- Email: [email protected]
Cardiovascular and behavioural components of conditioned fear to context after ganglionic and α-adrenergic blockade
Autonomic Neuroscience: Basic and Clinical 98 (2002) 90 – 93 www.elsevier.com/locate/autneu
Cardiovascular and behavioural components of conditioned fear to context after ganglionic and a-adrenergic blockade Pascal Carrive * Department of Anatomy, School of Medical Sciences, University of New South Wales, NSW 2052, Sydney, Australia
Abstract This study investigates the contribution of the peripheral nervous system to the cardiovascular component of long lasting (40 min) conditioned fear responses to context. The conditioned fear response evoked by reexposure to a footshock chamber was tested 10 min after intravenous injection of either the nicotinic ganglion blocker chlorisondamine (0.6 mg/kg) or the a-adrenoceptor antagonist phentolamine (10 mg/kg) in six rats implanted with telemetric probes. Compared to saline controls, chlorisondamine did not change the behavioural component of the response (freezing, ultrasonic vocalizations) but almost completely abolished its cardiovascular component (mean arterial pressure and heart rate). Phentolamine also abolished the pressor response but increased the cardiac response, and ultrasonic vocalizations were reduced by half. The results indicate that the long lasting pressor response of conditioned fear to context is sympathetically mediated like the much shorter pressor response of conditioned fear to a discrete stimulus. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Anxiety; Stress; Autonomic nervous system; Telemetry; Hypertension; Baroreflex
A normally innocuous environment or context will acquire aversive properties after it has been repetitively paired with an unpleasant or aversive stimulus. Subsequent reexposure to this same context then evokes a conditioned emotional response known as conditioned fear to context (Blanchard and Blanchard, 1969; Fendt and Fanselow, 1999). This can be obtained, for example, by reexposing a rat to the same box in which it has previously received electric footshocks. The fear of being shocked again is what produces the emotional response. The conditioned fear response to context is very similar to the better-known conditioned fear response to a discrete stimulus such as a light or a tone. Both evoke a characteristic freezing immobility and an increase in arterial blood pressure, together with other signs of autonomic activation such as defecation and urination (Antoniadis and McDonald, 1999; Carrive, 2000; Fanselow, 1980, for contextual fear, and Lawler et al., 1985; LeDoux et al., 1988; Sakaguchi et al., 1983, for fear to a discrete stimulus). There is, however, one important difference between the two types of fear: the duration of the response. Thus, while the conditioned fear response to a tone does not last for more than 1
*
Tel.: +61-2-9385-2467; fax: +61-2-9313-6252. E-mail address: [email protected] (P. Car r ive).
min (Sakaguchi et al., 1983), a conditioned fear response to context can be made to last for up to 40 min, depending on the duration of the conditioning sessions (Carrive, 2000). Because of its short duration, one would expect the pressor response of conditioned fear to a discrete stimulus to be sympathetically mediated. Indeed, it is abolished after combined chemosympathectomy and adrenal medullectomy or after intravenous injection of the a-adrenoceptor antagonist phentolamine (Sakaguchi et al., 1983). However, because the pressor response of contextual fear is much longer, its underlying mechanisms may not necessarily be as simple. The aim of this study was to test contextual fear after ganglionic or phentolamine blockade to see how much of its pressor response is of sympathetic origin. Blood pressure was recorded by radio-telemetry to avoid interference with the behavioural response that was also recorded at the same time. The subjects were six experimentally naive male Wistar rats (400 – 550 g) obtained from the colony of specific pathogen-free rats maintained by the University of New South Wales. They were housed in individual plastic home boxes (65 40 22 cm) during the whole duration of the experiment. All procedures 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.
