0013-7227/90/1262-0921$02.00/0 Endocrinology Copyright© 1990 by The Endocrine Society
Vol. 126, No. 2 Printed in U.S.A.
Different Control of the AdrenocorticotropinCorticosterone Response and of Prolactin Secretion during Cold Stress, Anesthesia, Surgery, and Nicotine Injection in the Rat: Involvement of Capsaicin-Sensitive Sensory Neurons* JOSEF DONNERERf AND FRED LEMBECK Department of Experimental and Clinical Pharmacology, University of Graz, Universitatsplatz 4, A-8010 Graz, Austria
ABSTRACT. The release of ACTH, corticosterone, and PRL was compared in capsaicin-pretreated rats, which lack afferent C-fibers, and their controls under somatosensory (cold, surgery) and central (restraint) forms of stress. Cold stress induced the release of ACTH and consequently that of corticosterone in the controls, but not in the capsaicinpretreated rats. Intravenous injection of ACTHi.24 was equally effective in releasing corticosterone in both groups. Whereas PRL was not released in response to cold stress, restraint stress did induce the release of both ACTH and PRL, in the capsaicinpretreated as well as in the control group. Pentobarbital anesthesia alone elicited PRL, but no ACTH release. ACTH release was evoked by surgery under pentobarbital anesthesia but was abolished by capsaicin pretreatment. PRL levels were not further increased by surgery.
Nicotine in a small dose (5 ng intra-arterially) evoked stimulation of afferent C-fibers as observed on a depressor reflex. Intraperitoneal injection of nicotine (250 MgAg) caused a marked rise in plasma ACTH both in the capsaicin-pretreated conscious rats and in their controls, probably resulting from central stimulation as this effect was shown to be inhibited during pentobarbital anesthesia. A moderate rise of PRL by nicotine was seen in conscious rats. The stimuli used, regarded as experimental models of stress, show essential differences in their ability to evoke the release of ACTH, corticosterone, and PRL. Those stimuli which cause the release of ACTH and corticosterone via afferent C-fiber stimulation do not release PRL, whereas emotional and cognitive stress causes the release of both ACTH and PRL. (Endocrinology 126: 921-926, 1990)
T
forms of stress (8, 9). The necessity for a sensory information carried via capsaicin-sensitive afferents has been proposed indirectly for the regulation of the PRL secretion during the neuroendocrine copulatory response and lactation in the rat (10-12). But no information is available as to whether the secretion of PRL during different forms of stress is dependent on afferent C-fibers. In the present study the secretion of ACTH and PRL and their dependence upon the afferent C-fiber system in the rat is compared during emotional and somatosensory forms of stress. This should contribute to our knowledge of the function of PRL during stress. Furthermore, since the main effect of ACTH is to control corticosterone secretion from the adrenal cortex, we also wanted to see if the corticosterone secretion itself was dependent on the afferent C-fiber system.
HE IDENTIFICATION and endogenous regulation of pituitary stress hormones have already been the object of several decades of investigations. The purpose of the elevated secretion of the typical stress hormone ACTH, and consequently of corticosterone, is well understood in the light of the regulatory effect of corticosterone on inflammatory and immune responses (1, 2). Of PRL however—another proposed stress hormone—it is only known that it is released during certain stressful conditions (3, 4), but the relevance of this release is not yet understood (5). In addition to being regulated by the central nervous system (6), ACTH release can be evoked via excitation of afferent C-fibers (7), while a lack of afferent C-fibers, as in the capsaicin pretreated rat, is connected with an impaired ACTH release during certain somatosensory
Materials and Methods
Received July 26, 1989. *This work was supported by the Austrian Scientific Research Funds, Grant P6646, the Austrian National Bank (Grant 2811), and the Pain Research Commission of the Austrian Academy of Sciences. fTo whom requests for reprints should be addressed.
Animals Pregnant female Sprague-Dawley rats were obtained from Himberg, Austria, to provide neonatal rats. Two days after 921
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 00:23 For personal use only. No other uses without permission. . All rights reserved.
