is consistent with the conditioned analgesia reaction first demonstrated by Williams, Worland et al. (1990). More relevant to the hypothesis of this experiment, there were no significant differences among the remaining groups. Therefore, diazepam and buspirone were very effective in blocking the conditioning of analgesia to the odor cues during the defeat sessions. These findings are not only consistent with the results of our previously described experiment using the shock-prod paradigm, but the pattern of results of freezing observed for the six groups in this study was also virtually identical to that found for the shock-prod experiment.
In conclusion, the findings of this research clearly demonstrate that both GABA and serotonin systems are involved in the conditioning of contextual odors at the time an animal is defeated by a conspecific. The ability of both diazepam and buspirone to alter these systems in such a way as to block fear conditioning is clearly consistent with the fact that these compounds have been shown to reduce fear reactions to aversive laboratory stimuli, such as electric shock. However, it is important to emphasize that similar mechanisms and drugs influence subjects when tested with a more natural type of stressor. Finally, this research is unique because it is the first to demonstrate that the use of the 5-H[T.sub.1A] autoreceptor agonist, buspirone, is capable of preventing fear and analgesia conditioning to contextual stimuli.
Effects of Repeated Sessions of Being Defeated by a Dominant Alpha Conspecific on Opioid Analgesia and Freezing
In contrast to the contextual specificity seen in terms of analgesia and defensive freezing to the odors of alpha-colony residents that we have observed following a single session of defeat (Hotsenpiller & Williams, 1996; Williams & Scott, 1989; Williams, Worland et al., 1990), it has been reported that repeated trials of uncontrollable, aversive stimuli (e.g., shock) produce alterations in behavior, physiology, and pain sensitivity when conditioned fear stimuli were not believed to be present during testing (e.g., Maier, 1986, 1989). In fact, researchers doing learned helplessness studies of this type often emphasize that transituational nature of behavioral and biological disruptions following exposure to inescapable stressors.
Prolonged exposure to conspecific attack and defeat has been reported to produce a profound and enduring analgesic reaction in male intruder mice when they are tested for tail-flick latencies to radiant heat (Miczek, Thompson, & Shuster, 1982, 1986; Rodgers & Randall, 1985). This reaction has also been shown to be mediated by an opioid system, because it can be completely blocked by opiate antagonists (Miczek et al., 1982, 1986; Rodgers & Hendrie, 1982; Rodgers & Randall, 1985), and shows cross-tolerance to and from morphine (Miczek et al., 1982; Rodgers & Randall, 1985). Using rats as subjects, Williams and Lierle (1988) reported that exposure to 25 consecutive sessions of being defeated as a resident-intruder, in contrast to nondefeated subjects, resulted in more defensive behavior, less open-field activity, and poorer escape learning from shock in a two-way shuttlebox. These investigators also reported that chronic defeat experience in rats did not result in analgesia when subjects were tested by examining the latency of flicking their tails to a radiant-heat source.
Based on the fact that the formalin test of analgesia was found to be an excellent assay for studying analgesia in rats to both conditioned stimuli (Williams, Worland et al., 1990) and unconditioned stimuli (Lester & Fanselow, 1985; Williams, 1991), an experiment was done in my laboratory by Marc Just, Paul Worland, and myself to examine if multiple sessions of being attacked and defeated by an alpha resident might result in analgesia in the presence and in the absence of alpha-colony odors using the formalin test procedure (Williams et al., 1994). The basic design and procedures used in this experiment were basically the same as those employed by Williams, Worland et al. (1990), except that the subjects were given repeated resident intruder sessions, as opposed to one session of intruder defeat. Specifically, two repeatedly defeated (RD) groups, of 10 male albino rats each, were given two resident-intruder sessions of 15 min each, 4 hr apart on 2 consecutive days. The alpha residents were previously judged to be aggressive because 1 of the males in each colony had attacked and bitten an intruder at least three times during sparring contests that were conducted a week prior to this experiment. To control for handling and being in a novel environment, the rats in two nondefeated (ND) control groups, of 10 subjects each, were placed in two empty cages with clean bedding for the same period of time, 4 hr apart on 2 consecutive days. As mentioned before, our previous research indicated that there was no difference between the behaviors of control subjects, during later testing, which had been exposed to clean bedding versus the bedding of a pair of nonaggressive male conspecific residents. One day following the last defeat or nondefeat session, all subjects were primed by being placed in the tub cage of an alpha colony for a 1-min period, without being attacked, and then were formalin tested in a chamber that contained the odors of bedding from an alpha colony (AO) or fresh bedding (NAO). Thus, this study involved a 2 x 2 factorial design with four independent groups: repeated defeat and later formalin testing with alpha-colony odors (RD/AO), repeated defeat and testing with no alpha-colony odors (RD/NAO), no defeat and testing with alpha-colony odors (ND/AO), and no defeat and testing with no alpha-colony odors (ND/NAO).
The mean number of observed time-sampling periods in which licking of the formalin-injected paw was recorded for each of the four groups is seen in Figure 11. Repeated sessions of defeat produced a striking suppression in paw licking (i.e., analgesia), regardless of whether the subjects were tested with the alpha-colony odors being present or not. The finding that alpha-colony odors are capable of eliciting a conditioned analgesia reaction following exposure to attack and defeat by a resident conspecific replicates the previous research (e.g., Williams, Worland et al., 1990). However, the fact that repeated defeat also produces analgesia when intruders were tested in the absence of alpha-colony odors indicates that this reaction is not specific to the contextual cues associated with the stressor (i.e., the alpha-colony odors). This type of transituational analgesic reaction, following repeated defeat, appears to be similar to the stress-induced analgesia that has been reported in "learned helplessness" studies in which rats were exposed to a series of inescapable shocks (Maier, 1989).