1566-0702/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 1 5 6 6 - 0 7 0 2 ( 0 2 ) 0 0 0 3 9 - 5
P. Carrive / Autonomic Neuroscience: Basic and Clinical 98 (2002) 90–93
The rats were first anaesthetised with a mixture of ketamine and xylazine (100 and 50 mg/kg, i.p., respectively) and implanted intraperitoneally with radio-telemetric probes (PA-C40, Data Sciences International) as described previously (Carrive, 2000). Animals were then given 1 week to recover before conditioning started. Preconditioning, conditioning and testing were done in footshock chambers (23 21 20 cm) made of clear Perspex walls on two sides with a grid floor wired to a shock generator. The chambers were cleaned before and after use with 0.05% acetic acid. Preconditioning consisted of two 5min-long preexposures to the footshock chamber, done on consecutive days. It was followed by four conditioning shock sessions done on separate days over a period of 7 days. Each shock session consisted of a 40-min-long exposure to the footshock chamber during which four electric footshocks (1 mA, 1 s) were delivered at approximately t = 5, 15, 25 and 35 min. A 40-min-long reexposure with no shock delivery (mock test session) was also made between the third and fourth shock sessions. Conditioning and test sessions were conducted during the light phase of the cycle, and there was never more than one session per day. The animals were re-anaesthetized with halothane (2.5 – 3% in O2) 2 days before testing began, and a chronic intravenous catheter was placed in the inferior vena cava via the left external jugular vein. The catheter was passed subcutaneously, externalised at the back of the neck and sealed with a stainless steel obturator. The catheters were flushed everyday with heparinized saline. The two drugs tested were the nicotinic cholinoceptor antagonist chlorisondamine (Ecolid, CIBA-Geigy, 0.6 mg/ kg), which blocks nicotinic cholinoceptors at autonomic ganglia as well as adrenal medullae (McCarty and Kopin, 1979), and the a-adrenoceptor antagonist phentolamine mesylate (CIBA-Geigy, 10 mg/kg). The testing procedure was as follows. Animals were first transferred from the colony room to the telemetry room in their home box and the probes were turned on. After at least 2 h of baseline recording, the i.v. catheter was connected to a 1-ml syringe, and a 0.5-ml bolus injection was made which contained either saline or the drug dissolved in saline. The animal was then returned to its home cage and 10 min later was transferred to the footshock chamber for the conditioned fear test (no shock during the test). Later, after 40 min, the animal was returned to its home box, and a few hours later, to the colony room. Phentolamine and its saline control were tested first, the day after a reinforcing (fifth) shock session. Phentolamine was either tested the day before saline (n = 4) or the day after saline (n = 2). Chlorisondamine and its saline control were tested a few days later after a reinforcing (sixth) shock session. Chlorisondamine was always tested the day after saline to avoid any confounding long-term effects of chlorisondamine on the saline control. Five parameters were recorded: heart rate (HR), mean arterial pressure (MAP), activity, freezing and ultrasonic vocalizations. HR and MAP (both sampled every 20 s from
91
3-s time windows) and activity (a continuous measure of body movements) were derived from the probe signal and acquired automatically by the LabPro software before, during and after the test reexposure. Freezing (sampled every 2 s and defined as the complete absence of movement while the animal assumed a characteristic tense posture) and ultrasonic vocalizations (detected by an Ultrasonic bat detector [Mini-3, Ultrasound advice] tuned at 22 kHz) were acquired manually by an experimenter sitting in the telemetry room. All parameters were finally averaged (MAP, HR, activity) or cumulated (freezing, ultrasonic vocalisations) over 1-min periods. Fig. 1 shows the effect of chlorisondamine and phentolamine and their respective saline controls. As can be seen, the two saline responses were identical, indicating that the conditioned response had reached its maximum and was stable throughout the experiment. Initially, the animals were at rest in their home cage (MAP, 101 mm Hg; HR, 289 bpm), but the injection procedure evoked an increase in activity when the animals were returned to their home box. This was associated with an increase in MAP and HR ( + 22 mm Hg, + 124 bpm), which gradually decreased over the next 10 min (ending at 117 mm Hg and 375 bpm). Normally, all parameters would have returned to baseline within 30 min (personal observations). Reexposure to the footshock box then produced an immediate cessation of activity and a complete freezing immobility that was associated with the emission of ultrasonic vocalizations (on average one every 2 s). At the same time, there was a further increase in MAP ( + 13 from 117 mm Hg) and a drop in HR ( 56 from 375 bpm). Later, after 20 min, i.e., halfway through the reexposure and at a time when the arousing effect of the injection procedure would have disappeared, the animals were still freezing and vocalizing, and MAP was still elevated ( + 13 mm Hg). However, HR had gradually recovered and increased ( + 35 from 375 bpm) and was now stabilizing. Other signs of autonomic activity included exophthalmos, defecation (total of 4 – 8 faecal boli) and urination. Finally, return to the home box after the reexposure evoked an immediate increase in activity that appeared to last longer than the postinjection, pre-reexposure period in the home box. Within 60– 70 min, Activity, MAP and HR were back to baseline (data not shown). An almost identical behavioural response was observed after i.v. administration of the ganglion blocker chlorisondamine (0.6 mg/kg). Pre-reexposure and post-reexposure activities were the same, as were the immobility and ultrasonic vocalizations during the reexposure. Freezing was also strong although it did not appear as intense as with saline in the second half of the reexposure. In contrast, the cardiovascular response was clearly reduced. MAP fell to a new baseline (70 mm Hg) where it remained throughout the reexposure, except for a very short initial burst ( + 16 mm Hg, first 3 min). HR also fell to a new baseline (269 bpm), and here too, there was a sudden increase ( + 51 bpm) at the beginning of the reexposure. This sudden rise in HR then
92
P. Carrive / Autonomic Neuroscience: Basic and Clinical 98 (2002) 90–93
Fig. 1. Time course of the changes in heart rate, mean arterial pressure, freezing, ultrasonic vocalization and activity evoked by fear to context after intravenous injections of chlorisondamine (0.6 mg/kg), phentolamine (10 mg/kg) and their respective saline controls. Contextual fear was evoked by a 40-min reexposure to the same footshock box in which animals had previously been conditioned with electric footshocks. No shock was given during the test. Mean F S.E.M., n = 6 in each group.