AFFERENT C-FIBERS AND STRESS RESPONSE
922
delivery of the litter the newborn rats were injected sc under ether anesthesia with either capsaicin (50 mg/kg) or an appropriate volume of the vehicle (0.9% NaCl containing 5% ethanol and 5% Tween 80). Rats were weaned when 4 weeks old and kept in groups of three in cages 42 X 22 X 15 cm with a 12-h light and dark cycle at a room temperature of 22 C. A standard laboratory diet and water were available ad libitum. Male animals were used for the experiments at the age of 8-12 weeks after the efficacy of the capsaicin-denervation had been confirmed by the wiping test (13). There was no difference in mean body weight between the capsaicin-pretreated and vehicle-pretreated rats (range: 250-350 g). On the 3 days preceding the experiment the rats were weighed and handled by the experimenter for adaptation. Experiments on conscious rats were done in the room in which the rats were housed or the animals were anesthetized in this room with pentobarbital (50 mg/kg ip) and then carried to the laboratory. Experimental protocol Rats were restrained by placement into a plastic cylinder of 6-cm diameter for 10 min. Exposure to cold stress was achieved by placing them in a lighted room at 6 C for various time periods. Immediately afterward the rats were decapitated and blood collected from the trunk in EDTA tubes. Colon temperature was determined with an electronic thermometer (Digimed, ETW, FRG) in a separate set of experiments. To test the integrity of adrenal cortical response in capsaicinpretreated rats, a dose-response study of the effect of iv injected ACTH was conducted in anesthetized rats. The biologically active synthetic tetracosactide ACTHi_24 analog was used (14). Rats were anesthetized with pentobarbital and ACTHi_24 in 0.3 ml saline or saline alone was injected into the tail vein. The rats were left undisturbed for 20 min and then decapitated and trunk blood collected for plasma corticosterone determination.
Endo • 1990 Vol 126 • No 2
RIA After centrifugation of the obtained blood samples, the plasma was pipetted off and stored at —60 C until the assays. For the RIA of ACTH a commercially available kit from Incstar (Stillwater, OK) was used. The concentration of PRL in plasma was determined by a specific RIA set up with the kit kindly provided by NIDDK using the iodination method of Lenox et al. (17) and the double antibody separation technique. The limit of detection of rat PRL was 0.2 ng/ml and the intra- and interassay coefficients of variation were 4 and 10%, respectively. Corticosterone in plasma was determined according to Woodward and Emery (18). Briefly, 1 ml of plasma was mixed with 150 ^1 of 0.3 M sodium hydroxide solution and corticosterone extracted with 5 ml diethylether-dichlormethane (60:40, vol/vol). The organic phase was washed once with 1 ml of water and evaporated at room temperature under nitrogen. The residue was redissolved in 300 n\ of methanol-water (55:45, vol/ vol). Two hundred microliters were injected onto a reversed phase column (Lichrosorb RP18, 7 nm, 125 x 4 mm), eluted with methanol-water (55:45, vol/vol) and corticosterone measured by UV detection (250 nm). Statistical analysis Data were analyzed by use of ANOVA and differences between treatments were determined by Scheffe's multiple comparisons test. Substances Sodium pentobarbital (Sanofi Sante Animali, Paris, France), ACTHi.24 (a gift from Ciba Geigy, Basel, Switzerland), nicotine-ditartrate (Sigma, St. Louis, MO), and capsaicin (Merck, Darmstadt, FRG) were used.
Results Surgical stress Under pentobarbital anesthesia the trachea was cannulated and a carotid artery and a jugular vein dissected free from the surrounding tissue and cannulated. Five minutes after this surgical procedure blood was collected for the determination of hormone levels. To investigate the proposed excitation of afferent C-fibers by nicotine (15), nicotine was injected intraarterially (ia) into the hind leg of anesthetized rats. A selective excitation of afferent C-fibers would be made evident by a reflex fall in blood pressure as it has been demonstrated for capsaicin (16). For this purpose 5 /xg of nicotine in 30 n\ of saline were injected retrogradely into the epigastric artery while the blood pressure was recorded from a carotid artery. The same amount of nicotine was also injected after the sciatic and the saphenous nerve had been cut or in capsaicin-denervated rats. For systemic application nicotine was dissolved in 0.3 ml of saline and injected in a dose of 250 Mg/kg into the tail vein of anesthetized animals or ip in awake animals. Rats were decapitated 5 min later.