The mean number of freezing occurrences during the formalin tests, for each of the four groups, is shown in Figure 12. The results of the statistical analyses indicated that Group RD/AO showed significantly more freezing than any of the other three groups. Thus, freezing was observed primarily for intruders that had been repeatedly defeated and later tested with the alpha-colony bedding on the floor of the test chamber. This finding is consistent with the results of studies in which subjects were given a single session of defeat and tested with alpha-colony odors during formalin tests (e.g., Williams, Worland et al., 1990) or shock-prod tests (e.g., Williams & Scott, 1989). This result also suggests that freezing, as an overt mode of defensive behavior, is controlled much more precisely by putative conditioned alpha-colony odors than is defeat-induced analgesia. In addition, it is important to emphasize that the subjects in Group RD/NAO, which showed a sensitized analgesic reaction when tested in the absence of alpha-colony odors, displayed very little freezing. Like the previously described findings (Williams, Worland et al., 1990), this result implies that freezing can occur independent of stress-induced suppressions in paw licking during formalin tests of analgesia. This disassociation between freezing and analgesia also rules out the possibility that the stress-induced suppressions in paw licking were simply a function of response competition, but rather that such suppressions were clearly indicative of reduced pain sensitivity or reactivity.
Because repeated exposure to shock (e.g., Maier, 1989) and defeat (e.g., Miczek et al., 1982, 1986) have been found to produce an analgesic reaction that can be completely reversed by the administration of an opiate antagonist, another experiment by Marc Just, Paul Worland, and myself was done to determine if an opioid system might be involved in mediating the previously described analgesia for defeated rats during subsequent formalin tests when alpha-colony odors were either present or absent (Williams et al., 1994). This experiment involved the same conspecific-defeat and formalin-test procedures as those described previously. A control group of 10 male rats was not given defeat experience and later received a saline injection before colony priming and formalin testing with no alpha-colony odors present in the bedding. In addition, four experimental groups, of 10 rats each, were given four defeat sessions and later given a 7-mg/kg injection of naltrexone or saline prior to priming and testing with the alpha-colony odors either present or absent.
Figure 13 presents the mean number of recorded occurrences of paw licking during the formalin tests for all of the five groups of subjects. Both groups of subjects that were exposed to repeated defeat and were given an injection of saline prior to formalin testing, with either the alpha-colony odors being present or absent, showed significantly lower levels of paw licking than the nondefeated control group of rats that was tested without the colony odors. In contrast, both of the repeatedly defeated groups that were injected with naltrexone, the opiate antagonist, showed levels of paw licking that were comparable to the control group. These naltrexone-produced reversals suggest that the conditioned analgesia to the alpha-colony odors, by Group RD/N/AO, and the sensitized analgesia seen during tests without these odors, by Group RD/N/NAO), were both mediated by opioid processes. Finally, when the freezing data from these same subjects were analyzed, it was found that freezing only occurred for the subjects that experienced the combination of repeated defeat by a conspecific and were later tested in the presence of alpha-colony odors. Neither conspecific defeat nor testing with alpha-colony odors, per se, was found to produce freezing. Whereas injections of naltrexone, prior to testing, reversed the suppressed levels of paw licking (i.e., analgesia) for the repeatedly defeated subjects, it did not result in an increase in freezing behavior. This finding provides further support for the fact that paw-lick analgesia can be manipulated independent of defensive freezing and that freezing appears to be under more stimulus control of contextual odor stimuli than does opioid-mediated analgesia. Finally, the results of a third experiment done by the same investigators (Williams et al., 1994) indicated that an injection of dexamethasone, given before the repeated defeat sessions, only prevented the occurrence of analgesia when intruders were tested without alpha-colony odors being present. This implies that this analgesic reaction relied on a hormonal system, as opposed to a neural mechanism (for more details, see Williams et al., 1994). In summary, the findings of this research on multiple sessions of conspecific defeat parallel many "learned helplessness" studies in terms of showing the transituational effects of sensitized analgesia, and yet they also demonstrate the stimulus specificity effects of odor cues in terms of freezing behavior.
Effects of Repeated Sessions of Being Defeated by a Dominant Alpha Conspecific on Naloxone-Precipitated Morphine Withdrawal Reactions
Repeated exposure to an uncontrollable stressor has been shown to augment a variety of reactions to the administration of morphine and other opiates (Belenky & Holaday, 1981; Grau, Hyson, Maier, Madden, & Barchas, 1981). For example, Williams, Drugan, and Maier (1984) reported that giving rats two daily sessions of inescapable shocks, in contrast to rats that were given escapable shocks or were restrained without shock, showed an enhanced series of correlated withdrawal behaviors (i.e., mouthing, teeth chattering, and head/body shakes) 1 day later when injected with a small dose of morphine that was followed by a naloxone-challenge injection. These investigators also found that the effect of inescapable shock on later naloxone-precipitated withdrawal to morphine was completely blocked when subjects were administered an opiate antagonist before each shock session. These results clearly implied that exposure to inescapable shock resulted in the activation of opioid processes or systems that were capable of potentiating later morphine withdrawal.
More recently, Marc Just, Charles Farmer, and I reported experiments that used withdrawal and opioid-blocking procedures similar to those of Williams et al. (1984) to determine if the number of intruder-defeat sessions would increase the levels of precipitated-morphine withdrawal, and if such reactions could be prevented by administering an opiate antagonist prior to these sessions (Williams, Farmer, & Just, 1992). Specifically, 60 male albino rats that were used as subjects in this experiment were randomly assigned to six groups of 10 subjects each. Pairs of groups were then exposed to one of the following conditions: two sessions of no defeat by being placed in a nonaggressive colony as an intruder for two 15-min resident-intruder sessions on 2 consecutive days (NONE), one session of being defeated as a resident intruder in an aggressive colony (ONE), or two sessions of being defeated as a resident intruder in different aggressive colonies for two consecutive days (FOUR). All aspects of the procedures used during the resident-intruder sessions were previously described in this paper. One day after their last resident-intruder session, all of the rats were given a 5-mg/kg (SC) injection of morphine sulfate. Thirty minutes later, they were given an injection (IP) of 5 mg/kg of naloxone, an opiate antagonist that was used as a morphine challenge to precipitate a morphine withdrawal reaction. These dosages of morphine and naloxone were previously found not to produce any withdrawal effects when given independently, but did result in moderate levels of withdrawal when administered successively to nonstressed rats (see Williams et al., 1984, for more details). Then, all of the rats were primed by being placed in an aggressive colony for a 1-min period, during which they were not attacked. Two minutes later, they were placed inside a 1-gallon glass jar with a lid that consisted of a perforated-metal odor box containing either clean bedding material (NAO) or the soiled bedding of an alpha colony (AO). Experimentally blind observers recorded bouts of withdrawal behaviors that the rats made while in the jar for a 15-min observation-test session. The most frequent and reliable measures of precipitated-morphine withdrawal were the number of bouts of head and body shakes (i.e., "wet-dog shakes") and mouthing (i.e., vigorous licking of the air).
