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The effects of ethanol on a measure of conditioned fear in mice
Alcohol,Vol. 14, No. 4, pp. 403407, 1997 Copyrighto 1997ElsevierScienceInc. Printedin the USA.All rightsreserved 0741-8329/97$17.00+ .00 ELSEVIER
PII
S0741-8329(96)0190-5
The Effects of Ethanol on a Measure of Conditioned Fear in Mice MICHAEL
F. S T R O M B E R G *
A N D L Y N N J. H A M M O N D t
*Center for Studies of Addiction, University of Pennsylvania, Philadelphia, PA 19104 ?Temple University, Philadelphia, PA 19140 R e c e i v e d 8 July 1996; A c c e p t e d 1 N o v e m b e r 1996 STROMBERG, M. F. AND L. J. HAMMOND. The effects of ethanol on a measure of conditioned fear in mice. ALCOHOL 14(4) 403-407, 1997.--The effects of ethanol on conditioned freezing, a species-specific defensive behavior used as an assay of fear, was examined in mice. In Experiment I, ethanol, 1.2 g/kg, significantly increased freezing compared to a saline control when the mice were reexposed to a context in which they were previously shocked. Experiment 2, which administered ethanol or saline to no-shock control animals, demonstrated that the potentiated freezing produced by ethanol in Experiment 1 was specific to the interaction of ethanol and the stress response. These results suggest that both the qualitative and quantitative dimensions of the environmental stressor, as well as the dose of ethanol used, may be critical for determining ethanol's effect on a stress response, o 1997 Elsevier Science Inc. Alcohol abuse
Ethanol
Stress
Opioids
A prominent theoretical interpretation of the relationship between ethanol and the behavioral response to environmental stressors suggests that ethanol functions to reduce or dampen the effects of stress on the organism (10,18,19). Reviews of the literature testing this hypothesis, however, suggest that the evidence supporting this relationship is equivocal (9). Traditionally, tests of ethanol's ability to alter the behavioral effects produced by exposure to environmental stressors in rodents have taken two forms. Ethanol is experimenter administered and its effects on either the learning or performance of an escape or avoidance task is evaluated. Alternatively, animals are trained to self-administer ethanol and a control solution and this choice behavior is measured in relation to the application of an environmental stressor. More recently, species-specific defensive behaviors have been utilized to evaluate the effects of various pharmacological agents on the expression of emotional responses in rodents. This ethoexperimental approach is argued to be sensitive to the biological basis of emotional behavior and, therefore, provides a base for the development of animal models generalizable to human emotional behavior (4). One advantage of this approach is that emotional responses to environmental stressors can be measured directly. As such, the relationship between ethanol and those experimental vari-
ables surrounding the stress response can be examined under controlled conditions. One behavior that has been widely studied is freezing. Freezing is a species-specific defensive behavior emanating from the activation of the fear motivational system (8). Freezing can be operationally defined as the absence of any noticeable movement with the exception of respiration. As a defensive behavior it was most likely selected because it reduced the probability of detection by predators. Freezing has been elicited as an unconditioned response by exposing rats to innately recognized predators such as cats (3,17) or as a conditioned response by exposing rats to cues that have been associated with foot shock (12). Because freezing is used as an assay of emotionality or fear, the effect of pharmacological agents on the expression of the freezing response can be utilized to assess their anxiolytic properties. Midazolam and chlordiazepoxide, benzodiazepines that produce anxiolytic action through their agonist effects at G A B A receptors, have been shown to reduce conditioned freezing in the rat following exposure to foot shock (15). Ethanol has also been shown to reduce freezing in rats that had been exposed to a cat (5,7); however, diazepam failed to reduce freezing in rats exposed to a cat odor stimulus (6). This suggests that the nature of the stressor has the poten-