gradually declined throughout the reexposure and 20 min later, i.e. halfway through the reexposure, HR was almost back to its new baseline. At this time in the saline controls, HR was close to its maximum. Finally, other signs of effective ganglionic blockade were observed such as ptosis
of the eyelids and absence of defecation and urination. There are good reasons to believe that the initial peaks in MAP and HR were due to incomplete ganglionic blockade at the time the reexposure started. Indeed, they were not observed in pilot experiments that used a higher dose of
P. Carrive / Autonomic Neuroscience: Basic and Clinical 98 (2002) 90–93
chlorisondamine (1 mg/kg, n = 2, data not shown) while they were stronger and lasted longer with a lower dose (0.25 mg/kg, n = 2 also pilot experiments). Although the 1-mg/kg dose was effective in completely blocking the cardiovascular response, it was considered too high for the present study because it produced side effects such as sneezing and itching/grooming which interfered with the freezing and vocalizations. Finally, it must be said that the 0.6 mg/kg is a relatively mild dose. Most studies using intravenous injections in the awake rat have doses ranging from 1 to 10 mg/ kg to achieve complete ganglionic blockade (McCarty and Kopin, 1979; Santajuliana et al., 1996). Administration of the a-adrenoceptor antagonist phentolamine (10 mg/kg) reduced MAP to the same level as chlorisondamine (75 mm Hg). A decrease ( 15 mm Hg) rather than an increase in MAP was then observed upon reexposure, but by the 20-min mark, MAP was at the same level as the new baseline. The initial decrease may have been due to a fear-related vasodilation mediated by intact beta-adrenergic receptors in the skeletal muscle vasculature. The slow increase in MAP observed throughout the reexposure may be attributed either to the gradual decline of this peripheral vasodilation or simply to the gradual extinction of the pharmacological effect of the drug on vasoconstrictor tone. The cardiac response was affected in the other direction, which is consistent with a baroreceptor reflex (secondary to the fall in MAP) mediated by intact beta-adrenergic receptors. From a new baseline value of 396 bpm, the reexposure evoked a further increase of + 50 mm Hg which then declined and stabilised ( + 22 mm Hg) to end at the same level as in the saline controls. At the 20-min mark, it was close to the saline controls, suggesting that at this stage, most of the cardiac acceleration was sympathetically mediated. However, this has to be verified with a beta-adrenergic antagonist. Perhaps the most important finding concerning the cardiac response after phentolamine was the complete absence of the initial bradycardia normally seen in saline controls. Since the main difference between the effects of phentolamine and saline was the MAP change, it is most likely that this drop in HR was a baroreceptor-mediated reflex secondary to the rise in blood pressure evoked by the fear response. As has been shown by others, this bradycardia is parasympathetically mediated (Iwata and LeDoux, 1988; Nijsen et al., 1998). However, it did not appear to be maintained throughout the entire reexposure since HR gradually increased after 10 min despite a maintained elevated MAP. All these changes were associated with the characteristic behavioural components of the fear response, i.e. freezing and ultrasonic vocalizations. Although freezing was less intense than with saline in the first half of the reexposure, the total amount of freezing was about the same (5% reduction only). Ultrasonic vocalizations, however, were reduced by 45%. In addition, activity was not as strong before and after the reexposure, and the animals were not as immobile during the reexposure (33% more activity). It is not known if the reduction in amplitude of the
93
behavioural response was due to a peripheral or central effect of the drug, but if it was the latter, it may be that the central drive on the cardiovascular component was also reduced. In conclusion, the results show that the cardiovascular component of conditioned fear to context is entirely mediated by the autonomic nervous system like the much shorter conditioned fear response to a discrete stimulus (Sakaguchi et al., 1983). The results also show (i) that it is possible to block the autonomic component of contextual fear without affecting its behavioural component, (ii) that the + 30 mm Hg increase in MAP is induced and maintained for up to 40 min by the sympathetic nervous system, and (iii) that the baroreflex is operating during the first 10 min of this fear response.