Figure 14 presents the mean number of shaking episodes of each of the six groups that had either no, one, or four defeat sessions as a resident intruder and were later tested with the alpha-colony odors being either present or absent. Shaking clearly increased as a function of the number of intruder-defeat sessions. In addition, the presence of the alpha-colony odors was found to be marginally significant (i.e., at the .05 level) for the group that had one defeat session, but there was no effect of odors for the other pairs of groups. The mouthing data that were observed for the same six groups also indicated a similar pattern of results with exaggerated levels of withdrawal occurring with greater defeat sessions and virtually no differences between the two odor conditions.
These findings are consistent with the results of the previously described study by Williams et al. (1994) which indicated that when rats are exposed to repeated sessions of defeat by a dominant conspecific they showed paw-lick analgesia during formalin tests 24 hr later with the odors of the alpha colony being present or absent. Because this analgesia reaction was found to be reversed by the administration of an opioid antagonist, this approach was again used in a follow-up study by Williams et al. (1992) to determine if augmentations in precipitated morphine reactions as a result of repeated defeat could also be prevented by an injection of an opiate antagonist prior to sessions of intruder defeat. Because naltrexone is known to be a long-term opiate antagonist, injections of 7 mg/kg were assumed to effectively block the activation of endogenous opioids if they were given 30 min before the first of two daily resident-intruder sessions, which were given on 2 consecutive days. In addition, two control groups were given saline injections prior to the same number (i.e., four) of defeat sessions. Then, all of the subjects in this experiment were tested for morphine sensitivity by means of the previously mentioned naloxone-challenge procedure for eliciting morphine withdrawal.
Figure 15 shows the mean number of shaking bouts for the four groups of rats that were injected with either saline or naltrexone prior to each pair of defeat sessions that they received on 2 separate days. As can be seen, the two groups of rats that were injected with naltrexone showed only moderate levels, as opposed to exaggerated levels, of withdrawal. These findings again make the point that repeated exposure to defeat as a colony intruder, as a natural conspecific stressor, increases the activation of endogenous opiates that, in turn influences reactions that such subjects show to exogenous morphine. At this point, it is not known whether the effects of repeated defeat are the result of an increase in the release of an endogenous opiate, a sensitization of opioid receptors, a proliferation of opioid receptors, or some combination of these and/or other neuronal changes.
Behavioral and Physiological Reactions to Cat Exposure and Cat Odors as Predatory Stressors
Our research previously presented in this paper has documented that exposing an animal to the social stressor of being attacked and defeated by an aggressive conspecific produces defensive behaviors and alterations in physiological responses, particularly when the subject is tested where the odors of a dominant conspecific are present. It has also been known for some time that animals show unconditioned fear reactions when exposed to natural predators or the odors of a predator. For example, rats display pronounced behavioral and physiological fear reactions when exposed to a cat. Such exposures to an actual cat have been found to produce freezing (Blanchard, R. J., Fukunaga, & Blanchard, 1976, Williams & Barber, 1990; Williams & Scott, 1989) and induce analgesia through an endogenous opioid mechanism (Lester & Fanselow, 1985). Other researchers have reported that the odors of cats are capable of eliciting a number of unconditioned fear reactions, such as place avoidance in a plus maze (Williams & Groux, 1993), risk assessment of the source of cat odors (Blanchard, D.C., Blanchard, & Rodgers, 1991; Blanchard, R. J., Blanchard, Weiss, & Mayer, 1990), or opioid-mediated analgesia during formalin tests (Lester & Fanselow, 1985).
Based on my past research using the shock-prod paradigm, David Scott and I hypothesized that the odors of cats might induce a moderate level of fear that would decrease prod burying and increase freezing to some degree. More substantial aberrations in behavior were expected to occur for rats that had been previously exposed to a cat and were later tested with cat odors. Therefore, we did a factorial-design experiment in which separate groups, of 8 rats each, were either exposed or not exposed to a caged male cat for 45 min and tested 24 hr later for defensive prod burying and freezing in the presence, or absence, of a 1-liter sample of cat bedding spread evenly on the floor of the chamber (Williams & Scott, 1989). The results of this experiment indicated that prior exposure to a cat neither suppressed prod burying nor increased freezing during later testing. In contrast, the presence of cat odors during the test session disrupted burying, produced only a moderate amount of freezing, and elicited a high level of risk-assessment behavior with the rats slowly exploring the cat-odor bedding with an outstretched posture. This type of defensive information-seeking behavior has been extensively studied by the Blanchards and their colleagues (e.g., Blanchard, D.C., et al., 1991; Blanchard, R. J., et al., 1990), who have shown that risk assessment can be effectively reduced by the administration of various anxiolytics, such as diazepam and buspirone. The pattern of results that were found in our study also suggests that changes in prod burying can be disassociated from freezing, and thus the observed suppressions in burying were not necessarily the result of freezing as a competing response. One reason the subjects in this study may not have shown much freezing to cat odors was because they were protected from being attacked by the cat during the pretest exposures, thus there was no opportunity to associate pain with the cat odors. This is in contrast to the aggression research, reported earlier, in which the resident intruder was attacked, defeated, and bitten by a dominant conspecific.