Requests for reprints should be addressed to Michael F. Stromberg, Ph.D., Center For Studies of Addiction, 3900 Chestnut Street, Philadelphia, PA 19104. Tel: (215) 823-4325: Fax: (215) 823-5171; E-mail: [email protected]
403
404
STROMBERG AND H A M M O N D
tial to interact differentially with the pharmacological agent being used. These results raise a question as to whether the anxiolytic properties of a drug are dependent upon the interaction of that drug with the nature of the threat stimulus used. The present experiments sought to evaluate the effects of ethanol on conditioned freezing following exposure to foot shock. Because benzodiazepines have been shown to reduce freezing under these conditions, a test of ethanol's effects under these conditions will extend our knowledge of how ethanol interacts with a stress response. EXPERIMENT
1
Method Subjects. The subjects were 16 Swiss Webster female mice obtained from the breeding colony of the Temple University Medical School and weighing 39-53 g at the time of the experiment. They were housed four to a cage under a 12:12-h reversed light:dark cycle with all experimental procedures conducted during the dark portion of the cycle. Food and water were continuously available throughout the experiment. Apparatus. A n observation chamber (20 x 23 × 20 cm) was utilized for both conditioning and testing. Its front and back walls were stainless steel and the sidewalls and ceiling were clear acrylic plastic. The chamber floor consisted of stainless steel rods 2.5 mm in diameter and spaced 1.5 cm apart center to center. The rods were wired to a Grayson-Stadler shock
generator-scrambler that provided a 2-s, 1.0-mA shock. A noise generator provided white noise during all trials. Procedure. Prior to the start of the experiment, all animals were handled for 30 s per day for 4 days. On the first day of the experiment mice were placed into the observation chamber and observed for freezing for 2 rain. Freezing was defined according to the description offered by Fanselow and Helmstetter (15) as "the lack of all visible movement of the body and vibrissae except for necessary respiration." A time sampling procedure was used such that each mouse was observed every 5 s. Behavior was classified as either freezing or nonfreezing following a 1-s observation. At the end of the 2-min observation period, a single shock was administered. The mouse remained in the chamber for an additional minute and was returned to its home cage. This procedure was repeated across three daily sessions. The mice were matched based on the amount of freezing observed during the 2 min prior to the third shock and assigned to either an Ethanol or Saline group with the lowest member of each pair being placed in the Ethanol group. Fifteen minutes before an extinction test, rats in the Ethanol group were injected IP with ethanol, 1.2 g/kg, in a volume of 1.0 ml/kg. Saline was injected in a volume of 1.0 ml/ kg. The mice were placed in the observation chamber and behavior was sampled every 5 s for 2 min. Again, behavior was recorded as either freezing or nonfreezing. Drugs. Ethanol (20% v/v) mixed with 0.09% physiological was injected IP in a dose of 1.2 g/kg. All injections were administered in a volume of 1.0 ml/kg. Ethanol, 1.2 g/kg, was chosen
100
• Freezing Before Third Shock U Freezing Following Injections
90
m 80 4'
•~
70
I O m
60 I"
"6
O
eL
50
40
I g II
30
! "
20 10
Ethanol Group
Saline Group
FIG. 1. Freezing, as a percent of total behavior in Experiment 1, for Ethanol and Saline groups. Solid bars represent freezing observed prior to third shock. Cross-hatched bars represent freezing observed following ethanol or saline injection.