Acknowledgements I wish to thank Peter Walker for excellent technical assistance and Dr. Samuel Leman for useful comments on the manuscript. The study was supported by the National Heart Foundation and the National Health and Medical Research Council of Australia.
References Antoniadis, E.A., McDonald, R.J., 1999. Discriminative fear conditioning to context expressed by multiple measures of fear in the rat. Behav. Brain Res. 101, 1 – 13. Blanchard, R.J., Blanchard, D.C., 1969. Crouching as an index of fear. J. Comp. Physiol. Psychol. 67, 370 – 375. Carrive, P., 2000. Conditioned fear to environmental context: cardiovascular and behavioral components in the rat. Brain Res. 858, 440 – 445. Fanselow, M.S., 1980. Conditioned and unconditional components of postshock freezing. Pavlovian J. Biol. Sci. 15, 177 – 182. Fendt, M., Fanselow, M.S., 1999. The neuroanatomical and neurochemical basis of conditioned fear. Neurosci. Biobehav. Rev. 23, 743 – 760. Iwata, J., LeDoux, J.E., 1988. Dissociation of associative and non-associative concomitants of classical conditioning in the freely behaving rat. Behav. Neurosci. 102, 66 – 76. Lawler, J.E., Cox, R.H., Hubbard, J.W., Mitchell, V.P., Barker, G.F., Trainor, W.P., Sanders, B.J., 1985. Blood pressure and heart rate responses to environmental stress in the spontaneously hypertensive rat. Physiol. Behav. 34, 973 – 976. LeDoux, J.E., Iwata, J., Cicchetti, P., Reis, D.J., 1988. Different projections of the central amygdaloid nucleus mediate autonomic and behavioral correlates of conditioned fear. J. Neurosci. 8, 2517 – 2529. McCarty, R., Kopin, I.J., 1979. Stress-induced alterations in plasma catecholamines and behavior of rats: effects of chlorisondamine and bretylium. Behav. Neural Biol. 27, 249 – 265. Nijsen, M.J., Croiset, G., Diamant, M., Stam, R., Delsing, D., de Wied, D., Wiegant, V.M., 1998. Conditioned fear-induced tachycardia in the rat: vagal involvement. Eur. J. Pharmacol. 350, 211 – 222. Sakaguchi, A., LeDoux, J.E., Reis, D.J., 1983. Sympathetic nerves and adrenal medulla: contributions to cardiovascular-conditioned emotional responses in spontaneously hypertensive rats. Hypertension 5, 728 – 738. Santajuliana, D., Hornfeldt, B.J., Osborn, J.W., 1996. Use of ganglionic blockers to assess neurogenic pressor activity in conscious rats. J. Pharmacol. Toxicol. Methods 35, 45 – 54.