In an attempt to differentiate the effects of fear versus pain, David Scott and I did a follow-up experiment involving the use of cat odors versus the odor of citronella, which has been shown to be an hedonically neutral odor for the rat (Lester & Fanselow, 1985), as a potential conditioned stimulus. Specifically, rats were given 1, 5, or 30 inescapable 1-s presentations of footshock in the presence of either cat odors or the odor of citronella, and they were tested 24 hr later for prod burying and freezing with or without these odors. Both the cat and the citronella odors resulted in a significant reduction in burying and increase in freezing for rats that received 5 and 30 conditioning trials, when they were tested later with these respective odors. For the groups that were given five trials, conditioning and testing in the presence of the cat odors resulted in significantly less prod burying and more freezing than for the subjects that were conditioned and tested in the presence of the odor of citronella. These findings support the concept of "biological preparedness" concerning the ease with which ethologically related stimuli can be associated (i.e., the odors of a predator and pain).
In light of the fear-inducing effects that exposure to social stressors have on later behaviors during testing with the odors of a dominant conspecific, a study was conducted by Amy Rogers, Alison Adler, and myself to see if a more intense predator exposure experience would result in a potentiation of fear-mediated freezing during later shock-prod testing. We were also interested in determining whether this augmented freezing reaction could be attenuated by giving a long period of exposure to cat odors, as a desensitization procedure, prior to testing (Williams, Rogers et al., 1990). Preliminary studies were done to establish that an increase in freezing reliably occurred for rats following protected exposure to a pair of cats along with the odors from their litter boxes. Then, separate groups of 8 cat-exposed rats were given a 12-hr exposure session with cat odors, a 12-hr exposure-control session with no cat odors, or a no-exposure treatment. Following these treatments, all three groups were tested for burying and freezing after prod shock, with a 1-liter sample of the soiled cat bedding spread on the floor of the chamber. Compared with the two control groups, the subjects that were given exposure to cat odors showed significantly less freezing during the shock-prod tests, but they did not show prod burying. Therefore, as we saw earlier with alpha-colony odor exposure, a prolonged period of exposure to the odors of a predator resulted in the reduction of fear, probably caused by a desensitization process rather than extinction because cat odors are innately feared. The fact that freezing was reduced, without an increase in prod burying, represents another example of the possible independence of these response mechanisms. Finally, the fact that prod burying appeared to be more resistant to desensitization also suggests that it may be a more sensitive measure of the reduced fear that was still elicited by predator odors after the desensitization session.
Repeated exposure to many types of aversive laboratory stressors has been shown to sensitize or potentiate changes in behaviors that reflect alterations of various neurotransmitters. We have already shown that repeated presentation of electric shock or sessions of conspecific defeat produce analgesic reactions that are mediated by endogenous opioids (Williams et al., 1984; Williams et al., 1994). Repeated sessions of defeat were also found to result in an enhancement in naloxone-precipitated withdrawal to a low dose of morphine (Williams et al., 1992). In addition, Williams and Foster (1999) demonstrated that exposure to repeated sessions of defeat produced an increase in response stereotypy when these intruders were later administered a 1-mg/kg dose of d-amphetamine prior to observational testing in a chamber with the odors of alpha-colony rats present. In light of these findings and the work that we had done on predatory stressors, Gary Barber and I were interested in examining the effects of cat exposure and cat odors on subsequent amphetamine-induced stereotypy (Williams & Barber, 1990). This study involved separate groups, of 8 male rats each, in a factorial design in which rats were either exposed to a cat and soiled cat litter (C) for a 15-min session while protected in a wire cage or given a no-catexposure control (NC) treatment. Twenty-four hours later, all of the subjects were given an injection of 1 mg/kg of d-amphetamine and tested 30 min later for stereotypy (i.e., typically, rapid repetitions of head/body movements) in the presence of cat odors from soiled cat litter (O) or with fresh litter that did not contain cat odors (NO).
The test results, over six postinjection intervals of 15 min after the 30-min absorption period, are shown in Figure 16. These findings and the statistical tests indicated that the group of rats that had the combination of being exposed to a cat, and later testing with cat odors present (i.e., Group C/O), displayed significantly higher levels of amphetamine-induced stereotypy than the subjects in the other three conditions. These results clearly demonstrate that amphetamine reactivity is influenced by prior exposure to a predator and the presence of predatory odors during later testing. Specifically, it appears that this type of predatory experience can potentiate reactions which have been shown to be mediated by the activation of mesocortical dopamine neurons that have been postulated to play an important role in amphetamine psychosis (Kokkinidis & Anisman, 1981).
Effects of Exposure to a Cat, as a Predatory Stressor, and Age on Spatial Working Memory
Chronic exposure to a stressor has been shown to result in a significant long-lasting impairment in spatial working memory, which is reflected in a loss of memory for places or response-generated cues that an organism has recently experienced. For example, it has been reported that, after receiving many sessions of physical restraint as a stressor, rats display an impairment in spatial memory when tested in a radial-arm maze (Luine, Villegas, Martinez, & McEwen, 1994). In addition, the chronic administration of corticosterone in rats has been shown to produce deficits in spatial memory in a radial maze (Luine, Spencer, & McEwen, 1993). Such memory impairments have been attributed to excessive levels of glucocorticoids, released by the hypothalamic-pituitary-adrenal axis, that have been found to produce atrophy of neurons in the hippocampus, a structure that is integral to the establishment of spatial working memory (McEwen & Sapolsky, 1995). Furthermore, the effect of increasing age, with associated stress, has also been reported to result in neuronal degeneration in the hippocampus (Coleman & Flood, 1987) and corresponding deficits in spatial memory (e.g., Barnes, 1979; Gallagher & Pellymounter, 1988). Several researchers have attempted to explain these age-related memory losses in terms of what has been referred to as the "glucocorticoid cascade hypothesis," which refers to the inability of the hippocampus to attenuate the release of corticosterone in old rats, particularly following exposure to a chronic stressor (e.g., McEwen, 1992; Sapolsky, 1986).
