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Conditioned fear to environmental context: cardiovascular and behavioral components in the rat
Brain Research 858 Ž2000. 440–445 www.elsevier.comrlocaterbres
Interactive report
Conditioned fear to environmental context: cardiovascular and behavioral components in the rat 1 Pascal Carrive
)
School of Anatomy, UniÕersity of New South Wales, Sydney, NSW 2052, Australia Accepted 19 January 2000
Abstract This study compares the time course of the cardiovascular changes Žmean arterial blood pressure, heart rate. and behavioral changes Žfreezing, rearing, grooming and activity. evoked by 30 min long exposures to a footshock chamber before and after conditioning with footshocks. The main finding is that the conditioned fear evoked by re-exposure to the footshock chamber after conditioning is associated with a prolonged freezing response, a marked rise in mean arterial pressure Žq35 mm Hg above a resting baseline of 105 mm Hg. and a delayed rise in heart rate. The pattern of behavioral and cardiovascular changes is the same as with conditioned fear to a discrete stimulus but the effect is a lot longer. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Conditioned fear; Blood pressure; Heart rate; Radio-telemetry; Freezing; Exploratory behavior
1. Introduction Conditioned fear to context, or contextual conditioned fear, is the fear evoked by re-exposure to an environment that has previously been paired with an aversive or unpleasant stimulus. This is observed, for example, when a rat is re-exposed to a footshock chamber in which it has previously received electric footshocks. Traditionally, the fear evoking stimulus — or conditioning stimulus — is a discrete stimulus such as a light or tone. The conditioning stimulus is initially neutral. It acquires its aversive properties through repetitive pairing with the unpleasant footshocks. In the case of contextual fear, there is no light or tone; here, the conditioning stimulus is the environmental context, that is, the footshock chamber itself w3x. Conditioning to context is best when the footshocks are unsignaled and inescapable. The behavioral response of conditioned fear has been well described w1–3,6x. It is characterized by an immobile and tense posture known as freezing. Qualitatively, the freezing response is the same for contextual conditioned fear and conditioned fear to a discrete stimulus. There is, however, a marked difference in the duration of the two
1
Published on the World Wide Web on 1 February 2000. Tel.: q61-2-9385-2467; Fax: q61-2-9313-6252; [email protected] )
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freezing responses. Thus, the duration of the conditioned response to a discrete stimulus such as a tone is short lived Žless than 2 min. w6x, whereas the conditioned response to context can last for much longer, e.g. 8 min w3x or 10 min w8x. The cardiovascular component of conditioned fear has been described also but only for the short responses evoked by a discrete stimulus Žtone. w4,5,9x. It consists of a transient Žless than 10 s. increase in mean arterial pressure and heart rate. Little is known about the cardiovascular changes that accompany the long freezing responses evoked by contextual fear. A recent paper by Nijsen et al. w8x shows that it is associated with an increase in heart rate and a release of circulating catecholamines, but the blood pressure component of the response has not yet been investigated. The aim of this study is to describe the concurrent cardiovascular and behavioral changes that occur during the long conditioned fear responses to context. To avoid interference between the recording of cardiovascular parameters and the behavioral response, blood pressure was recorded in freely moving rats with radio-telemetric probes. This also allowed for simultaneous recording of cardiovascular and behavioral changes. The main idea behind this investigation is that the longer response of conditioned fear to context may provide a better model for studying the cardiovascular readjustments that occur during anxious states and semi-chronic forms of psychological stress.
0006-8993r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 0 0 . 0 2 0 2 9 - 1
P. CarriÕer Brain Research 858 (2000) 440–445
2. Material and methods The subjects were 6 experimentally naive male Wistar rats Ž300–500 g. obtained from the colony of specific pathogen free rats maintained by the University of New South Wales. All experiments were approved by the Animal ethics committee of the University of New South Wales and conformed to the rules and guidelines on animal experimentation in Australia. The colony room was maintained at a 12-h lightrdark cycle and the experiments were conducted during the light phase of the cycle. Mean arterial pressure, heart rate and activity Žbody movements. were recorded by radio-telemetry. The telemetric probes ŽPA-C40, Data Sciences International. were implanted in the peritoneal cavity under anesthesia with a mixture of ketamine Ž100 mgrkg. and xylazine Ž50 mgrkg. injected intraperitoneally. Briefly, a midline incision was made in the abdomen and the descending aorta was exposed at the level of the iliac bifurcation. The artery was punctured at this level and the tip of the catheter Ž1 cm long. was inserted. The puncture was then glued with the catheter in place and the probe stitched