In all of the following experimental series, the measured parameters were at the same time determined in capsaicin-pretreated rats and their controls. Corticosterone release by ACTH ACTHi_24, injected iv in pentobarbital-anesthetized rats, caused a dose-dependent increase in plasma corticosterone levels as measured after 20 min (Fig. 1). There was no significant difference in the response between controls and capsaicin-pretreated rats. Cold stress
When animals were exposed to a cold environment, the plasma ACTH and corticosterone levels reached a maximum at 30 min then dropped over the following 60 min period (Fig. 2). The release of ACTH and corticosterone as a response to cold stress was at its peak at 30 min 55 ± 4% and 66 ± 10%, respectively, lower in
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 00:23 For personal use only. No other uses without permission. . All rights reserved.
AFFERENT C-FIBERS AND STRESS RESPONSE ACTH 1 _ 24
923
300
Cold Stress
400r
200
3
300
200
« 100 o.
o O
100 30
Saline 0.5/ug/kg 2.5/ug/kg 25.0yug/kg FlG. 1. Plasma concentrations of corticosterone 20 min after the iv injection of saline or increasing doses of ACTHi_24 in anesthetized rats. Rats were pretreated as neonates either with capsaicin (hatched columns) or the capsaicin vehicle [open columns). Means ± SEM, n = 57.
capsaicin-pretreated rats. There was no secretion of PRL induced by the same cold exposure in both groups of animals (Fig. 3). Colon temperature was 37.5 ± 0.2 C (n = 10) in both groups of rats before cold exposure. After 30 min of cold exposure there was no change in colon temperature, but after 90 min of cold exposure it was lower in capsaicinpretreated rats (36.4 ± 0.2 C) than in vehicle-pretreated rats (37.2 ± 0.1 C, n = 10 for each group, P < 0.05). Both groups of rats exhibited thermogenic behavior (piloerection, shivering) from the beginning of the cold exposure as has been previously reported (8). Restraint stress Isolation and restricting the movements of the rats in a plastic cylinder induced a similar rise in ACTH and PRL secretion in controls and capsaicin-pretreated rats (Fig. 3). Likewise, the corticosterone levels increased from a basal level of 30-60 ng/ml in both groups to 570 ± 90 ng/ml in controls and to 450 ± 80 ng/ml in capsaicin-pretreated rats (n = 5 for each group). Pentobarbital anesthesia and surgery
Pentobarbital anesthesia did not raise ACTH levels noticeably as has been shown previously (7). Surprisingly, however, PRL levels increased 5-6 times in both groups of rats following pentobarbital anesthesia (Fig. 3). Whereas additional surgery induced a 6-fold increase in ACTH secretion in control rats, but not in capsaicinpretreated rats, PRL levels were not affected further (Fig. 3).
90
60
600 r
? 500
400
*
I 300 o I 200
100
Controls
30 min
60min
90 min
FIG. 2. Time course of plasma ACTH and corticosterone levels during cold exposure (6 C) of rats pretreated with vehicle (open circles and open columns) or capsaicin (filled circles and hatched columns) as neonates. Means ± SEM, n = 6-8. Significance of differences: *P < 0.05 compared to the control condition; OP < 0.05 compared to capsaicinpretreated rats.
Depressor reflex by nicotine
Injection of nicotine (5 ng, intra-arterially into the hind leg of rats) caused a fall in blood pressure which was absent after nerve section of the leg or following capsaicin pretreatment (Table 1). The same dose of nicotine injected into the systemic circulation (jugular vein) did not have any significant effect on the blood pressure (n = 3). ACTH and PRL release by nicotine Nicotine (250 ng/kg) injected ip in conscious rats caused a pronounced ACTH surge (7-8 times basal levels) in both groups. However, iv injection of the same dose of nicotine during anesthesia caused only a slight ACTH increase by 60% in control rats but no increase at all in capsaicin-pretreated rats, resulting in significant
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 00:23 For personal use only. No other uses without permission. . All rights reserved.
AFFERENT C-FIBERS AND STRESS RESPONSE
924 No stress
Cold stress
Restraint
800
i
Endo • 1990 Voll26«No2
CONSCIOUS
Pentobarbital Pentobarbital Anaesthesia Surgery •o
900
Saline
Nicotine
PENTOBARBITAL ANAESTHESIA Saline
Nicotine
600 E
1 600 x o
Vol. 126, No. 2 Printed in U.S.A.
