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Proconflict effect of carbon dioxide inhalation in rats
Life Sciences, Vol. 56, h’o. 16 pp. PL 321-324, 1995 Copyright Q 1595 Elsevier Science Ltd Printed in the USA. All rights reserved 0024-3205/95 $9.50 t .oO
Pergamon 0024.3205(95)00093-3
Ph54RAUCOLOGY LETTERS Accelerated Comnttrrticafiott
PROCONFLICT
EFFECT OF CARBON DIOXIDE INHALATION IN RATS
Tonino Cuccheddu,
Department
Stefania Floris, Mariangela Serra, Maria Luisa Porceddu, Enrico Sanna, and Giovanni Biggio of Experimental Biology, Chair of Pharmacology, University of Cagliari, Italy
(Submitted
December 15, 1994; accepted January received in final form January 30, 1995)
4, 1995;
Abstract. The effect of brief inhalation of carbon dioxide (COJ was studied in a conflict situation (Vogel test) in the rat. This treatment, which inhibits y-aminobutyric acid (GABA)mediated transmission in rat brain and induces anxiety and panic attacks in humans, elicited a proconflict effect. Exposure of rats for 1 min to CO2 decreased by - 40 % the number of licking periods in the test. This effect was abolished by prior administration of alprazolam (0.5 mg per kilogram of body mass, i.p.). Although these results may support a role for GABA-mediated the possibility that different transmission in the anxiogenic effect of CO2 inhalation, neurotransmitters other than GABA are involved in the action of COP can not be ruled out.
Key Words: carbon
dioxide,
conflict
behavior,
GABA-mediated
transmission
Introduction y-Aminobutyric acid (GABA)-mediated transmission has been suggested to play an important role in the modulation of emotional state and fear that is exerted by anxiolytic and anxiogenic drugs as well as by environmental stimuli in rats (1). Anxiolytic and anxiogenic ligands of central benzodiazepine recognition sites as well as stress have been shown to modulate the function of GABA, receptors in rat brain (1); anxiogenic ll-carbolines and stress reduce the ability of GABA to enhance the receptor-associated Cl-channel conductance, whereas anxiolytic benzodiazepines elicit an opposite effect. Recently, we showed that brief inhalation of carbon dioxide (CO?), a treatment known to induce anxiety in healthy subjects and anxious patients as well as panic attacks in individuals with panic disorder (2), results in a rapid inhibition of the function of GABA, receptor-coupled Cl- channels (3,4). This action is similar to the effect elicited by various anxiogenic ligands of benzodiazepine recognition sites, which is antagonized by the previous administration of anxiolytic benzodiazepines ( 1) . These observations prompted us to investigate further the pharmacology of the anxiogenic effect of CO, inhalation. Thus, we evaluated the effect of this treatment on the emotional state of rats, as assessed by its ability to facilitate the suppression of drinking induced by punishment (proconflict effect).
PL-322
Proconflict
Effect of CO, Inhalation
Vol. 56, No. 16, 1995
Methods Male Sprague-Dawley rats (Charles River, Como, Italy) with body masses of 200 to 250 g were housed under conditions of controlled temperature and lighting (light period: 0800 - 2000) and were given free access to food and water. Test groups of 15 to 20 rats that had been habituated for 4 to 5 days to the handling maneuvers that precede the experiment were deprived of water for 24 h before the conflict session. The conflict test was performed as previously described (5). Briefly, rats were placed in a clear Plexiglas box (20 by 28 by 20 cm) with a stainless steel grid floor, and the box was enclosed in a sound-attenuated ventilated chamber (Lafayette Instruments, Lafayette, IN, USA). Water was provided through a stainless steel drinking tube that extended 1 cm into the box, 3 cm above the floor. The drinking tube and the grid floor were connected to a constant-current shock generator and to a “drinkometer”. The shock generator delivered one shock (0.4 mA), lasting 0.5 s, for a cumulative period of 15 licks. This period of cumulative drinking was termed the “licking period”. Experiments were performed between 1400 and 1900. Animals were exposed for 1 min to a mixture of 35 % CO, and 65 % O2 that was delivered to the hermetically closed box at the rate of 4 liters/min; CO, content was monitored with a commercial analyzer. Drugs were suspended in distilled water with one drop of Tween 80 per 5 ml and injected intraperitoneally. Each rat was allowed to habituate to the new environment for 15 min, after which the drinking tube was inserted into the cage and the animal was allowed to lick for three licking periods (training period) before the start of the test. Data were analyzed by analysis of variance (ANOVA) followed by Scheffe’s test.