to the abdominal wall while closing the midline incision. The animals were given antibiotics and moved to individual plastic home boxes Ž65 = 40 = 22 cm. in which they remained until the end of the experiment. During the recovery period Ž1 week. the animals were handled every day to habituate to the experimenter. Pre-conditioning, conditioning and testing were done in footshock chambers Ž23 = 21 = 20 cm. made of clear Perspex walls on two sides with a grid floor composed of 18 stainless steel rods Ž2 mm in diameter., spaced 1.5 cm apart and wired to a shock generator ŽGrason Stadler.. The chambers were cleaned before and after use with 0.05% acetic acid. The experiment started with 2 pre-exposures of 30 min to the shock chamber on Days 1 and 2. No shock was given during the pre-exposures. Day 3 was a rest day. The first conditioning session was done on Day 4: it consisted of a 30-min long exposure to the footshock chamber with 3 electric footshocks Ž1 mA, 1 s. delivered at t s 5, 15 and 25 min. Four h later, the animals were re-exposed to the footshock chamber for 30 min; no shock was delivered: it was the first test re-exposure for the conditioned response. Day 5 was similar to Day 4: animals had a second conditioning session and 4 h later, the second test re-exposure. Day 6 was a rest day. Finally, on Day 7, the rats were exposed for 30 min to a new environment, a plastic box similar to the home box but with transparent walls and clean bedding. On each of these days the general procedure was the same: in the morning, the animals were transferred in their home box from the colony room to the telemetry room and their probes were turned on. After a few hours of baseline recording, they were gently taken out of their home box and placed in the shock chamber or the new environment. Thirty min later the animals were returned to their home box. They stayed in the room for at
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least another 4 h and were either returned to the colony room or re-exposed to the shock chamber a second time ŽDay 4 and Day 5.. There were six parameters measured simultaneously: two cardiovascular Žmean arterial pressure, heart rate. and four behavioral Žactivity, freezing, rearing and grooming.. Mean arterial pressure ŽMAP. was extracted from the pulsatile blood pressure signal and sampled for 3 s every 10 s with the DataQuest IV software ŽData Sciences International. running on a PC computer. Heart rate ŽHR. was calculated manually by counting the number of beats in 3 s blocks of the pulsatile trace Ždisplayed on the screen of the computer.. It was sampled regularly every min during the test exposures but irregularly before and after. Activity was a continuous measure of body movements, which the software automatically extracted from changes in orientation of the radio-telemetric probe. In addition to these 3 measures provided directly or indirectly by the radio-telemetric probes, an experimenter sitting in the telemetry room recorded the time spent freezing, the time spent grooming and the number of rearings during exposures. Freezing was defined as the complete absence of movement while the animal assumed a characteristic tense posture. Rearing was defined as rearing along the walls of the footshock chamber or the new environment box. All measures were averaged ŽMAP, HR, activity. or cumulated Žrearing, grooming, freezing. over 1 min periods. Data were analyzed using a 2 way ANOVA with one repeated factor Žtime.. The five different types of exposure were treated as between-subjects factors. Post-hoc analysis was carried out using the Games–Howell procedure for comparing the effects of types of exposure over the entire response and the Tukey–Kramer procedure for comparing the effects of types of exposure for each minute of the re-exposure. The analysis was done with Statview 5 ŽSAS institute..
3. Results Fig. 1 shows the averaged minute to minute changes in HR, MAP, freezing, rearing, grooming and activity evoked by the two pre-exposures before conditioning, the two test re-exposures after conditioning and finally, the exposure to a new environment. As can be seen, baseline MAP and HR were stable and did not change throughout the 7 days. Average readings taken around the 5th min before exposures, when the animals were at rest in their home box, were: MAP, 105 " 1 mm Hg ŽMean " SEM.; HR, 357 " 7 bpm. In contrast, there were clear differences in the cardiovascular and behavioral responses between the different types of exposure. A repeated measure ANOVA on the 30 min of testing revealed a significant main effect of the type of exposure for all 6 parameters ŽMAP, F Ž4,25. s 10.56, p - 0.0001; HR, F Ž4,25. s 4.31, p s 0.008; activity, F Ž4,25. s 6.84, p - 0.0007; freezing, F Ž4,25. s 29.63, p
442
P. CarriÕer Brain Research 858 (2000) 440–445
Fig. 1. Time course of the changes in heart rate, mean arterial pressure, freezing, rearing, grooming and activity evoked by the 2 pre-exposures, the 2 test re-exposures and finally the exposure to the new environment. The first and second pre-exposures were done in the footshock chamber before conditioning. The first and second test re-exposures were also done in the footshock chamber but after conditioning Ž4 h after the first and second conditioning session, respectively.. Exposure to the new environment was done last, after conditioning. Mean " SEM, n s 6 rats.