Acute exposure, as opposed to chronic exposure, to an uncontrollable stressor for repeated trials has been shown to result in "learned helplessness effects." As was noted earlier in this paper, exposure to a series of inescapable shocks has been found to produce deficits in a variety of learning tasks, alterations in the levels of numerous neurotransmitters, and an activation of endogenous opiates and immunosuppression (see Peterson, Maier, & Seligman, 1993, for a review of this literature). Finally, exposure to inescapable shock has been reported to disrupt mechanisms that are central to working memory, such as hippocampal plasticity and hippocampal long-term potentiation (Foy, Stanton, Levine, & Thompson, 1987; Shors, Levine, & Thompson, 1990).
In light of the effects of stress on memory and my previous research with ethological stressors, Shelley Baker, Jennifer Gress, Ben Givens, and I were interested in determining if using a cat as an acute stressor, in contrast to painful electric shock, may disrupt the cognitive processes involved in the rat remembering response-dependent events (Williams, Baker, Gress, & Givens, 1998). The basic design of this study involved giving young adult and old rats initial training-to-criterion on a delayed-alternation task (i.e., a task that is often used to assess spatial working memory), then exposing them intermittently to a caged cat or a neutral environment, and finally testing them on the original delay-alternation task to determine if a decrement in performance occurred for the catexposed subjects. Two age groups were used to examine if this type of predator stressor might produce different degrees of disruption in spatial working memory for young versus old rats.
Eighteen 3-month-old (i.e., young adult) and eighteen 20-month-old (i.e., old) male rats were used as subjects in this experiment. All of the rats were maintained, in individual cages, on a regular light-dark cycle, and were run as subjects in the light phase. They received food on an ad libitum basis, but were water restricted to 85% of the pretraining weight so that water could be used as a reinforcer during maze-training sessions. Elevated T mazes, with two arms of 60 x 10 cm at the end of a 60- x 10-cm runway, were used to train and assess the performance of the rats on a 20-s delayed-alternation task. The start position of the T maze was indicated by a strip of tape located 15 cm from the end of the runway or stem of the maze. Recessed water cups were located at the end of each choice arm, and the water reinforcer was not visible by the rat from the choice-point location. The mazes were located in separate cubicles that were illuminated by a 60-W bulb, placed behind the right arm of the T maze on top of a speaker from which white noise was emitted. Because the walls of the maze were low (i.e., 3 cm), the rats could easily use room cues and the white noise to determine their location in the maze. During shaping sessions, two drops of water were put in the water cup at the end of either the right or left arm of the maze, which was determined randomly for each trial. The subjects were first placed directly in front of the water cup, and then they were moved farther away on subsequent trials. Eventually, all the rats learned to run down the runway of the maze and were forced to go randomly to either the right or left arm to receive water as a reinforcer.
Following shaping sessions, all of the rats were given delayed-alternation training sessions that began with one forced-choice trial, followed by 20 choice trials during which the rat was free to choose to go down either the right or left arm. On the initial forced-choice trial, water was randomly placed in the cup of either the right or left arm, and a barrier forced the rat to go to the rewarded arm. On the subsequent 20 choice trials, water was placed in the cup of the arm that was opposite to the one that the rat entered on the previous trial. After each trial, the subject was placed in its home cage and could not see the maze during a delay interval of 20 s between trials. A correct response was recorded when the rat went to the arm opposite to its choice on the previous trial, regardless of whether it was rewarded or not on that trial. The raw data were converted to "percent choice accuracy" scores by dividing the number of correct responses that each rat made by 20 and multiplying that value by 100. All of the subjects were given daily sessions until they met the following learning criterion: either 85% choice accuracy for 2 consecutive days or an average of 80% over 5 consecutive days. The results of the analysis of this training phase of the experiment indicated that there was a significant increase in choice accuracy for both the young and the old groups of rats. A two-sample t test indicated that the old rats required significantly more trials to reach the previously mentioned training criterion than the younger rats. However, after 10 daily training sessions, all of the old and young rats had reached the same training criterion for the delayed-alternation task.
Twenty-four hours after their last training session, separate groups of 9 young and 9 old rats were matched on the basis of their training scores and then were randomly assigned to either a no-stress condition or a stress condition. Thus, this experiment involved a 2 x 2 factorial design, comprised of the following four independent groups: Young/No Stress (Y/NS), Young/Stress (Y/S), Old/No Stress (O/NS), and Old/Stress (O/S). The rats in the stressed groups were individually placed in a small wire cage that was set in a room with two 5-yr-old male neutered cats that had resided there since they were kittens. The cats were free to roam around this room, which also contained food and water bowls and two cat-litter boxes. The cats were deprived of food for a 4-hr period prior to being presented with a rat. A cat-exposure session was given for 30 min on each of 3 consecutive days. The rats in the no-stress groups were given the same duration and number of sessions, except they were individually placed in a small wire cage that was set in a neutral room to control for moving of the subjects and exposing them to a novel environment.
Twenty-four hours after the last stress or no-stress exposure session, all of the rats were given a 5-min period of stressor priming by being exposed to the cats as previously described. This type of priming procedure has typically been employed in studies where subjects are tested a day after exposure to a stressor. Then, subjects were given further training/testing on the 20-s delayed-alternation task for 4 daily sessions, using the identical procedure as was employed during the previous training sessions. The results during the 4 days of testing indicated that there were significant differences between groups, in terms of choice accuracy, on the 1st day of testing but not on the remaining three sessions.
A more powerful analysis for examining these group differences on the first test session involved the use of a mixed-analysis of variance and post hoc tests on the changes in choice accuracy, for each subject, from the last session of the initial delayed-alternation training to the first posttreatment testing session. Figure 17 presents these data for the old and the young rats that were in the no-stress and stress conditions. Somewhat unexpectedly, it can be seen that the stress resulting from exposure to a cat produced greater reductions in choice accuracy for the young rats. However, it should be noted that there was a significant decrease in choice accuracy for both the young and the old groups of stressed rats (i.e., Groups Y/S and O/S) from the last pretreatment to the first posttreatment session. Further data analyses indicated that the changes in choice accuracy, seen in Figure 17, were not the result of differences in the choice latencies for different age groups. Although the older rats showed longer choice latencies than young rats, exposure to the cats resulted in a comparable increase in choice latencies for both age groups. In addition, the within-group correlations between these two measures were not found to be significant, further supporting the argument that the differences in choice accuracy were not related to choice latency.