ETHANOL AND FEAR
405
as the dose because it has been demonstrated to increase motor behavior in mice (20). This dose was considered to be a conservative test and would avoid any potential confound produced by the sedative effects of higher doses of ethanol. RESULTS AND DISCUSSION The results of this experiment demonstrate that ethanol administered prior to an extinction test in a context that had been associated with shock increases freezing relative to a saline control. Figure 1 shows the amount of freezing expressed as a percent of total behavior. Prior to the third shock session, the final session before administration of drug or vehicle, freezing comprised 28.1% of total behavior of the Ethanol group compared to 34.9% for the Saline group. During the extinction test following administration of ethanol or saline, freezing comprised 63% of the total behavior of the Ethanol group and 48.4% of the total behavior of the Saline group, an increase of 34.9% and 13.5%, respectively. A paired t-test of freezing during the extinction test revealed that the Ethanol group froze significantly more than the Saline group, t(7) = 2.632, p < 0.04. The pretest difference in freezing is in the opposite direction of the observed results and cannot serve to explain the outcome and, therefore, introduces a conservative bias. EXPERIMENT 2 Despite the fact that the ethanol dose chosen for the first experiment has been demonstrated to increase motor activity (20), it could be argued that ethanol's effect on freezing in Experiment 1 may have been due to sedative properties that reduced motor activity, thereby producing a behavior that was similar in form to the freezing response. If this were the case, the behavior scored as freezing would have been artefactual and ethanol may have actually reduced fear to the shock-associated context. This experiment was designed to replicate Experiment 1 with animals that had the same handling and context exposure but without exposure to shock. This allowed for the evaluation of the same dose of ethanol on motor behavior in similar subjects with the same basic procedure. METHOD
Subjects. Subjects were 16 additional female Swiss Webster mice obtained and maintained identically to those used in Experiment 1. Apparatus. The observation chamber and noise generator were identical to those used in Experiment 1; however, the shock generator was turned off. Procedure. The procedure was identical to that employed in Experiment 1 with two exceptions. No shock was administered to the animals and they were placed in the observation chamber for 4 days prior to ethanol or saline injections rather than the 3 days in Experiment 1. RESULTS AND DISCUSSION No freezing was observed in any of the mice during the first 4 context exposure days. Figure 2 shows freezing data following injections of saline or ethanol. Both groups showed a small amount of freezing during the context exposure following injections. Mice in the Ethanol group froze 6.9% of the time whereas mice in the Saline group froze 5.4%. The difference between these groups did not approach significance. Because mice froze following both saline and ethanol injections,
this can be attributed to a nonspecific effect produced by exposure to the injection procedure rather than any specific drug effect of ethanol. A t the same time, the increase in freezing seen following ethanol injections in Experiment 1 was significantly greater than the freezing seen following saline injections and much larger than following ethanol injections in Experiment 2. These results suggest that the effect of ethanol seen in Experiment 1 is specific to the interaction of ethanol and the stress response. GENERAL DISCUSSION The results of Experiment 1 suggest that ethanol potentiates the conditioned freezing response in mice exposed to a context in which they had previously received brief exposure to uncontrollable foot shock. Further, the results of Experiment 2 demonstrate that these results are not due to any sedative properties of ethanol but are specific to the interaction of ethanol with the motivational state elicited by reexposure to the shock context. The results of Experiment 1 suggest almost paradoxically that ethanol increases the fear of the mouse, as measured by the conditional freezing assay. It is apparent from these results that ethanol does not exert any anxiolytic properties under the parameters used in the present experiment. Ethanol has been demonstrated to reduce crouching in rats reexposed to the same environment in which they had been exposed to a cat 5 days earlier; however, this effect was reliable only at the highest dose tested, 1.2 g/kg (7). A second demonstration that ethanol reduces cat-induced freezing is complicated by the fact that it included a conspecific attack procedure immediately following exposure to the cat. Although freezing was reliably reduced at the two highest ethanol doses used, it failed to reliably increase offensive behavior, which is hypothesized to emerge from ethanol's suppression of fear (5). Other studies of the effects of ethanol on aggression in mice have suggested that ethanol has little effect on the reduction of fear mediated behaviors and, in fact, actually