P. CarriÕer Brain Research 858 (2000) 440–445
- 0.0001; rearing, F Ž4,23. s 4.71, p - 0.0063; grooming, F Ž4,23. s 8.78, p - 0.0002.. There was also a significant main effect for time and a significant interaction between time and type of exposure for all 6 parameters Ž F Ž29, 725. ) 2.5, p - 0.0001 and F Ž116, 725. ) 1.5, p 0.0005, respectively.. Usually, it was during the first half of the responses Ž0–15 min. that the differences were most obvious. The two pre-exposures before conditioning evoked comparable levels of activity and rearing as the animals actively explored the footshock chamber. This activity gradually diminished over time and was marked by a peak in grooming midway through the re-exposure. There was no freezing. The exploratory activity was associated with a clear rise in MAP Žmaximum 128 " 2 mm Hg and 122 " 1 mm Hg, for first and second pre-exposure respectively, representing a q23 and q17 mm Hg increase from baseline.. There was also a rapid rise in HR Žmaximum 493 " 7 bpm and 469 " 9 bpm, respectively, i.e. a maximum of q136 and q112 bpm increase from baseline.. The two responses were very similar behaviorally, but the cardiovascular response appeared weaker the second time. Games–Howell post-hoc analysis revealed a statistically significant difference for MAP and HR between the two conditions Ž p - 0.05.. In contrast to the pre-exposures, the two test re-exposures done after conditioning were characterized by a reduction in activity, rearing and grooming. Behavioral activity was replaced by freezing. In the first test re-exposure, the effect was observed mainly during the first 10 min. In the second test re-exposure the effect extended over the entire 30 min period with almost 100% freezing in the first 10 min. Both conditioned fear responses were associated with a marked rise in MAP which peaked at the 4th min Ž137 " 3 mm Hg and 140 " 4 mm Hg, for first and second re-exposure respectively, representing a q32 and q35 mm Hg increase from baseline.. Moreover, this increase was q15 and q18 mm Hg above the second pre-exposure. The HR response was completely different: it was not augmented but reduced compared to the pre-exposures. There was no initial rapid increase in HR; instead, the rise in HR was late and slow. The effect was particularly obvious in the second test re-exposure, suggesting a relationship between the slowness of the rise in HR and the level of conditioning. A close examination of the time course of the response also suggests a relationship between HR, activity and freezing. For example, the response to the first test re-exposure shows that HR stayed low as long as the animal froze and remained immobile, but when activity resumed Žafter the 5th min., HR gradually rose to levels comparable to the pre-exposures. Thus, the longer and more intense the freezing immobility, the slower the rise in HR. Post-hoc analysis showed that both HR and freezing were significantly increased on the second test re-exposure compared to the first one ŽGames–Howell, p - 0.05.. The difference was statistically significant from the 9th to the
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20th min for HR and from the 9th to the 28th min for Freezing ŽTukey–Kramer, p - 0.05.. Interestingly, the MAP response was the same for both test-re-exposures. Nevertheless, the effect of conditioning on the behavioral and cardiovascular response to the footshock chamber were very clear: when the two test re-exposures were compared to the second pre-exposure before conditioning, post-hoc analysis revealed statistically significant differences for all 6 parameters ŽGames–Howell, p - 0.05.. For MAP and HR, the difference between the second pre-exposure and the second test re-exposure was statistically significant between the 2nd and the 12th min ŽTukey– Kramer, p - 0.05.. The conditioned animals were finally tested in a new environment. The response to this different context resembled in many ways the response observed during the pre-exposures before conditioning. Although grooming was reduced and there was some freezing for the first 20 min, there was as much activity and rearing as during the pre-exposures. The changes in MAP and HR were also the same as with the pre-exposures Žmaximum of 127 " 2 mm Hg and 463 " 22 bpm, representing a q22 mm Hg and a q106 bpm increase from baseline., and the same fast rise in HR was observed. There was no statistically significant difference for MAP, HR and activity between the new environment and the second pre-exposure. In contrast, there were statistically significant differences for all 6 parameters between the new environment and the second test re-exposure ŽGames–Howell, p - 0.05.. For MAP and HR, the difference was statistically significant between the 2nd and the 12th min ŽTukey–Kramer, p - 0.05.. 4. Discussion The most important finding of this study is that conditioned fear to context evokes a marked rise in mean arterial pressure. The change we observed was q35 mm Hg above a resting baseline of 105 mm Hg. It was also at least 12 mm Hg greater than the change in MAP evoked by pre-exposure before conditioning or by the exposure to a new environment after conditioning. Further, it was associated with high levels of freezing immobility whereas in the pre-exposures and new exposure the animals were actively exploring their environment. Thus, this marked hypertensive response is clearly a component of the conditioned fear response to the aversive context of the footshock chamber. For a normotensive animal housed individually, a q35 mm Hg rise from baseline is a very significant physiological change. We have done continuous recording over a period of a few days Žday and night. and rarely observed such a rise. The increases observed during the night period were comparable to those observed during the pre-exposures or in the new environment, i.e. q21 to q25 mm Hg from baseline. These values are similar to those reported in a telemetric study of circadian variations of
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P. CarriÕer Brain Research 858 (2000) 440–445