Although the use of a cat as a stressor was found to interfere with working memory in both the young and the old rats, it is necessary to comment briefly on why the young rats seemed to be more responsive to this type of stressor than the old rats. McEwen and Sapolsky (1995) postulated that transitory disruptions of long-term potentiation may involve endogenous opioids. In light of this, it is particularly interesting to note that aged rats have been found to show reduced levels of opioid-mediated analgesia (Crisp et al., 1994; Hoskins, Burton, & Ho, 1986; Saksida, Galea, & Kavaliers, 1993). One reason for this reduced level of analgesia is because aged rats have been shown to have fewer opioid receptors (Frolkis, 1993; Piva, Maggi, Limonta, Dondi, & Martini, 1986). Assuming that activation of opioid systems is critical in producing transitory disruptions in memory processes, it may well be that the cat stressor for the old rats in this study did not result in a sufficient level of opioid involvement to produce as great a temporary deficit in working memory as was seen for the young rats.
The results of this experiment provide a novel and unique demonstration of the fact that exposure to a predator, without any experience of pain, can result in a significant short-term interference in spatial working memory. Perhaps, more intense types of predator exposure to other natural predators for the rat (e.g., a fox) may have greater and longer lasting detrimental effects on working memory, particularly if the behavioral assessment involves a more difficult test than a delayed-alternation task. Finally, future studies clearly need to establish the specific physiological mechanisms (e.g., the activation of opioid processes) that are involved in mediating the influence that predatory stressors have on performance of animals on various memory and cognitive tasks.
Effects of a Synthetic Predator Odor on Defensive Behavior, Analgesia, Amphetamine-Induced Stereotypy, and Spatial Working Memory
Much of the previous research done in my laboratory has demonstrated that natural ethologically relevant stressors, like exposing a rat to a cat and/or cat odors, results in such fear-mediated reactions as opioid-mediated analgesia, freezing, and enhanced levels of stereotypy to a small dose of amphetamine. However, in contrast to electric shock, a major problem with using exposure to a cat and cat odors as a stressor is that such stimuli are difficult to quantify and thus can not be precisely controlled or held constant during an experiment. For example, a cat does not react consistently when it is exposed to a rat; rather the cat's behavior varies depending on the age, sex, and the responses emitted by the rat as well as on the number and duration of sessions the cat has previously observed rats. Furthermore, the cat odors used in experiments are typically obtained from soiled litter boxes containing cat urine, feces, and hair. This heterogeneous source of odors makes it virtually impossible to define and manipulate this type of stress odor very precisely. Because of these problems with using a cat and cat odors as a stressor, our more recent research has involved the use of 2,5,-dihydro-2,4,5-trimethylthiazoline (TMT), a chemical compound that has been isolated in the laboratory from the feces of the red fox (Vulpes, vulpes), which is known to be a natural predator for the rat (Vernet-Maury, 1980). This synthetic compound has been found to produce fear reactions equivalent to those elicited by whole fecal extracts of fox, in terms of suppressing open-field activity and increasing the corticosterone levels in rats (Vernet-Maury, Polak, & Demael, 1984). In addition, it has been speculated that these reactions are innate because rat pups were found to respond in a fear-like manner to the odor of TMT from birth to adulthood, without evidence of habituation of freezing during open-field tests (Vernet-Maury, Constant, & Chanel, 1992).
Catherine Baez and I were interested in finding out if the odor of TMT, like cat odors, might elicit opioid-mediated analgesia and freezing when present during paw-lick formalin tests (Williams & Baez, 1997). Specifically, this experiment used 40 male rats that were handled for 3 days and habituated for two 30-min daily sessions in previously described test chambers. Each test chamber had a 3-cm layer of corncob on the floor, and a Plexiglas top so that the rat's behavior could be recorded by a video camera. A white noise was presented in the experimental rooms containing the chamber, with a monitor and video recorder located in an adjacent room. On the day of testing, 10 rats were randomly assigned to one of four groups involving a 2 x 2 factorial design, with subjects receiving either an injection (IP) of saline or a 7-mg/kg injection of naltrexone (an opiate antagonist) and 15 min later given a paw injection of formalin 20 min prior to observational tests with the odor of TMT or citronella present in the test chamber. Thus, this study involved the following groups: injected with saline and tested with citronella (SAL/CIT), injected with saline and tested with TMT (SAL/TMT), injected with naltrexone and tested with citronella (NALTR/CIT) and injected with naltrexone and tested with TMT (NALTR/TMT). More specifically, the odor manipulations were done by placing a small sample of TMT (obtained from PheroTech Inc.) or citronella (citronellal, obtained from Sigma Chemical Co.) in the middle of a piece of fast absorbent filter paper that was suspended from the middle of the ceiling of the test chamber by a 4-cm metal hook. Previous research in our laboratory had established that the amounts of each odorant used in this experiment were equal in terms of the numbers of molecules absorbed by activated charcoal placed inside the test chamber (see Hotsenpiller & Williams, 1997, for more details about equating the amounts of test odors available to the rat while inside the test chamber). As noted before in this paper, the odor of citronella has been shown by researchers to be a salient, but hedonically neutral, odor for the rat (Lester & Fanselow, 1985; Williams & Scott, 1989). The bedding of the chamber was changed after each rat was tested, and the different odor conditions were used on separate days in different rooms. From videotapes of the 16-min test sessions, each subject's behavior was scored, every 8 s, for the occurrence of recuperative paw licking and freezing using the operational definitions of Williams, Worland et al. (1990).