Cardiovascular and behavioural components of conditioned fear to context after ganglionic and a-adrenergic blockade Pascal Carrive * Department of Anatomy, School of Medical Sciences, University of New South Wales, NSW 2052, Sydney, Australia
Abstract This study investigates the contribution of the peripheral nervous system to the cardiovascular component of long lasting (40 min) conditioned fear responses to context. The conditioned fear response evoked by reexposure to a footshock chamber was tested 10 min after intravenous injection of either the nicotinic ganglion blocker chlorisondamine (0.6 mg/kg) or the a-adrenoceptor antagonist phentolamine (10 mg/kg) in six rats implanted with telemetric probes. Compared to saline controls, chlorisondamine did not change the behavioural component of the response (freezing, ultrasonic vocalizations) but almost completely abolished its cardiovascular component (mean arterial pressure and heart rate). Phentolamine also abolished the pressor response but increased the cardiac response, and ultrasonic vocalizations were reduced by half. The results indicate that the long lasting pressor response of conditioned fear to context is sympathetically mediated like the much shorter pressor response of conditioned fear to a discrete stimulus. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Anxiety; Stress; Autonomic nervous system; Telemetry; Hypertension; Baroreflex
A normally innocuous environment or context will acquire aversive properties after it has been repetitively paired with an unpleasant or aversive stimulus. Subsequent reexposure to this same context then evokes a conditioned emotional response known as conditioned fear to context (Blanchard and Blanchard, 1969; Fendt and Fanselow, 1999). This can be obtained, for example, by reexposing a rat to the same box in which it has previously received electric footshocks. The fear of being shocked again is what produces the emotional response. The conditioned fear response to context is very similar to the better-known conditioned fear response to a discrete stimulus such as a light or a tone. Both evoke a characteristic freezing immobility and an increase in arterial blood pressure, together with other signs of autonomic activation such as defecation and urination (Antoniadis and McDonald, 1999; Carrive, 2000; Fanselow, 1980, for contextual fear, and Lawler et al., 1985; LeDoux et al., 1988; Sakaguchi et al., 1983, for fear to a discrete stimulus). There is, however, one important difference between the two types of fear: the duration of the response. Thus, while the conditioned fear response to a tone does not last for more than 1
*
Tel.: +61-2-9385-2467; fax: +61-2-9313-6252. E-mail address: [email protected] (P. Car r ive).
min (Sakaguchi et al., 1983), a conditioned fear response to context can be made to last for up to 40 min, depending on the duration of the conditioning sessions (Carrive, 2000). Because of its short duration, one would expect the pressor response of conditioned fear to a discrete stimulus to be sympathetically mediated. Indeed, it is abolished after combined chemosympathectomy and adrenal medullectomy or after intravenous injection of the a-adrenoceptor antagonist phentolamine (Sakaguchi et al., 1983). However, because the pressor response of contextual fear is much longer, its underlying mechanisms may not necessarily be as simple. The aim of this study was to test contextual fear after ganglionic or phentolamine blockade to see how much of its pressor response is of sympathetic origin. Blood pressure was recorded by radio-telemetry to avoid interference with the behavioural response that was also recorded at the same time. The subjects were six experimentally naive male Wistar rats (400 – 550 g) obtained from the colony of specific pathogen-free rats maintained by the University of New South Wales. They were housed in individual plastic home boxes (65 40 22 cm) during the whole duration of the experiment. All procedures 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.
1566-0702/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 1 5 6 6 - 0 7 0 2 ( 0 2 ) 0 0 0 3 9 - 5
P. Carrive / Autonomic Neuroscience: Basic and Clinical 98 (2002) 90–93
The rats were first anaesthetised with a mixture of ketamine and xylazine (100 and 50 mg/kg, i.p., respectively) and implanted intraperitoneally with radio-telemetric probes (PA-C40, Data Sciences International) as described previously (Carrive, 2000). Animals were then given 1 week to recover before conditioning started. Preconditioning, conditioning and testing were done in footshock chambers (23 21 20 cm) made of clear Perspex walls on two sides with a grid floor wired to a shock generator. The chambers were cleaned before and after use with 0.05% acetic acid. Preconditioning consisted of two 5min-long preexposures to the footshock chamber, done on consecutive days. It was followed by four conditioning shock sessions done on separate days over a period of 7 days. Each shock session consisted of a 40-min-long exposure to the footshock chamber during which four electric footshocks (1 mA, 1 s) were delivered at approximately t = 5, 15, 25 and 35 min. A 40-min-long reexposure with no shock delivery (mock test session) was also made between the third and fourth shock sessions. Conditioning and test sessions were conducted during the light phase of the cycle, and there was never more than one session per day. The animals were re-anaesthetized with halothane (2.5 – 3% in O2) 2 days before testing began, and