blood pressure in Wistar–Kyoto and Sprague–Dawley rats w7x. The maximum MAP value obtained with fear to context was 105 q 35 s 140 mm Hg. Interestingly, despite different baseline values and different changes from baseline, the same maximum is obtained with conditioned fear to a discrete stimulus: 122 q 15 s 137 mm Hg w9x, 117 q 21 s 138 mm Hg w5x and 129 q 10 s 139 mm Hg w4x. Thus, the magnitude of the pressor response is approximately the same for both forms of conditioned fear Ž137–140 mm Hg.. There is a marked difference, however, in the duration of the response. In the case of a discrete stimulus, the response typically lasts for less than 10 s w4,5,9x, whereas with context, it remained above the 130 mm Hg mark for more than 10 min. This is a big difference. It shows that the physiological impact of the fear response is much greater with context than with a discrete stimulus. Fear to context is a very potent psychological stressor. Still, the pressor effect was not maintained throughout the entire re-exposure. It peaked in the first 5 min and then gradually declined to more moderate levels. The same observation was made for freezing. This decline in MAP and freezing could be due to the fact that the conditioning to context was perhaps not optimal Ži.e. the absence of the first shock reduced the anticipation of the subsequent ones.. Alternatively, it may be that the fear response cannot stay up for more than 5 min and then habituates. However, recent experiments conducted in the laboratory show that MAP and freezing will stay at peak level for the entire 30 min of re-exposure if one or two more conditioning sessions are given and if the pre-exposures are reduced to 5 min Žinstead of 30 min.. Thus, the fear response does not habituate even over long periods of time. The decline in MAP and freezing is more likely to have been due to weak conditioning. Surprisingly, the MAP responses of the first and second test re-exposures were not different from each other. This is surprising because the level of conditioning and the amount of freezing were clearly different between the first and second test re-exposures. The problem here may be that the MAP response of the first test re-exposure was due in part to the increased in activity that gradually replaced freezing as the level of fear dropped Žthe effect started after the fifth min and peaked between the 10th and 20th min.. Since activity also causes MAP to rise Žas shown by the pre-exposures., it is likely that the MAP response of the first test re-exposure was higher than should have been. Unfortunately, this cannot be verified unless the animal remains immobile. Thus, MAP is high when freezing is high, but the relationship between MAP and freezing cannot be clearly established because of the confounding effect of activity on MAP. Another important finding of this study is that HR did not change in the same direction as MAP during conditioned fear. Thus, when fear was greatest, i.e. when MAP and freezing were at their peak in the beginning of the test
re-exposures, HR remained low, only marginally higher than baseline Žthere was no statistically significant difference until the 4th min of re-exposure for the first test re-exposure and until the 13th min for the second test re-exposure, Games–Howell, p - 0.05.. Then, as freezing and MAP declined, HR gradually rose. Most importantly, comparison of the first and second test re-exposures shows that the better the conditioning and the longer the freezing, the slower the rise in HR. Thus, increasing the level of conditioned fear appears to delay and reduce the rise in HR, and it seems that this decelerative effect is coupled to the freezing immobility. In contrast, increased activity and movement was always associated with an acceleration in HR. This was observed not only during the pre-exposures and the exposure to the new environment, but also in the first pre-exposure when activity returned after the 5th min. Thus activity is coupled with an accelerative effect on the heart, the opposite of freezing. These observations are in good agreement with a recent report by Nijsen et al. w8x who showed that the HR response during conditioned fear to context is the result of co-activation of the parasympathetic and sympatheticrsympathoadrenal systems, whereas the HR response to novelty and exploratory activity is mainly the result of sympathetic activation. It was also noticed in this report that the decelerative parasympathetic effect was associated with freezing immobility w8x. Thus, the slow rise in HR observed in the present study almost certainly corresponds to a decelerative parasympathetic effect superimposed on an accelerative sympatheticrsympathoadrenal effect. Interestingly, Iwata and Ledoux w5x made similar observations with conditioned fear to a discrete stimulus. The origin of this effect is unclear: it could be a direct central effect of fear, a baroreceptor mediated reflex secondary to the rise in MAP, or an indirect effect secondary to the freezing immobility. In any case, it is important to note that this presumed parasympathetic activation disappears as soon as the animal moves. Its role could be to act as a brake on the heart, holding back the accelerative effect of the background sympathetic activation, only to release it in the eventuality of a sudden somatomotor response Že.g. an escape response to the footshock.. In conclusion, this study shows for the first time that freezing evoked by conditioned fear to context is associated with a marked and sustained rise in blood pressure, a change that is of major physiological significance. It shows also that freezing is coupled with a decelerative effect on the heart. Finally, fear to context appears to be a strong stressor. Its physiological impact is likely to be of much greater significance than that of fear to a discrete stimulus.
Acknowledgements This work was supported by research grants from the National Heart Foundation of Australia, the National Health
P. CarriÕer Brain Research 858 (2000) 440–445
and Medical Research Council of Australia and the Merck Sharp and Dohme Foundation. I wish to thank Prof F. Westbrook for his help in the initial purchase of the radio-telemetry equipment and Dr. Michael Morgan for his useful comments on the manuscript.
References w1x E.A. Antoniadis, R.J. McDonald, Discriminative fear conditioning to context expressed by multiple measures of fear in the rat, Behav. Brain Res. 101 Ž1999. 1–13. w2x R.J. Blanchard, D.C. Blanchard, Crouching as an index of fear, J. Comp. Physiol. Psychol. 67 Ž1969. 370–375. w3x M.S. Fanselow, Conditioned and unconditional components of postshock freezing, Pav. J. Biol. Sci. 15 Ž1980. 177–182.
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w4x F.J. Helmstetter, S.A. Tershner, Lesions of the periaqueductal gray and rostral ventromedial medulla disrupt antinociceptive but not cardiovascular aversive conditional responses, J. Neurosci. 14 Ž1994. 7099–7108. w5x J. Iwata, J.E. Ledoux, Dissociation of associative and non associative concomitants of classical conditioning in the freely behaving rat, Behav. Neurosci. 102 Ž1988. 66–76. w6x J.E. LeDoux, J. Iwata, P. Cicchetti, D.J. Reis, Different projections of the central amygdaloid nucleus mediate autonomic and behavioral correlates of conditioned fear, J. Neurosci. 8 Ž1988. 2517–2529. w7x B. Lemmer, A. Mattes, M. Bohm, D. Ganten, Circadian blood ¨ pressure variation in transgenic hypertensive rats, Hypertension 22 Ž1993. 97–101. w8x M.J.M.A. Nijsen, G. Croiset, M. Diamant, R. Stam, D. Delsing, D. de Wied, V.M. Wiegant, Conditioned fear-induced tachycardia in the rat; vagal involvement, Europ. J. Pharmacol. 350 Ž1998. 211–222. w9x A. Sakaguchi, J.E. LeDoux, D.J. Reis, Sympathetic nerves and adrenal medulla: contributions to cardiovascular-conditioned emotional responses in spontaneously hypertensive rats, Hypertension 5 Ž1983. 728–738.