Different Control of the AdrenocorticotropinCorticosterone Response and of Prolactin Secretion during Cold Stress, Anesthesia, Surgery, and Nicotine Injection in the Rat: Involvement of Capsaicin-Sensitive Sensory Neurons* JOSEF DONNERERf AND FRED LEMBECK Department of Experimental and Clinical Pharmacology, University of Graz, Universitatsplatz 4, A-8010 Graz, Austria
ABSTRACT. The release of ACTH, corticosterone, and PRL was compared in capsaicin-pretreated rats, which lack afferent C-fibers, and their controls under somatosensory (cold, surgery) and central (restraint) forms of stress. Cold stress induced the release of ACTH and consequently that of corticosterone in the controls, but not in the capsaicinpretreated rats. Intravenous injection of ACTHi.24 was equally effective in releasing corticosterone in both groups. Whereas PRL was not released in response to cold stress, restraint stress did induce the release of both ACTH and PRL, in the capsaicinpretreated as well as in the control group. Pentobarbital anesthesia alone elicited PRL, but no ACTH release. ACTH release was evoked by surgery under pentobarbital anesthesia but was abolished by capsaicin pretreatment. PRL levels were not further increased by surgery.
Nicotine in a small dose (5 ng intra-arterially) evoked stimulation of afferent C-fibers as observed on a depressor reflex. Intraperitoneal injection of nicotine (250 MgAg) caused a marked rise in plasma ACTH both in the capsaicin-pretreated conscious rats and in their controls, probably resulting from central stimulation as this effect was shown to be inhibited during pentobarbital anesthesia. A moderate rise of PRL by nicotine was seen in conscious rats. The stimuli used, regarded as experimental models of stress, show essential differences in their ability to evoke the release of ACTH, corticosterone, and PRL. Those stimuli which cause the release of ACTH and corticosterone via afferent C-fiber stimulation do not release PRL, whereas emotional and cognitive stress causes the release of both ACTH and PRL. (Endocrinology 126: 921-926, 1990)
T
forms of stress (8, 9). The necessity for a sensory information carried via capsaicin-sensitive afferents has been proposed indirectly for the regulation of the PRL secretion during the neuroendocrine copulatory response and lactation in the rat (10-12). But no information is available as to whether the secretion of PRL during different forms of stress is dependent on afferent C-fibers. In the present study the secretion of ACTH and PRL and their dependence upon the afferent C-fiber system in the rat is compared during emotional and somatosensory forms of stress. This should contribute to our knowledge of the function of PRL during stress. Furthermore, since the main effect of ACTH is to control corticosterone secretion from the adrenal cortex, we also wanted to see if the corticosterone secretion itself was dependent on the afferent C-fiber system.
HE IDENTIFICATION and endogenous regulation of pituitary stress hormones have already been the object of several decades of investigations. The purpose of the elevated secretion of the typical stress hormone ACTH, and consequently of corticosterone, is well understood in the light of the regulatory effect of corticosterone on inflammatory and immune responses (1, 2). Of PRL however—another proposed stress hormone—it is only known that it is released during certain stressful conditions (3, 4), but the relevance of this release is not yet understood (5). In addition to being regulated by the central nervous system (6), ACTH release can be evoked via excitation of afferent C-fibers (7), while a lack of afferent C-fibers, as in the capsaicin pretreated rat, is connected with an impaired ACTH release during certain somatosensory
Materials and Methods
Received July 26, 1989. *This work was supported by the Austrian Scientific Research Funds, Grant P6646, the Austrian National Bank (Grant 2811), and the Pain Research Commission of the Austrian Academy of Sciences. fTo whom requests for reprints should be addressed.
Animals Pregnant female Sprague-Dawley rats were obtained from Himberg, Austria, to provide neonatal rats. Two days after 921
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 00:23 For personal use only. No other uses without permission. . All rights reserved.