Figure 18 presents the mean number of recorded occurrences during which the rats in each group were licking the paw that was injected with formalin prior to the observational test. As seen in this figure and confirmed by an analysis of variance along with post hoc tests, the odor of TMT, for the saline injected rats, resulted in a suppression in paw licking which is indicative of analgesia. The fact that the presence of TMT, during formalin testing, is capable of inducing pawlick analgesia was previously reported by Hotsenpiller and Williams (1997). Of particular interest is the finding that this analgesic reaction to TMT was found to be completely blocked for rats that were injected with the opiate antagonist, naltrexone, prior to being tested with the odor of TMT. That is, the amount of paw licking by the rats in Group NALTR/TMT was not significantly different from the levels of paw licking shown by the subjects in the two CIT groups. In addition, statistical tests of the freezing data from these four groups indicated that there was significantly greater freezing by the rats that were tested with TMT, as opposed to those tested with the control odor of citronella; and naltrexone was seen to have no effect on TMT-induced freezing. Therefore, the results of this study clearly imply that odor of the synthetic compound TMT, which has been isolated from fox, has similar behavioral effects as cat odors in terms of eliciting defensive freezing and an analgesia reaction that was found to be regulated by an opioid system.
Williams and Baez also conducted a separate experiment to determine if exposure to TMT would augment the stereotypic motor responses to amphetamine that rats have been previously found to show following exposure to cats and cat odors (Williams & Barber, 1990) and following conspecific defeat (Williams & Foster, 1999). Four groups of male rats were used as subjects in this 2 x 2 factorial-design experiment. Following two habituation sessions in an activity monitor, two groups of rats were given an injection of 1 mg/kg of d-amphetamine or saline, and later subgroups were tested in the activity monitor with the odors of TMT or citronella presented by means of a piece of filter paper suspended from the ceiling of the monitor. The specific details concerning the procedures for presenting these odors and testing for amphetamine-induced stereotypy were described in studies cited earlier in this paper. The statistical findings of this experiment revealed that neither amphetamine nor TMT, per se, had a significant effect on behavior. However, a significant interaction effect was found, with only the group that had been injected with a small dose of amphetamine and tested with TMT displaying a very high level of stereotypy throughout the 1-hr test session. In summary, the results of the two previously described experiments indicate that TMT seems to produce similar types of behavioral and neurophysiological reactions in rats as those found following exposure to cats and/or cat odors. However, unlike the use of cat odors, it should be noted that TMT is a synthetic compound that can be more easily controlled and manipulated in an experiment.
As described earlier, Williams, Baker et al. (1998) demonstrated that a rat's performance of a delayed-alternation task was temporarily disrupted after having been exposed to a cat for a 30-min session while the subject was in a protective cage. In an effort to more precisely examine the effects of exposure to a predator odor on spatial working memory, Katie Hladky and I conducted a recent study in which rats were trained, and later tested, on a delayed-alternation task that was interrupted by exposing the subjects to the odor of TMT or citronella (Williams & Hladky, 1998). In this experiment, the interval between trials was varied by having a 0-s delay (i.e., no explicit delay) or a 30-s delay. In addition, the amount of TMT that the stressed subjects were exposed to was systematically varied for three separate groups. Therefore, it was possible to examine the effects of exposing rats to a low or a high level of TMT versus a novel, but hedonically neutral, control odor (i.e., citronellal) on subsequent tests of performance (0-s delay trials) and memory (30-s delay trials), using a response-alternation task in a T maze.
Specifically, this experiment used 36 adult male rats as subjects that were water deprived and given shaping and training on a spatial-alternation task, using a T maze and procedure that were described previously in this paper (see Williams, Baker et al., 1998, for details). The subjects were trained on the spatial-alternation task for 13 consecutive daily sessions in a room that contained many extra-maze cues. During each session, the subjects completed 30 trials during which the subject was reinforced with water for choosing to run down the arm opposite the one it had chosen on the previous trial. Between each trial, the subject was returned to its home cage, which was positioned so that the rat could not view the maze. On 15 of the 30 trials, subjects were returned directly to the maze following the previous trial. The other 15 trials included a 30-s delay between trials. The 0- and 30-s delay trials were randomly given throughout a daily training session. Prior to each training session, the rats were placed individually in a metal box, referred to as an odor-exposure box, for a 30-min period. This box had fresh bedding on the floor and no experimental odors were presented until the last training/test session, at which time filter paper containing the test odors was suspended from the ceiling of the box. A training criterion of 80% correct choice accuracy on the 30-s delay trials was used to ensure that subjects had learned the task to an equivalent level before testing. After Day 13, 10 of the 12 subjects in each of the three groups of randomly assigned subjects had achieved this criterion.
The procedure for the test session was identical to that used during the previous training sessions except for the addition of the test odors to the filter paper that was hung from the top of the odor-exposure box. The control group had 132 [[micro]liter] of citronellal (citronella), reapplied after every 4 subjects. The TMT-low group was exposed to 132 [[micro]liter] of TMT, replenished every 4 subjects; whereas the TMT-high group was exposed to 264 [[micro]liter] of TMT replenished every 2 subjects. As with training, each subject's performance on each 0-s and 30-s delay trial was recorded as correct or incorrect, depending on whether the rat alternated or not, respectively, from the choice that it made on the previous trial.
The data used to assess an individual subject's change in accuracy because of the odor to which it was exposed and the delay interval were obtained by calculating the difference in performance, in terms of the percentage of correct alternation trials, between the test session (Day 14) and the final session of training (Day 13). Figure 19 presents the mean of the change, or "odor effect," scores for each of three odor-exposed groups on the 0-s and 30-s trials. Correlated within-subjects t tests indicated that the change in performance for the control group did not differ significantly on the 0-s and the 30-s delay trials. There was no significant change in the performance of the TMT-low group on the 0-s trials, but a significant change was seen for this group on the 30-s delay trials. The TMT-high group showed a significant disruption of choice performance on both the 0-s and 30-s delay trials. In addition, an analysis of variance, followed by post hoc tests, indicated that there were significant differences between the performance of the control group and the TMT-low group on the 30-s delay trials. Finally, the TMT-high group showed a significantly greater disruption in performance than observed for the other two groups, but performance on the 0-s and 30-s delay trials was not statistically different for this group.