Interactive report
Conditioned fear to environmental context: cardiovascular and behavioral components in the rat 1 Pascal Carrive
)
School of Anatomy, UniÕersity of New South Wales, Sydney, NSW 2052, Australia Accepted 19 January 2000
Abstract This study compares the time course of the cardiovascular changes Žmean arterial blood pressure, heart rate. and behavioral changes Žfreezing, rearing, grooming and activity. evoked by 30 min long exposures to a footshock chamber before and after conditioning with footshocks. The main finding is that the conditioned fear evoked by re-exposure to the footshock chamber after conditioning is associated with a prolonged freezing response, a marked rise in mean arterial pressure Žq35 mm Hg above a resting baseline of 105 mm Hg. and a delayed rise in heart rate. The pattern of behavioral and cardiovascular changes is the same as with conditioned fear to a discrete stimulus but the effect is a lot longer. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Conditioned fear; Blood pressure; Heart rate; Radio-telemetry; Freezing; Exploratory behavior
1. Introduction Conditioned fear to context, or contextual conditioned fear, is the fear evoked by re-exposure to an environment that has previously been paired with an aversive or unpleasant stimulus. This is observed, for example, when a rat is re-exposed to a footshock chamber in which it has previously received electric footshocks. Traditionally, the fear evoking stimulus — or conditioning stimulus — is a discrete stimulus such as a light or tone. The conditioning stimulus is initially neutral. It acquires its aversive properties through repetitive pairing with the unpleasant footshocks. In the case of contextual fear, there is no light or tone; here, the conditioning stimulus is the environmental context, that is, the footshock chamber itself w3x. Conditioning to context is best when the footshocks are unsignaled and inescapable. The behavioral response of conditioned fear has been well described w1–3,6x. It is characterized by an immobile and tense posture known as freezing. Qualitatively, the freezing response is the same for contextual conditioned fear and conditioned fear to a discrete stimulus. There is, however, a marked difference in the duration of the two
1
Published on the World Wide Web on 1 February 2000. Tel.: q61-2-9385-2467; Fax: q61-2-9313-6252; [email protected] )
E-mail:
freezing responses. Thus, the duration of the conditioned response to a discrete stimulus such as a tone is short lived Žless than 2 min. w6x, whereas the conditioned response to context can last for much longer, e.g. 8 min w3x or 10 min w8x. The cardiovascular component of conditioned fear has been described also but only for the short responses evoked by a discrete stimulus Žtone. w4,5,9x. It consists of a transient Žless than 10 s. increase in mean arterial pressure and heart rate. Little is known about the cardiovascular changes that accompany the long freezing responses evoked by contextual fear. A recent paper by Nijsen et al. w8x shows that it is associated with an increase in heart rate and a release of circulating catecholamines, but the blood pressure component of the response has not yet been investigated. The aim of this study is to describe the concurrent cardiovascular and behavioral changes that occur during the long conditioned fear responses to context. To avoid interference between the recording of cardiovascular parameters and the behavioral response, blood pressure was recorded in freely moving rats with radio-telemetric probes. This also allowed for simultaneous recording of cardiovascular and behavioral changes. The main idea behind this investigation is that the longer response of conditioned fear to context may provide a better model for studying the cardiovascular readjustments that occur during anxious states and semi-chronic forms of psychological stress.
0006-8993r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 0 0 . 0 2 0 2 9 - 1
P. CarriÕer Brain Research 858 (2000) 440–445
2. Material and methods The subjects were 6 experimentally naive male Wistar rats Ž300–500 g. obtained from the colony of specific pathogen free rats maintained by the University of New South Wales. All experiments were approved by the Animal ethics committee of the University of New South Wales and conformed to the rules and guidelines on animal experimentation in Australia. The colony room was maintained at a 12-h lightrdark cycle and the experiments were conducted during the light phase of the cycle. Mean arterial pressure, heart rate and activity Žbody movements. were recorded by radio-telemetry. The telemetric probes ŽPA-C40, Data Sciences International. were implanted in the peritoneal cavity under anesthesia with a mixture of ketamine Ž100 mgrkg. and xylazine Ž50 mgrkg. injected intraperitoneally. Briefly, a midline incision was made in the abdomen and the descending aorta was exposed at the level of the iliac bifurcation. The artery was punctured at this level and the tip of the catheter Ž1 cm long. was inserted. The puncture was then glued with the catheter in place and the probe stitched to the abdominal wall while closing the midline incision. The animals were given antibiotics and moved to