AFFERENT C-FIBERS AND STRESS RESPONSE
922
delivery of the litter the newborn rats were injected sc under ether anesthesia with either capsaicin (50 mg/kg) or an appropriate volume of the vehicle (0.9% NaCl containing 5% ethanol and 5% Tween 80). Rats were weaned when 4 weeks old and kept in groups of three in cages 42 X 22 X 15 cm with a 12-h light and dark cycle at a room temperature of 22 C. A standard laboratory diet and water were available ad libitum. Male animals were used for the experiments at the age of 8-12 weeks after the efficacy of the capsaicin-denervation had been confirmed by the wiping test (13). There was no difference in mean body weight between the capsaicin-pretreated and vehicle-pretreated rats (range: 250-350 g). On the 3 days preceding the experiment the rats were weighed and handled by the experimenter for adaptation. Experiments on conscious rats were done in the room in which the rats were housed or the animals were anesthetized in this room with pentobarbital (50 mg/kg ip) and then carried to the laboratory. Experimental protocol Rats were restrained by placement into a plastic cylinder of 6-cm diameter for 10 min. Exposure to cold stress was achieved by placing them in a lighted room at 6 C for various time periods. Immediately afterward the rats were decapitated and blood collected from the trunk in EDTA tubes. Colon temperature was determined with an electronic thermometer (Digimed, ETW, FRG) in a separate set of experiments. To test the integrity of adrenal cortical response in capsaicinpretreated rats, a dose-response study of the effect of iv injected ACTH was conducted in anesthetized rats. The biologically active synthetic tetracosactide ACTHi_24 analog was used (14). Rats were anesthetized with pentobarbital and ACTHi_24 in 0.3 ml saline or saline alone was injected into the tail vein. The rats were left undisturbed for 20 min and then decapitated and trunk blood collected for plasma corticosterone determination.
Endo • 1990 Vol 126 • No 2
RIA After centrifugation of the obtained blood samples, the plasma was pipetted off and stored at —60 C until the assays. For the RIA of ACTH a commercially available kit from Incstar (Stillwater, OK) was used. The concentration of PRL in plasma was determined by a specific RIA set up with the kit kindly provided by NIDDK using the iodination method of Lenox et al. (17) and the double antibody separation technique. The limit of detection of rat PRL was 0.2 ng/ml and the intra- and interassay coefficients of variation were 4 and 10%, respectively. Corticosterone in plasma was determined according to Woodward and Emery (18). Briefly, 1 ml of plasma was mixed with 150 ^1 of 0.3 M sodium hydroxide solution and corticosterone extracted with 5 ml diethylether-dichlormethane (60:40, vol/vol). The organic phase was washed once with 1 ml of water and evaporated at room temperature under nitrogen. The residue was redissolved in 300 n\ of methanol-water (55:45, vol/ vol). Two hundred microliters were injected onto a reversed phase column (Lichrosorb RP18, 7 nm, 125 x 4 mm), eluted with methanol-water (55:45, vol/vol) and corticosterone measured by UV detection (250 nm). Statistical analysis Data were analyzed by use of ANOVA and differences between treatments were determined by Scheffe's multiple comparisons test. Substances Sodium pentobarbital (Sanofi Sante Animali, Paris, France), ACTHi.24 (a gift from Ciba Geigy, Basel, Switzerland), nicotine-ditartrate (Sigma, St. Louis, MO), and capsaicin (Merck, Darmstadt, FRG) were used.
Results Surgical stress Under pentobarbital anesthesia the trachea was cannulated and a carotid artery and a jugular vein dissected free from the surrounding tissue and cannulated. Five minutes after this surgical procedure blood was collected for the determination of hormone levels. To investigate the proposed excitation of afferent C-fibers by nicotine (15), nicotine was injected intraarterially (ia) into the hind leg of anesthetized rats. A selective excitation of afferent C-fibers would be made evident by a reflex fall in blood pressure as it has been demonstrated for capsaicin (16). For this purpose 5 /xg of nicotine in 30 n\ of saline were injected retrogradely into the epigastric artery while the blood pressure was recorded from a carotid artery. The same amount of nicotine was also injected after the sciatic and the saphenous nerve had been cut or in capsaicin-denervated rats. For systemic application nicotine was dissolved in 0.3 ml of saline and injected in a dose of 250 Mg/kg into the tail vein of anesthetized animals or ip in awake animals. Rats were decapitated 5 min later.