The results of this experiment reveal that TMT disrupts spatial working memory, as seen by the decrease in accuracy on the 30-s delay trials for the rats that were exposed to a low level (or exposure dose) of TMT compared to those exposed to citronella as a novel, control odor. A low dose of TMT also seemed to disrupt only the memory of the rats on the 30-s trials, without significantly affecting their performance on the 0-s delay trials. Finally, a high dose seemed to induce a fear reaction which disrupted the general performance of the rats, and not just their memory, because it affected their scores on both the 0- and the 30-s delay trials. As noted earlier, Hotsenpiller and Williams (1997) and Williams and Baez (1997) reported that TMT exposure leads to a release of endogenous opiates as evidenced by naltrexone-reversible analgesia. Other studies also indicate that exposure to a natural predator (a cat) results in a short-term interference in spatial working memory (Williams, Baker et al., 1998) and that infusion of endogenous opiates into the medial area of the hippocampus changes theta activity and alters working memory as assessed by a delayed-alternation task (Wan, Givens, & Olton, 1995). These findings suggest that the activation of opioid processes may be a biological mechanism that is capable of disrupting spatial working memory during or immediately following exposure to a nonpainful stressor, such as the odor of TMT. Further studies, using various pharmacological agents that influence endogenous opiates, need to examine the effects of TMT on theta-wave activity, long-term potentiation, and spatial working memory.
Summary, Conclusions, and Implications
The overall objective of the research discussed in this paper was to demonstrate that unconditioned fear, conditioned fear, and fear sensitization are processes that are not unique to artificial-laboratory stimuli (e.g., tones) and stressors (e.g., electric shock), but they also occur when animals are subjects in experiments using natural, or ethologically relevant, stimuli and stressors. The present findings indicate that the unconditioned fear odors from shocked-donor conspecifics and predators, including certain synthetic compounds (i.e., TMT), result in freezing as the dominant mode of defensive behavior, analgesia due to the activation of endogenous opiates, and specific reactions to various drugs (e.g., morphine, amphetamine). These fear-mediated behaviors were shown to be sensitized, or potentiated, if subjects have been exposed to a repeated and/or a long-term stressor, such as sessions of defeat by a dominant conspecific. It was also reported that exposure to a predator (e.g., a cat) or the odors of a predator (e.g., cat odors, odor of TMT) produces a temporary disruption in cognitive processing, as assessed by the spatial working memory of rats performing a delayed-alternation task.
It appears that the odors of alpha-colony rats become effective conditioned fear stimuli after a single session during which a resident intruder was attacked and defeated by a dominant male conspecific. This type of conditioning results in defensive freezing and opioid-mediated analgesia which were shown to be dissociated when subjects were administered an opiate antagonist prior to testing in the presence of these odors. Evidence supporting the fact that alpha-colony odors are conditioned stimuli came from studies demonstrating that a prolonged-exposure session to these odors resulted in the extinction of freezing and analgesia during subsequent odor tests. This finding contrasts with the results of our experiments showing that a similar exposure duration to unconditioned predator odors is only moderately effective in reducing freezing (i.e., desensitization), without having any effect on analgesia. Furthermore, as was found by other researchers using shock, the associative processes involved in the conditioning of alpha-colony odors, during the time animals were attacked and defeated, were found to be prevented by the administration of anxiolytics that are known to independently affect GABA and serotonin mechanisms.
Reactions to a brief exposure to a stressor, or an associated odor, undoubtedly have important consequences in terms of the animal's survival in its natural environment. As has been noted by other investigators, analgesia is beneficial to an animal in terms of helping it to maintain defensive behaviors, such as freezing, when in the vicinity of a dominant conspecific or predator. However, the fact that conditioned fear reactions to the odors of a dominant alpha conspecific can readily be extinguished suggests that this source of social stress is somewhat malleable. This is probably advantageous to the animal because the dominance hierarchy of male rats in a colony is constantly changing as a function of age and the number of agonistic encounters. In contrast, exposure to predators and to odors associated with a predator were found to be much more resistant to change, thus increasing the animal's chances of survival. However, it should be noted that exposure to predator stressors were found to disrupt spatial working memory, which might result in interfering temporarily with the animal's ability to forage for food or water in its natural environment.
In conclusion, it is important to emphasize that repeated exposure to a stressor (e.g., shock, defeat, predator) has been shown in the present research to be particularly devastating for two reasons. The first reason is that such exposures elicit long-term stress reactions that are not restricted to the context in which the organism experienced the stressor. For example, animals that experienced repeated sessions of attack and defeat by a dominant conspecific showed subsequent analgesic reactions, regardless of whether they were tested in the presence or absence of the odors of the alpha colony. However, it is also interesting to note that the odors of the alpha colony were necessary for rats to show freezing behavior, which may indicate that this type of defensive reaction is controlled more precisely by the stimuli present in the test environment. Finally, the second reason for the devastating effects of repeated exposure to a stressor is that they are not only transituational, but we have known for some time that they also involve a wide range of physiological mechanisms (e.g., drug reactions, immune function) and behaviors (e.g., sexual, maternal, aggressive, appetitive, and cognitive).
This article was presented as a paper in a symposium, entitled "Experience-Produced Odor Emissions in Rats," chaired by Wayne Ludvigson, at the Southwestern Comparative Psychological Association of the Southwestern Psychological Association, on April 17, 1997, in Fort Worth, Texas.
This research was supported by grants from NIMH, NIH, NSF, Kenyon College Summer Science Fellowships, and endowment funds from the Samuel B. Cummings Professorship in Psychology to Jon L. Williams. Reprint requests should be sent to Jon L. Williams, Department of Psychology, Kenyon College, Gambler, OH 43022.
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|Title Annotation:||Special Issue: Odorous, Behavioral, and Physiological Reactions of Rats to Episodes of Reward, Frustration, Illness, Attack and Threat|
|Author:||Williams, Jon L.|
|Publication:||The Psychological Record|
|Date:||Jun 22, 1999|
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