individual plastic home boxes Ž65 = 40 = 22 cm. in which they remained until the end of the experiment. During the recovery period Ž1 week. the animals were handled every day to habituate to the experimenter. Pre-conditioning, conditioning and testing were done in footshock chambers Ž23 = 21 = 20 cm. made of clear Perspex walls on two sides with a grid floor composed of 18 stainless steel rods Ž2 mm in diameter., spaced 1.5 cm apart and wired to a shock generator ŽGrason Stadler.. The chambers were cleaned before and after use with 0.05% acetic acid. The experiment started with 2 pre-exposures of 30 min to the shock chamber on Days 1 and 2. No shock was given during the pre-exposures. Day 3 was a rest day. The first conditioning session was done on Day 4: it consisted of a 30-min long exposure to the footshock chamber with 3 electric footshocks Ž1 mA, 1 s. delivered at t s 5, 15 and 25 min. Four h later, the animals were re-exposed to the footshock chamber for 30 min; no shock was delivered: it was the first test re-exposure for the conditioned response. Day 5 was similar to Day 4: animals had a second conditioning session and 4 h later, the second test re-exposure. Day 6 was a rest day. Finally, on Day 7, the rats were exposed for 30 min to a new environment, a plastic box similar to the home box but with transparent walls and clean bedding. On each of these days the general procedure was the same: in the morning, the animals were transferred in their home box from the colony room to the telemetry room and their probes were turned on. After a few hours of baseline recording, they were gently taken out of their home box and placed in the shock chamber or the new environment. Thirty min later the animals were returned to their home box. They stayed in the room for at
441
least another 4 h and were either returned to the colony room or re-exposed to the shock chamber a second time ŽDay 4 and Day 5.. There were six parameters measured simultaneously: two cardiovascular Žmean arterial pressure, heart rate. and four behavioral Žactivity, freezing, rearing and grooming.. Mean arterial pressure ŽMAP. was extracted from the pulsatile blood pressure signal and sampled for 3 s every 10 s with the DataQuest IV software ŽData Sciences International. running on a PC computer. Heart rate ŽHR. was calculated manually by counting the number of beats in 3 s blocks of the pulsatile trace Ždisplayed on the screen of the computer.. It was sampled regularly every min during the test exposures but irregularly before and after. Activity was a continuous measure of body movements, which the software automatically extracted from changes in orientation of the radio-telemetric probe. In addition to these 3 measures provided directly or indirectly by the radio-telemetric probes, an experimenter sitting in the telemetry room recorded the time spent freezing, the time spent grooming and the number of rearings during exposures. Freezing was defined as the complete absence of movement while the animal assumed a characteristic tense posture. Rearing was defined as rearing along the walls of the footshock chamber or the new environment box. All measures were averaged ŽMAP, HR, activity. or cumulated Žrearing, grooming, freezing. over 1 min periods. Data were analyzed using a 2 way ANOVA with one repeated factor Žtime.. The five different types of exposure were treated as between-subjects factors. Post-hoc analysis was carried out using the Games–Howell procedure for comparing the effects of types of exposure over the entire response and the Tukey–Kramer procedure for comparing the effects of types of exposure for each minute of the re-exposure. The analysis was done with Statview 5 ŽSAS institute..
3. Results Fig. 1 shows the averaged minute to minute changes in HR, MAP, freezing, rearing, grooming and activity evoked by the two pre-exposures before conditioning, the two test re-exposures after conditioning and finally, the exposure to a new environment. As can be seen, baseline MAP and HR were stable and did not change throughout the 7 days. Average readings taken around the 5th min before exposures, when the animals were at rest in their home box, were: MAP, 105 " 1 mm Hg ŽMean " SEM.; HR, 357 " 7 bpm. In contrast, there were clear differences in the cardiovascular and behavioral responses between the different types of exposure. A repeated measure ANOVA on the 30 min of testing revealed a significant main effect of the type of exposure for all 6 parameters ŽMAP, F Ž4,25. s 10.56, p - 0.0001; HR, F Ž4,25. s 4.31, p s 0.008; activity, F Ž4,25. s 6.84, p - 0.0007; freezing, F Ž4,25. s 29.63, p
442
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Fig. 1. Time course of the changes in heart rate, mean arterial pressure, freezing, rearing, grooming and activity evoked by the 2 pre-exposures, the 2 test re-exposures and finally the exposure to the new environment. The first and second pre-exposures were done in the footshock chamber before conditioning. The first and second test re-exposures were also done in the footshock chamber but after conditioning Ž4 h after the first and second conditioning session, respectively.. Exposure to the new environment was done last, after conditioning. Mean " SEM, n s 6 rats.