a chronic intravenous catheter was placed in the inferior vena cava via the left external jugular vein. The catheter was passed subcutaneously, externalised at the back of the neck and sealed with a stainless steel obturator. The catheters were flushed everyday with heparinized saline. The two drugs tested were the nicotinic cholinoceptor antagonist chlorisondamine (Ecolid, CIBA-Geigy, 0.6 mg/ kg), which blocks nicotinic cholinoceptors at autonomic ganglia as well as adrenal medullae (McCarty and Kopin, 1979), and the a-adrenoceptor antagonist phentolamine mesylate (CIBA-Geigy, 10 mg/kg). The testing procedure was as follows. Animals were first transferred from the colony room to the telemetry room in their home box and the probes were turned on. After at least 2 h of baseline recording, the i.v. catheter was connected to a 1-ml syringe, and a 0.5-ml bolus injection was made which contained either saline or the drug dissolved in saline. The animal was then returned to its home cage and 10 min later was transferred to the footshock chamber for the conditioned fear test (no shock during the test). Later, after 40 min, the animal was returned to its home box, and a few hours later, to the colony room. Phentolamine and its saline control were tested first, the day after a reinforcing (fifth) shock session. Phentolamine was either tested the day before saline (n = 4) or the day after saline (n = 2). Chlorisondamine and its saline control were tested a few days later after a reinforcing (sixth) shock session. Chlorisondamine was always tested the day after saline to avoid any confounding long-term effects of chlorisondamine on the saline control. Five parameters were recorded: heart rate (HR), mean arterial pressure (MAP), activity, freezing and ultrasonic vocalizations. HR and MAP (both sampled every 20 s from
91
3-s time windows) and activity (a continuous measure of body movements) were derived from the probe signal and acquired automatically by the LabPro software before, during and after the test reexposure. Freezing (sampled every 2 s and defined as the complete absence of movement while the animal assumed a characteristic tense posture) and ultrasonic vocalizations (detected by an Ultrasonic bat detector [Mini-3, Ultrasound advice] tuned at 22 kHz) were acquired manually by an experimenter sitting in the telemetry room. All parameters were finally averaged (MAP, HR, activity) or cumulated (freezing, ultrasonic vocalisations) over 1-min periods. Fig. 1 shows the effect of chlorisondamine and phentolamine and their respective saline controls. As can be seen, the two saline responses were identical, indicating that the conditioned response had reached its maximum and was stable throughout the experiment. Initially, the animals were at rest in their home cage (MAP, 101 mm Hg; HR, 289 bpm), but the injection procedure evoked an increase in activity when the animals were returned to their home box. This was associated with an increase in MAP and HR ( + 22 mm Hg, + 124 bpm), which gradually decreased over the next 10 min (ending at 117 mm Hg and 375 bpm). Normally, all parameters would have returned to baseline within 30 min (personal observations). Reexposure to the footshock box then produced an immediate cessation of activity and a complete freezing immobility that was associated with the emission of ultrasonic vocalizations (on average one every 2 s). At the same time, there was a further increase in MAP ( + 13 from 117 mm Hg) and a drop in HR ( 56 from 375 bpm). Later, after 20 min, i.e., halfway through the reexposure and at a time when the arousing effect of the injection procedure would have disappeared, the animals were still freezing and vocalizing, and MAP was still elevated ( + 13 mm Hg). However, HR had gradually recovered and increased ( + 35 from 375 bpm) and was now stabilizing. Other signs of autonomic activity included exophthalmos, defecation (total of 4 – 8 faecal boli) and urination. Finally, return to the home box after the reexposure evoked an immediate increase in activity that appeared to last longer than the postinjection, pre-reexposure period in the home box. Within 60– 70 min, Activity, MAP and HR were back to baseline (data not shown). An almost identical behavioural response was observed after i.v. administration of the ganglion blocker chlorisondamine (0.6 mg/kg). Pre-reexposure and post-reexposure activities were the same, as were the immobility and ultrasonic vocalizations during the reexposure. Freezing was also strong although it did not appear as intense as with saline in the second half of the reexposure. In contrast, the cardiovascular response was clearly reduced. MAP fell to a new baseline (70 mm Hg) where it remained throughout the reexposure, except for a very short initial burst ( + 16 mm Hg, first 3 min). HR also fell to a new baseline (269 bpm), and here too, there was a sudden increase ( + 51 bpm) at the beginning of the reexposure. This sudden rise in HR then
92
P. Carrive / Autonomic Neuroscience: Basic and Clinical 98 (2002) 90–93
Fig. 1. Time course of the changes in heart rate, mean arterial pressure, freezing, ultrasonic vocalization and activity evoked by fear to context after intravenous injections of chlorisondamine (0.6 mg/kg), phentolamine (10 mg/kg) and their respective saline controls. Contextual fear was evoked by a 40-min reexposure to the same footshock box in which animals had previously been conditioned with electric footshocks. No shock was given during the test. Mean F S.E.M., n = 6 in each group.