Results Exposure of rats for 1 min to CO2 elicited a proconflict effect. According to previous biochemical studies (4), the animals were tested 10 min after CO, exposure. This treatment decreased by - 40% the number of licking periods during punishment (Table l), without having any effect on unpunished drinking periods (data not shown). This effect of CO, was similar to that exerted by the anxiogenic benzodiazepine receptor ligand FG 7142; as expected (6), at a dose of 10 mg per kilogram of body mass (i.p.), this compound decreased by - 50 % the number of licking periods during punishment (Table 1). The effect of FG 7142, but not that of CQ, was completely prevented by pretreatment with the benzodiazepine recognition site antagonist flumazenil (Table 2), which itself failed to affect punished behavior (Table 1).
TABLE 1 Effect of CO, and the anxiogenic B-carboline FG 7142 on proconflict behavior in rats.
Treatment
Shocks/3 min
No. of animals
Control
31 + 3 (100%)
50
CO,
19 f
l* (61%)
72
FG 7142
16 ) 2* (52%)
16
Flumazenil
32 +_ 1 (103%)
16
Alprazolam
49 + 2*(158%)
16
Rats were exposed to CO, for 1 min and tested 10 min later. FG 7142 (10 mg per kilogram of body mass), alprazolam (0.5 mg/kg), and flumazenil (5 mg/kg) were administered intraperitoneally 30 or 35 min before the start of experiments. Values are means + S.E.M. of two to five experiments with eight to 12 rats per group. *p < 0.01 vs. control group (ANOVA followed by Scheffe’s test).
Vol. 56, No. 16, 1995
PL-323
Proconflict Effect of CO, Inhalation
To investigate further the proconflict action of CO2 inhalation, we examined the effect of prior administration of the anxiolytic and antipanic drug alpmzolam (0.5 mg/kg, i.p.) (2,7). Alprazolam completely prevented the proconflict effect elicited by CO2 (Table 2).
TABLE 2 Effects of alurazolam and flumazenil on the orocontlict Treatment Control
Shocks/3 min
effect of CO, inhalation No. of antmals
31 &- 3 (100%)
50
Alprazolam
+ CO,
41 k 2*(132%)
30
Flumazenil
+ CO,
I8 + 2% (58%)
20
Flumazenil
+ FG 7142
29 + 2 (94%)
I6
Rats were exposed to CO, for I min and tested IO min later. FG 7142 (10 mgikg), alprazolam (0.5 mg/kg), and flumazenil (5 mg/kg) were administered intraperitoneally 30 or 35 min before the start of experiments. Value are means + S.E.M. of two to five experiments with eight to 12 rats per group. *p < 0.01 vs control group (ANOVA followed by Scheffe’s test).