In all of the following experimental series, the measured parameters were at the same time determined in capsaicin-pretreated rats and their controls. Corticosterone release by ACTH ACTHi_24, injected iv in pentobarbital-anesthetized rats, caused a dose-dependent increase in plasma corticosterone levels as measured after 20 min (Fig. 1). There was no significant difference in the response between controls and capsaicin-pretreated rats. Cold stress
When animals were exposed to a cold environment, the plasma ACTH and corticosterone levels reached a maximum at 30 min then dropped over the following 60 min period (Fig. 2). The release of ACTH and corticosterone as a response to cold stress was at its peak at 30 min 55 ± 4% and 66 ± 10%, respectively, lower in
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 00:23 For personal use only. No other uses without permission. . All rights reserved.
AFFERENT C-FIBERS AND STRESS RESPONSE ACTH 1 _ 24
923
300
Cold Stress
400r
200
3
300
200
« 100 o.
o O
100 30
Saline 0.5/ug/kg 2.5/ug/kg 25.0yug/kg FlG. 1. Plasma concentrations of corticosterone 20 min after the iv injection of saline or increasing doses of ACTHi_24 in anesthetized rats. Rats were pretreated as neonates either with capsaicin (hatched columns) or the capsaicin vehicle [open columns). Means ± SEM, n = 57.
capsaicin-pretreated rats. There was no secretion of PRL induced by the same cold exposure in both groups of animals (Fig. 3). Colon temperature was 37.5 ± 0.2 C (n = 10) in both groups of rats before cold exposure. After 30 min of cold exposure there was no change in colon temperature, but after 90 min of cold exposure it was lower in capsaicinpretreated rats (36.4 ± 0.2 C) than in vehicle-pretreated rats (37.2 ± 0.1 C, n = 10 for each group, P < 0.05). Both groups of rats exhibited thermogenic behavior (piloerection, shivering) from the beginning of the cold exposure as has been previously reported (8). Restraint stress Isolation and restricting the movements of the rats in a plastic cylinder induced a similar rise in ACTH and PRL secretion in controls and capsaicin-pretreated rats (Fig. 3). Likewise, the corticosterone levels increased from a basal level of 30-60 ng/ml in both groups to 570 ± 90 ng/ml in controls and to 450 ± 80 ng/ml in capsaicin-pretreated rats (n = 5 for each group). Pentobarbital anesthesia and surgery
Pentobarbital anesthesia did not raise ACTH levels noticeably as has been shown previously (7). Surprisingly, however, PRL levels increased 5-6 times in both groups of rats following pentobarbital anesthesia (Fig. 3). Whereas additional surgery induced a 6-fold increase in ACTH secretion in control rats, but not in capsaicinpretreated rats, PRL levels were not affected further (Fig. 3).
90
60
600 r
? 500
400
*
I 300 o I 200
100
Controls
30 min
60min
90 min
FIG. 2. Time course of plasma ACTH and corticosterone levels during cold exposure (6 C) of rats pretreated with vehicle (open circles and open columns) or capsaicin (filled circles and hatched columns) as neonates. Means ± SEM, n = 6-8. Significance of differences: *P < 0.05 compared to the control condition; OP < 0.05 compared to capsaicinpretreated rats.
Depressor reflex by nicotine
Injection of nicotine (5 ng, intra-arterially into the hind leg of rats) caused a fall in blood pressure which was absent after nerve section of the leg or following capsaicin pretreatment (Table 1). The same dose of nicotine injected into the systemic circulation (jugular vein) did not have any significant effect on the blood pressure (n = 3). ACTH and PRL release by nicotine Nicotine (250 ng/kg) injected ip in conscious rats caused a pronounced ACTH surge (7-8 times basal levels) in both groups. However, iv injection of the same dose of nicotine during anesthesia caused only a slight ACTH increase by 60% in control rats but no increase at all in capsaicin-pretreated rats, resulting in significant
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 00:23 For personal use only. No other uses without permission. . All rights reserved.
AFFERENT C-FIBERS AND STRESS RESPONSE
924 No stress
Cold stress
Restraint
800
i
Endo • 1990 Voll26«No2
CONSCIOUS
Pentobarbital Pentobarbital Anaesthesia Surgery •o
900
Saline
Nicotine
PENTOBARBITAL ANAESTHESIA Saline
Nicotine
600 E
1 600 x o