gradually declined throughout the reexposure and 20 min later, i.e. halfway through the reexposure, HR was almost back to its new baseline. At this time in the saline controls, HR was close to its maximum. Finally, other signs of effective ganglionic blockade were observed such as ptosis
of the eyelids and absence of defecation and urination. There are good reasons to believe that the initial peaks in MAP and HR were due to incomplete ganglionic blockade at the time the reexposure started. Indeed, they were not observed in pilot experiments that used a higher dose of
P. Carrive / Autonomic Neuroscience: Basic and Clinical 98 (2002) 90–93
chlorisondamine (1 mg/kg, n = 2, data not shown) while they were stronger and lasted longer with a lower dose (0.25 mg/kg, n = 2 also pilot experiments). Although the 1-mg/kg dose was effective in completely blocking the cardiovascular response, it was considered too high for the present study because it produced side effects such as sneezing and itching/grooming which interfered with the freezing and vocalizations. Finally, it must be said that the 0.6 mg/kg is a relatively mild dose. Most studies using intravenous injections in the awake rat have doses ranging from 1 to 10 mg/ kg to achieve complete ganglionic blockade (McCarty and Kopin, 1979; Santajuliana et al., 1996). Administration of the a-adrenoceptor antagonist phentolamine (10 mg/kg) reduced MAP to the same level as chlorisondamine (75 mm Hg). A decrease ( 15 mm Hg) rather than an increase in MAP was then observed upon reexposure, but by the 20-min mark, MAP was at the same level as the new baseline. The initial decrease may have been due to a fear-related vasodilation mediated by intact beta-adrenergic receptors in the skeletal muscle vasculature. The slow increase in MAP observed throughout the reexposure may be attributed either to the gradual decline of this peripheral vasodilation or simply to the gradual extinction of the pharmacological effect of the drug on vasoconstrictor tone. The cardiac response was affected in the other direction, which is consistent with a baroreceptor reflex (secondary to the fall in MAP) mediated by intact beta-adrenergic receptors. From a new baseline value of 396 bpm, the reexposure evoked a further increase of + 50 mm Hg which then declined and stabilised ( + 22 mm Hg) to end at the same level as in the saline controls. At the 20-min mark, it was close to the saline controls, suggesting that at this stage, most of the cardiac acceleration was sympathetically mediated. However, this has to be verified with a beta-adrenergic antagonist. Perhaps the most important finding concerning the cardiac response after phentolamine was the complete absence of the initial bradycardia normally seen in saline controls. Since the main difference between the effects of phentolamine and saline was the MAP change, it is most likely that this drop in HR was a baroreceptor-mediated reflex secondary to the rise in blood pressure evoked by the fear response. As has been shown by others, this bradycardia is parasympathetically mediated (Iwata and LeDoux, 1988; Nijsen et al., 1998). However, it did not appear to be maintained throughout the entire reexposure since HR gradually increased after 10 min despite a maintained elevated MAP. All these changes were associated with the characteristic behavioural components of the fear response, i.e. freezing and ultrasonic vocalizations. Although freezing was less intense than with saline in the first half of the reexposure, the total amount of freezing was about the same (5% reduction only). Ultrasonic vocalizations, however, were reduced by 45%. In addition, activity was not as strong before and after the reexposure, and the animals were not as immobile during the reexposure (33% more activity). It is not known if the reduction in amplitude of the
93
behavioural response was due to a peripheral or central effect of the drug, but if it was the latter, it may be that the central drive on the cardiovascular component was also reduced. In conclusion, the results show that the cardiovascular component of conditioned fear to context is entirely mediated by the autonomic nervous system like the much shorter conditioned fear response to a discrete stimulus (Sakaguchi et al., 1983). The results also show (i) that it is possible to block the autonomic component of contextual fear without affecting its behavioural component, (ii) that the + 30 mm Hg increase in MAP is induced and maintained for up to 40 min by the sympathetic nervous system, and (iii) that the baroreflex is operating during the first 10 min of this fear response.
Acknowledgements I wish to thank Peter Walker for excellent technical assistance and Dr. Samuel Leman for useful comments on the manuscript. The study was supported by the National Heart Foundation and the National Health and Medical Research Council of Australia.
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