potentiates timidity or defensive behaviors (2). One approach to understanding how pharmacological agents may interact with behavior is to determine what neural systems underlie and affect the expression of that behavior and how a drug may interact with those systems to modify the behavior. One model that has been offered to explain the emergence of defensive behaviors like freezing is the perceptual-defensive-recuperative model of fear and pain (8). This model hypothesizes that the endogenous opioid system, acting as a nociceptive detection or pain mediated motivational system, and a fear motivational system, responsible for generating species-specific defense behaviors, exist in parallel. The endogenous opioid system further serves to select and organize the expression of defensive behavior by suppressing recuperative behavior. The correlation between freezing and endogenous opioid activity has been established both indirectly and directly. The aversiveness of a noxious stimulus, defined by both shock intensity (14) and the number of shock exposures (13), increases the occurrence of the freezing response when the animal is reexposed to the context associated with the shock. Naloxone, a nonselective opioid antagonist, has also been demonstrated to increase freezing when administered prior to shock exposure. The potentiated freezing seen following naloxone is hypothesized to occur as a result of an increase in the perceived intensity of the shock (14,16). The mechanism for these effects emerging from the interaction of stress responses and ethanol remains unclear; how-
406
STROMBERG AND H A M M O N D 10
[] Freezing Following Injections 9 A
=E 14.1 ¢n 8 ÷
0
~6 0
I--
~5 .s m m
3
II
UL
Ethanol Group
Saline Group
FIG. 2. Freezing, as a percent of total behavior in Experiment 2, for Ethanol and Saline groups. No freezing was observed during four context exposures. Cross-hatched bars represent freezing observed following ethanol or saline injection. ever, one suggestion offered is that ethanol may interact with endogenous opioid mechanisms activated either directly or indirectly by exposure to environmental stressors (21). One such investigation has shown that ethanol administered to rats prior to 15-rain restraint stress increases later open field activity compared to saline controls (1). Ethanol's interaction with stress in this case is hypothesized as due to recruitment of opioid mechanisms because naltrexone blocks this effect while having no similar effect on motor behavior when administered in the absence of ethanol. In the same series of experiments, a similar test of rats exposed to 60 rain of restraint stress failed to yield similar effects for ethanol. It is interesting that variations in either the quantitative or qualitative dimensions of the stressor appear to interact differentially with the pharmacological properties of ethanol. One interpretation offered for these differences is that different stressors elicit either opioid or nonopioid responses and that ethanol may then interact with the opioid response (1). The direction of ethanol's effects on fear motivated behavior may also be determined by the dose employed. For example,
lower stimulating doses of ethanol are thought to activate opioidergic and dopaminergic transmission, whereas higher doses of ethanol activate G A B A pathways (11). The results from Experiment 1 of the present series may reflect these differences. If ethanol, at the dose utilized, has its primary effect at opioidergic sites, it may operate to increase freezing to the shock-paired context. This is consistent with increases in freezing seen following manipulations of the endogenous opioid system with naloxone prior to shock exposure (14,16). The results of the present experiments are not consistent with the reported effects of ethanol on defensive behavior following exposure to innate threat stimuli, cats or cat odor (5,7). These differences may be due to the nature of the aversive stimulus used, the intensity of those stimuli, the physiological response elicited by the stimuli or differences in the doses of ethanol used. The results of the present series of experiments, when taken together with the action of ethanol with innate threat stimuli, suggest that the interaction of ethanol with stress is not a unitary phenomenon and may be specific to the particular stressor used.
REFERENCES
1. Aragon, C. M. G.; Trudeau, L-E.; Amit, Z.: Stress ethanol interaction: Involvement of endogenous opioid mechanisms. Neurosci. Biobehav. Rev. 14:535-541;1990. 2. Berry, M. S.: Ethanol-induced enhancement of defensive behavior in different models of murine aggression. J. Stud. Alcohol Suppl. 11:156-162;1993. 3. Blanchard, R. J.; Blanchard, D. C.: Defensive reactions in the
albino rat. Learn. Motiv. 2:351-362; 1971. 4. Blanchard, D. C.; Blanchard, R. J.: Ethoexperimental approaches to the biology of emotion. Annu. Rev. Psychol. 39:43~58;1988. 5. Blanchard, R. J.; Blanchard, D. C.; Flannelly, K. J.; Hori, K.: Ethanol effects on freezing and conspecific attack in rats previously exposed to a cat. Behav. Process. 16:193-201; 1988. 6. Blanchard, R. J.; Blanchard, D. C.; Weiss, S. M.; Meyer, S.: The
ETHANOL
7. 8. 9. 10. 11. 12. 13. 14.