P. CarriÕer Brain Research 858 (2000) 440–445
- 0.0001; rearing, F Ž4,23. s 4.71, p - 0.0063; grooming, F Ž4,23. s 8.78, p - 0.0002.. There was also a significant main effect for time and a significant interaction between time and type of exposure for all 6 parameters Ž F Ž29, 725. ) 2.5, p - 0.0001 and F Ž116, 725. ) 1.5, p 0.0005, respectively.. Usually, it was during the first half of the responses Ž0–15 min. that the differences were most obvious. The two pre-exposures before conditioning evoked comparable levels of activity and rearing as the animals actively explored the footshock chamber. This activity gradually diminished over time and was marked by a peak in grooming midway through the re-exposure. There was no freezing. The exploratory activity was associated with a clear rise in MAP Žmaximum 128 " 2 mm Hg and 122 " 1 mm Hg, for first and second pre-exposure respectively, representing a q23 and q17 mm Hg increase from baseline.. There was also a rapid rise in HR Žmaximum 493 " 7 bpm and 469 " 9 bpm, respectively, i.e. a maximum of q136 and q112 bpm increase from baseline.. The two responses were very similar behaviorally, but the cardiovascular response appeared weaker the second time. Games–Howell post-hoc analysis revealed a statistically significant difference for MAP and HR between the two conditions Ž p - 0.05.. In contrast to the pre-exposures, the two test re-exposures done after conditioning were characterized by a reduction in activity, rearing and grooming. Behavioral activity was replaced by freezing. In the first test re-exposure, the effect was observed mainly during the first 10 min. In the second test re-exposure the effect extended over the entire 30 min period with almost 100% freezing in the first 10 min. Both conditioned fear responses were associated with a marked rise in MAP which peaked at the 4th min Ž137 " 3 mm Hg and 140 " 4 mm Hg, for first and second re-exposure respectively, representing a q32 and q35 mm Hg increase from baseline.. Moreover, this increase was q15 and q18 mm Hg above the second pre-exposure. The HR response was completely different: it was not augmented but reduced compared to the pre-exposures. There was no initial rapid increase in HR; instead, the rise in HR was late and slow. The effect was particularly obvious in the second test re-exposure, suggesting a relationship between the slowness of the rise in HR and the level of conditioning. A close examination of the time course of the response also suggests a relationship between HR, activity and freezing. For example, the response to the first test re-exposure shows that HR stayed low as long as the animal froze and remained immobile, but when activity resumed Žafter the 5th min., HR gradually rose to levels comparable to the pre-exposures. Thus, the longer and more intense the freezing immobility, the slower the rise in HR. Post-hoc analysis showed that both HR and freezing were significantly increased on the second test re-exposure compared to the first one ŽGames–Howell, p - 0.05.. The difference was statistically significant from the 9th to the
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20th min for HR and from the 9th to the 28th min for Freezing ŽTukey–Kramer, p - 0.05.. Interestingly, the MAP response was the same for both test-re-exposures. Nevertheless, the effect of conditioning on the behavioral and cardiovascular response to the footshock chamber were very clear: when the two test re-exposures were compared to the second pre-exposure before conditioning, post-hoc analysis revealed statistically significant differences for all 6 parameters ŽGames–Howell, p - 0.05.. For MAP and HR, the difference between the second pre-exposure and the second test re-exposure was statistically significant between the 2nd and the 12th min ŽTukey– Kramer, p - 0.05.. The conditioned animals were finally tested in a new environment. The response to this different context resembled in many ways the response observed during the pre-exposures before conditioning. Although grooming was reduced and there was some freezing for the first 20 min, there was as much activity and rearing as during the pre-exposures. The changes in MAP and HR were also the same as with the pre-exposures Žmaximum of 127 " 2 mm Hg and 463 " 22 bpm, representing a q22 mm Hg and a q106 bpm increase from baseline., and the same fast rise in HR was observed. There was no statistically significant difference for MAP, HR and activity between the new environment and the second pre-exposure. In contrast, there were statistically significant differences for all 6 parameters between the new environment and the second test re-exposure ŽGames–Howell, p - 0.05.. For MAP and HR, the difference was statistically significant between the 2nd and the 12th min ŽTukey–Kramer, p - 0.05.. 4. Discussion The most important finding of this study is that conditioned fear to context evokes a marked rise in mean arterial pressure. The change we observed was q35 mm Hg above a resting baseline of 105 mm Hg. It was also at least 12 mm Hg greater than the change in MAP evoked by pre-exposure before conditioning or by the exposure to a new environment after conditioning. Further, it was associated with high levels of freezing immobility whereas in the pre-exposures and new exposure the animals were actively exploring their environment. Thus, this marked hypertensive response is clearly a component of the conditioned fear response to the aversive context of the footshock chamber. For a normotensive animal housed individually, a q35 mm Hg rise from baseline is a very significant physiological change. We have done continuous recording over a period of a few days Žday and night. and rarely observed such a rise. The increases observed during the night period were comparable to those observed during the pre-exposures or in the new environment, i.e. q21 to q25 mm Hg from baseline. These values are similar to those reported in a telemetric study of circadian variations of
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blood pressure in Wistar–Kyoto and Sprague–Dawley rats w7x. The maximum MAP value obtained with fear to context was 105 q 35 s 140 mm Hg. Interestingly, despite different baseline values and different changes from baseline, the same maximum is obtained with conditioned fear to a discrete stimulus: 122 q 15 s 137 mm Hg w9x, 117 q 21 s 138 mm Hg w5x and 129 q 10 s 139 mm Hg w4x. Thus, the magnitude of the pressor response is approximately the same for both forms of conditioned fear Ž137–140 mm Hg.. There is a marked difference, however, in the duration of the response. In the case of a discrete stimulus, the response typically lasts for less than 10 s w4,5,9x, whereas with context, it remained above the 130 mm Hg mark for more than 10 min. This is a big difference. It shows that the physiological impact of the fear response is much greater with context than with a discrete stimulus. Fear to context is a very potent psychological stressor. Still, the pressor effect was not maintained throughout the entire re-exposure. It peaked in the first 5 min and then gradually declined to more moderate levels. The same observation was made for freezing. This decline in MAP and freezing could be due to the fact that the conditioning to context was perhaps not optimal Ži.e. the absence of the first shock reduced the anticipation of the subsequent ones.. Alternatively, it may be that the fear response cannot stay up for more than 5 min and then habituates. However, recent experiments conducted in the laboratory show that MAP and freezing will stay at peak level for the entire 30 min of re-exposure if one or two more conditioning sessions are given and if the pre-exposures are reduced to 5 min Žinstead of 30 min.. Thus, the fear response does not habituate even over long periods of time. The decline in MAP and freezing is more likely to have been due to weak conditioning. Surprisingly, the MAP responses of the first and second test re-exposures were not different from each other. This is surprising because the level of conditioning and the amount of freezing were clearly different between the first and second test re-exposures. The problem here may be that the MAP response of the first test re-exposure was due in part to the increased in activity that gradually replaced freezing as the level of fear dropped Žthe effect started after the fifth min and peaked between the 10th and 20th min.. Since activity also causes MAP to rise Žas shown by the pre-exposures., it is likely that the MAP response of the first test re-exposure was higher than should have been. Unfortunately, this cannot be verified unless the animal remains immobile. Thus, MAP is high when freezing is high, but the relationship between MAP and freezing cannot be clearly established because of the confounding effect of activity on MAP. Another important finding of this study is that HR did not change in the same direction as MAP during conditioned fear. Thus, when fear was greatest, i.e. when MAP and freezing were at their peak in the beginning of the test
re-exposures, HR remained low, only marginally higher than baseline Žthere was no statistically significant difference until the 4th min of re-exposure for the first test re-exposure and until the 13th min for the second test re-exposure, Games–Howell, p - 0.05.. Then, as freezing and MAP declined, HR gradually rose. Most importantly, comparison of the first and second test re-exposures shows that the better the conditioning and the longer the freezing, the slower the rise in HR. Thus, increasing the level of conditioned fear appears to delay and reduce the rise in HR, and it seems that this decelerative effect is coupled to the freezing immobility. In contrast, increased activity and movement was always associated with an acceleration in HR. This was observed not only during the pre-exposures and the exposure to the new environment, but also in the first pre-exposure when activity returned after the 5th min. Thus activity is coupled with an accelerative effect on the heart, the opposite of freezing. These observations are in good agreement with a recent report by Nijsen et al. w8x who showed that the HR response during conditioned fear to context is the result of co-activation of the parasympathetic and sympatheticrsympathoadrenal systems, whereas the HR response to novelty and exploratory activity is mainly the result of sympathetic activation. It was also noticed in this report that the decelerative parasympathetic effect was associated with freezing immobility w8x. Thus, the slow rise in HR observed in the present study almost certainly corresponds to a decelerative parasympathetic effect superimposed on an accelerative sympatheticrsympathoadrenal effect. Interestingly, Iwata and Ledoux w5x made similar observations with conditioned fear to a discrete stimulus. The origin of this effect is unclear: it could be a direct central effect of fear, a baroreceptor mediated reflex secondary to the rise in MAP, or an indirect effect secondary to the freezing immobility. In any case, it is important to note that this presumed parasympathetic activation disappears as soon as the animal moves. Its role could be to act as a brake on the heart, holding back the accelerative effect of the background sympathetic activation, only to release it in the eventuality of a sudden somatomotor response Že.g. an escape response to the footshock.. In conclusion, this study shows for the first time that freezing evoked by conditioned fear to context is associated with a marked and sustained rise in blood pressure, a change that is of major physiological significance. It shows also that freezing is coupled with a decelerative effect on the heart. Finally, fear to context appears to be a strong stressor. Its physiological impact is likely to be of much greater significance than that of fear to a discrete stimulus.
Acknowledgements This work was supported by research grants from the National Heart Foundation of Australia, the National Health
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and Medical Research Council of Australia and the Merck Sharp and Dohme Foundation. I wish to thank Prof F. Westbrook for his help in the initial purchase of the radio-telemetry equipment and Dr. Michael Morgan for his useful comments on the manuscript.
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w4x F.J. Helmstetter, S.A. Tershner, Lesions of the periaqueductal gray and rostral ventromedial medulla disrupt antinociceptive but not cardiovascular aversive conditional responses, J. Neurosci. 14 Ž1994. 7099–7108. w5x J. Iwata, J.E. Ledoux, Dissociation of associative and non associative concomitants of classical conditioning in the freely behaving rat, Behav. Neurosci. 102 Ž1988. 66–76. w6x J.E. LeDoux, J. Iwata, P. Cicchetti, D.J. Reis, Different projections of the central amygdaloid nucleus mediate autonomic and behavioral correlates of conditioned fear, J. Neurosci. 8 Ž1988. 2517–2529. w7x B. Lemmer, A. Mattes, M. Bohm, D. Ganten, Circadian blood ¨ pressure variation in transgenic hypertensive rats, Hypertension 22 Ž1993. 97–101. w8x M.J.M.A. Nijsen, G. Croiset, M. Diamant, R. Stam, D. Delsing, D. de Wied, V.M. Wiegant, Conditioned fear-induced tachycardia in the rat; vagal involvement, Europ. J. Pharmacol. 350 Ž1998. 211–222. w9x A. Sakaguchi, J.E. LeDoux, D.J. Reis, Sympathetic nerves and adrenal medulla: contributions to cardiovascular-conditioned emotional responses in spontaneously hypertensive rats, Hypertension 5 Ž1983. 728–738.