AND FEAR
effects of ethanol diazepam on reactions to predatory odors. Pharmacol. Biochem. Behav. 35:775-780; 1990. Blanchard, R. J.; Blanchard, D. C.; Weiss, S. M.: Ethanol effects in an anxiety/defense test battery. Alcohol 7:375-381; 1990. Bolles, R. C.; Fanselow, M. S.: A perceptual-defensive-recuperative model of fear and pain. Behav. Brain Sci. 3:291-323; 1980. Cappell, H.; Herman, C. P.: Alcohol and tension reduction: A review. Q. J. Stud. Alcohol 33:33-64; 1972. Conger, J. J.: Reinforcement theory and the dynamics of alcoholism. Q. J. Stud. Alcohol 17:296-305; 1956. DiChiara, G.; Acquas, E.; Tanda, G.: Ethanol as a neurochemical surrogate of conventional reinforcers: The dopamine-opioid link. Alcohol 13:13-17; 1996. Fanselow, M. S.: Conditional and unconditional components of postshock freezing. Pavlovian J. Biol. Sci. 15:177-182; 198ll. Fanselow, M. S.: Naloxone and Pavlovian fear conditioning. Learn. Motiv, 12:398-419; 1981. Fanselow, M. S.; Bolles, R. C.: (1979). Naloxone and shock-elicited freezing in the rat. J. Comp. Physiol. Psychol. 93:736-744: 1979.
407 15. Fanselow, M. S.; Helmstetter, F. J.: Conditioned analgesia, Defensive freezing and benzodiazepines. Behav. Neurosci. 102: 233-243; 1988. 16. Lester, L. S.; Fanselow, M. S.: Naloxone's enhancement of freezing: Modulation of perceived intensity or memory processes. Physiol. Psychol. 14:5-10; 1986. 17. Lichtman, A. H.; Fanselow, M. S.: Cats produce analgesia in rats on the tail-flick test: Naltrexone sensitivity is determined by the nociceptive test stimulus. Brain Res. 533:91-94: 1990. 18. Masserman~ J. H.; Jacques, M. E.; Nicholson, M. R.: Alcohol as a preventative of experimental neurosis. Q. J. Stud. Alcohol 6:281289; 1945. 19. Sher, K. J.: Stress-response dampening. In Blane, H. T.; Leonard, K., eds. Psychological theories of drinking and alcoholism. New York: Guilford: 1987. 20. Smoothy, R.; Berry, M. S.: Alcohol increases both locomotion and immobility in mice: An ethological analysis of spontaneous motor behavior. Psychopharmacology (Berlin) 83:272-276; 1984. 21. Volpicelli, J. R.: Uncontrollable events and alcohol drinking. Br. J. Addict. 82:385-396; 1987.
PII
S0741-8329(96)0190-5
The Effects of Ethanol on a Measure of Conditioned Fear in Mice MICHAEL
F. S T R O M B E R G *
A N D L Y N N J. H A M M O N D t
*Center for Studies of Addiction, University of Pennsylvania, Philadelphia, PA 19104 ?Temple University, Philadelphia, PA 19140 R e c e i v e d 8 July 1996; A c c e p t e d 1 N o v e m b e r 1996 STROMBERG, M. F. AND L. J. HAMMOND. The effects of ethanol on a measure of conditioned fear in mice. ALCOHOL 14(4) 403-407, 1997.--The effects of ethanol on conditioned freezing, a species-specific defensive behavior used as an assay of fear, was examined in mice. In Experiment I, ethanol, 1.2 g/kg, significantly increased freezing compared to a saline control when the mice were reexposed to a context in which they were previously shocked. Experiment 2, which administered ethanol or saline to no-shock control animals, demonstrated that the potentiated freezing produced by ethanol in Experiment 1 was specific to the interaction of ethanol and the stress response. These results suggest that both the qualitative and quantitative dimensions of the environmental stressor, as well as the dose of ethanol used, may be critical for determining ethanol's effect on a stress response, o 1997 Elsevier Science Inc. Alcohol abuse
Ethanol
Stress
Opioids
A prominent theoretical interpretation of the relationship between ethanol and the behavioral response to environmental stressors suggests that ethanol functions to reduce or dampen the effects of stress on the organism (10,18,19). Reviews of the literature testing this hypothesis, however, suggest that the evidence supporting this relationship is equivocal (9). Traditionally, tests of ethanol's ability to alter the behavioral effects produced by exposure to environmental stressors in rodents have taken two forms. Ethanol is experimenter administered and its effects on either the learning or performance of an escape or avoidance task is evaluated. Alternatively, animals are trained to self-administer ethanol and a control solution and this choice behavior is measured in relation to the application of an environmental stressor. More recently, species-specific defensive behaviors have been utilized to evaluate the effects of various pharmacological agents on the expression of emotional responses in rodents. This ethoexperimental approach is argued to be sensitive to the biological basis of emotional behavior and, therefore, provides a base for the development of animal models generalizable to human emotional behavior (4). One advantage of this approach is that emotional responses to environmental stressors can be measured directly. As such, the relationship between ethanol and those experimental vari-
ables surrounding the stress response can be examined under controlled conditions. One behavior that has been widely studied is freezing. Freezing is a species-specific defensive behavior emanating from the activation of the fear motivational system (8). Freezing can be operationally defined as the absence of any noticeable movement with the exception of respiration. As a defensive behavior it was most likely selected because it reduced the probability of detection by predators. Freezing has been elicited as an unconditioned response by exposing rats to innately recognized predators such as cats (3,17) or as a conditioned response by exposing rats to cues that have been associated with foot shock (12). Because freezing is used as an assay of emotionality or fear, the effect of pharmacological agents on the expression of the freezing response can be utilized to assess their anxiolytic properties. Midazolam and chlordiazepoxide, benzodiazepines that produce anxiolytic action through their agonist effects at G A B A receptors, have been shown to reduce conditioned freezing in the rat following exposure to foot shock (15). Ethanol has also been shown to reduce freezing in rats that had been exposed to a cat (5,7); however, diazepam failed to reduce freezing in rats exposed to a cat odor stimulus (6). This suggests that the nature of the stressor has the poten-
Requests for reprints should be addressed to Michael F. Stromberg, Ph.D., Center For Studies of Addiction, 3900 Chestnut Street, Philadelphia, PA 19104. Tel: (215) 823-4325: Fax: (215) 823-5171; E-mail: [email protected]
403
404
STROMBERG AND H A M M O N D
tial to interact differentially with the pharmacological agent being used. These results raise a question as to whether the anxiolytic properties of a drug are dependent upon the interaction of that drug with the nature of the threat stimulus used. The present experiments sought to evaluate the effects of ethanol on conditioned freezing following exposure to foot shock. Because benzodiazepines have been shown to reduce freezing under these conditions, a test of ethanol's effects under these conditions will extend our knowledge of how ethanol interacts with a stress response. EXPERIMENT
1
Method Subjects. The subjects were 16 Swiss Webster female mice obtained from the breeding colony of the Temple University Medical School and weighing 39-53 g at the time of the experiment. They were housed four to a cage under a 12:12-h reversed light:dark cycle with all experimental procedures conducted during the dark portion of the cycle. Food and water were continuously available throughout the experiment. Apparatus. A n observation chamber (20 x 23 × 20 cm) was utilized for both conditioning and testing. Its front and back walls were stainless steel and the sidewalls and ceiling were clear acrylic plastic. The chamber floor consisted of stainless steel rods 2.5 mm in diameter and spaced 1.5 cm apart center to center. The rods were wired to a Grayson-Stadler shock
generator-scrambler that provided a 2-s, 1.0-mA shock. A noise generator provided white noise during all trials. Procedure. Prior to the start of the experiment, all animals were handled for 30 s per day for 4 days. On the first day of the experiment mice were placed into the observation chamber and observed for freezing for 2 rain. Freezing was defined according to the description offered by Fanselow and Helmstetter (15) as "the lack of all visible movement of the body and vibrissae except for necessary respiration." A time sampling procedure was used such that each mouse was observed every 5 s. Behavior was classified as either freezing or nonfreezing following a 1-s observation. At the end of the 2-min observation period, a single shock was administered. The mouse remained in the chamber for an additional minute and was returned to its home cage. This procedure was repeated across three daily sessions. The mice were matched based on the amount of freezing observed during the 2 min prior to the third shock and assigned to either an Ethanol or Saline group with the lowest member of each pair being placed in the Ethanol group. Fifteen minutes before an extinction test, rats in the Ethanol group were injected IP with ethanol, 1.2 g/kg, in a volume of 1.0 ml/kg. Saline was injected in a volume of 1.0 ml/ kg. The mice were placed in the observation chamber and behavior was sampled every 5 s for 2 min. Again, behavior was recorded as either freezing or nonfreezing. Drugs. Ethanol (20% v/v) mixed with 0.09% physiological was injected IP in a dose of 1.2 g/kg. All injections were administered in a volume of 1.0 ml/kg. Ethanol, 1.2 g/kg, was chosen
100
• Freezing Before Third Shock U Freezing Following Injections
90
m 80 4'
•~
70
I O m
60 I"
"6
O
eL
50
40
I g II
30
! "
20 10
Ethanol Group
Saline Group
FIG. 1. Freezing, as a percent of total behavior in Experiment 1, for Ethanol and Saline groups. Solid bars represent freezing observed prior to third shock. Cross-hatched bars represent freezing observed following ethanol or saline injection.
ETHANOL AND FEAR
405
as the dose because it has been demonstrated to increase motor behavior in mice (20). This dose was considered to be a conservative test and would avoid any potential confound produced by the sedative effects of higher doses of ethanol. RESULTS AND DISCUSSION The results of this experiment demonstrate that ethanol administered prior to an extinction test in a context that had been associated with shock increases freezing relative to a saline control. Figure 1 shows the amount of freezing expressed as a percent of total behavior. Prior to the third shock session, the final session before administration of drug or vehicle, freezing comprised 28.1% of total behavior of the Ethanol group compared to 34.9% for the Saline group. During the extinction test following administration of ethanol or saline, freezing comprised 63% of the total behavior of the Ethanol group and 48.4% of the total behavior of the Saline group, an increase of 34.9% and 13.5%, respectively. A paired t-test of freezing during the extinction test revealed that the Ethanol group froze significantly more than the Saline group, t(7) = 2.632, p < 0.04. The pretest difference in freezing is in the opposite direction of the observed results and cannot serve to explain the outcome and, therefore, introduces a conservative bias. EXPERIMENT 2 Despite the fact that the ethanol dose chosen for the first experiment has been demonstrated to increase motor activity (20), it could be argued that ethanol's effect on freezing in Experiment 1 may have been due to sedative properties that reduced motor activity, thereby producing a behavior that was similar in form to the freezing response. If this were the case, the behavior scored as freezing would have been artefactual and ethanol may have actually reduced fear to the shock-associated context. This experiment was designed to replicate Experiment 1 with animals that had the same handling and context exposure but without exposure to shock. This allowed for the evaluation of the same dose of ethanol on motor behavior in similar subjects with the same basic procedure. METHOD
Subjects. Subjects were 16 additional female Swiss Webster mice obtained and maintained identically to those used in Experiment 1. Apparatus. The observation chamber and noise generator were identical to those used in Experiment 1; however, the shock generator was turned off. Procedure. The procedure was identical to that employed in Experiment 1 with two exceptions. No shock was administered to the animals and they were placed in the observation chamber for 4 days prior to ethanol or saline injections rather than the 3 days in Experiment 1. RESULTS AND DISCUSSION No freezing was observed in any of the mice during the first 4 context exposure days. Figure 2 shows freezing data following injections of saline or ethanol. Both groups showed a small amount of freezing during the context exposure following injections. Mice in the Ethanol group froze 6.9% of the time whereas mice in the Saline group froze 5.4%. The difference between these groups did not approach significance. Because mice froze following both saline and ethanol injections,
this can be attributed to a nonspecific effect produced by exposure to the injection procedure rather than any specific drug effect of ethanol. A t the same time, the increase in freezing seen following ethanol injections in Experiment 1 was significantly greater than the freezing seen following saline injections and much larger than following ethanol injections in Experiment 2. These results suggest that the effect of ethanol seen in Experiment 1 is specific to the interaction of ethanol and the stress response. GENERAL DISCUSSION The results of Experiment 1 suggest that ethanol potentiates the conditioned freezing response in mice exposed to a context in which they had previously received brief exposure to uncontrollable foot shock. Further, the results of Experiment 2 demonstrate that these results are not due to any sedative properties of ethanol but are specific to the interaction of ethanol with the motivational state elicited by reexposure to the shock context. The results of Experiment 1 suggest almost paradoxically that ethanol increases the fear of the mouse, as measured by the conditional freezing assay. It is apparent from these results that ethanol does not exert any anxiolytic properties under the parameters used in the present experiment. Ethanol has been demonstrated to reduce crouching in rats reexposed to the same environment in which they had been exposed to a cat 5 days earlier; however, this effect was reliable only at the highest dose tested, 1.2 g/kg (7). A second demonstration that ethanol reduces cat-induced freezing is complicated by the fact that it included a conspecific attack procedure immediately following exposure to the cat. Although freezing was reliably reduced at the two highest ethanol doses used, it failed to reliably increase offensive behavior, which is hypothesized to emerge from ethanol's suppression of fear (5). Other studies of the effects of ethanol on aggression in mice have suggested that ethanol has little effect on the reduction of fear mediated behaviors and, in fact, actually potentiates timidity or defensive behaviors (2). One approach to understanding how pharmacological agents may interact with behavior is to determine what neural systems underlie and affect the expression of that behavior and how a drug may interact with those systems to modify the behavior. One model that has been offered to explain the emergence of defensive behaviors like freezing is the perceptual-defensive-recuperative model of fear and pain (8). This model hypothesizes that the endogenous opioid system, acting as a nociceptive detection or pain mediated motivational system, and a fear motivational system, responsible for generating species-specific defense behaviors, exist in parallel. The endogenous opioid system further serves to select and organize the expression of defensive behavior by suppressing recuperative behavior. The correlation between freezing and endogenous opioid activity has been established both indirectly and directly. The aversiveness of a noxious stimulus, defined by both shock intensity (14) and the number of shock exposures (13), increases the occurrence of the freezing response when the animal is reexposed to the context associated with the shock. Naloxone, a nonselective opioid antagonist, has also been demonstrated to increase freezing when administered prior to shock exposure. The potentiated freezing seen following naloxone is hypothesized to occur as a result of an increase in the perceived intensity of the shock (14,16). The mechanism for these effects emerging from the interaction of stress responses and ethanol remains unclear; how-
406
STROMBERG AND H A M M O N D 10
[] Freezing Following Injections 9 A
=E 14.1 ¢n 8 ÷
0
~6 0
I--
~5 .s m m
3
II
UL
Ethanol Group
Saline Group
FIG. 2. Freezing, as a percent of total behavior in Experiment 2, for Ethanol and Saline groups. No freezing was observed during four context exposures. Cross-hatched bars represent freezing observed following ethanol or saline injection. ever, one suggestion offered is that ethanol may interact with endogenous opioid mechanisms activated either directly or indirectly by exposure to environmental stressors (21). One such investigation has shown that ethanol administered to rats prior to 15-rain restraint stress increases later open field activity compared to saline controls (1). Ethanol's interaction with stress in this case is hypothesized as due to recruitment of opioid mechanisms because naltrexone blocks this effect while having no similar effect on motor behavior when administered in the absence of ethanol. In the same series of experiments, a similar test of rats exposed to 60 rain of restraint stress failed to yield similar effects for ethanol. It is interesting that variations in either the quantitative or qualitative dimensions of the stressor appear to interact differentially with the pharmacological properties of ethanol. One interpretation offered for these differences is that different stressors elicit either opioid or nonopioid responses and that ethanol may then interact with the opioid response (1). The direction of ethanol's effects on fear motivated behavior may also be determined by the dose employed. For example,
lower stimulating doses of ethanol are thought to activate opioidergic and dopaminergic transmission, whereas higher doses of ethanol activate G A B A pathways (11). The results from Experiment 1 of the present series may reflect these differences. If ethanol, at the dose utilized, has its primary effect at opioidergic sites, it may operate to increase freezing to the shock-paired context. This is consistent with increases in freezing seen following manipulations of the endogenous opioid system with naloxone prior to shock exposure (14,16). The results of the present experiments are not consistent with the reported effects of ethanol on defensive behavior following exposure to innate threat stimuli, cats or cat odor (5,7). These differences may be due to the nature of the aversive stimulus used, the intensity of those stimuli, the physiological response elicited by the stimuli or differences in the doses of ethanol used. The results of the present series of experiments, when taken together with the action of ethanol with innate threat stimuli, suggest that the interaction of ethanol with stress is not a unitary phenomenon and may be specific to the particular stressor used.
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