Discussion We have shown that a brief exposure to CO, has a proconflict effect in rats. To our knowledge, this observation constitutes the first experimental evidence that CO1 inhalation alters the emotional state of laboratory animals. This effect was similar to that elicited by the anxiogenic B-carboline derivative FG 7142 and was abolished by prior administration of alprazolam. Given that the enhancement of shock-induced suppression of drinking in rats is predictive of an anxiogenic action (6), our results are consistent with the clinical evidence that CO, inhalation induces panic attacks in patients with anxiety disorders and that this effect is sensitive to the action of benzodiazepines (2,7). Although our data do not clarify the mechanism of the panic-inducing action of COz inhalation, the observations that prior administration of anxiolytic benzodiazepines prevent the CO*-induced proconflict effect in rats (Table 2), the CO,-induced reduction in GABA, receptor function in rat brain (3,4), as well as the CO,-induced anxiety and panic attacks in humans (2,7) may support the idea that central GABA-mediated transmission is involved in the pharmacology of CO, inhalation. However, the finding that CO,-induced proconflict effect is attenuated by alprazolam does not exclude the possibility that different neurotransmitters other than GABA may be involved in the behavioural effect elicited by CO, inhalation. Accordingly, alprazolam is able to attenuate panic attack induced by agents acting through a variety of pharmacologic mechanisms (8). Experiments are now in progress to evaluate whether serotoninergic anxiolytics mimick the action of alprazolam. Finally, the present results, together with previous biochemical data (3,4) suggest that rats exposed to different concentrations of CO, may be a new useful model with which to study the neurochemistry and pharmacology of changes in emotional state as well as the efficacy of new anxiolytic drugs. Acknowledgments Supported by grant 92.00022.PF41 from the National Research Council (CNR) (Targeted Project, “Prevention and Control Disease Factors’); Subproject, “Stress”.
PL-324
Proeonflict
Effect of CO, Inhalation
Vol. 56, No. 16, YXk5
References 1. 2. 3. 4.
G. BIGGIO, A. CONCAS, M.G. CORDA, 0. GIORGI, E. SANNA and M. SERRA, Pharmacol. Ther. 48, 121-142 (1990). S.W. WOODS, D.S. CHARNEY, J. LOKE, W.K. GOODMAN, D.E. REDMOND and G.R. HENINGER, Arch. Gen. Psychiatry 43, 900-905 (1986). E. SANNA, T. CUCCHEDDU, M. SERRA, A. CONCAS and G. BIGGIO, Eur. J. Pharmacol. 216, 457-458 (1992). A. CONCAS, E. SANNA, T. CUCCHEDDU, M.P. MASCIA, G. SANTORO, E. MACIOCCO and G. BIGGIO, Prog. Neuro-Psychopharmacol. Biol. Psychiatry 1z, 651-661 (1993). M.G. CORDA, 0. GIORGI, B. LONGONI and G. BIGGIO, Eur. J. Pharmacol. 159, 233238 (1989). M.G. CORDA, W.D. BLAKER, W.B. MENDELSON, A. GUIDOTTI and E. COSTA, Proc. Natl. Acad. Sci. USA, 80, 2072-2076 (1983). H. POLS, J. ZANDBERGEN, C. DE LOOF and E. GRIEZ, Acta Psychiatry Stand. 84, 585-590 (1991). M.R. LIEBOWITZ, A.J. FYER, J.M. GORMAN, R. CAMPEAS, A. LEVIN, S.R. DAVIES, D. GOETZ and D.F. KLEIN. J. Clin. Psychophartnacol., 6, 13-20 (1986).
Pergamon 0024.3205(95)00093-3
Ph54RAUCOLOGY LETTERS Accelerated Comnttrrticafiott
PROCONFLICT
EFFECT OF CARBON DIOXIDE INHALATION IN RATS
Tonino Cuccheddu,
Department
Stefania Floris, Mariangela Serra, Maria Luisa Porceddu, Enrico Sanna, and Giovanni Biggio of Experimental Biology, Chair of Pharmacology, University of Cagliari, Italy
(Submitted
December 15, 1994; accepted January received in final form January 30, 1995)
4, 1995;
Abstract. The effect of brief inhalation of carbon dioxide (COJ was studied in a conflict situation (Vogel test) in the rat. This treatment, which inhibits y-aminobutyric acid (GABA)mediated transmission in rat brain and induces anxiety and panic attacks in humans, elicited a proconflict effect. Exposure of rats for 1 min to CO2 decreased by - 40 % the number of licking periods in the test. This effect was abolished by prior administration of alprazolam (0.5 mg per kilogram of body mass, i.p.). Although these results may support a role for GABA-mediated the possibility that different transmission in the anxiogenic effect of CO2 inhalation, neurotransmitters other than GABA are involved in the action of COP can not be ruled out.
Key Words: carbon
dioxide,
conflict
behavior,
GABA-mediated
transmission
Introduction y-Aminobutyric acid (GABA)-mediated transmission has been suggested to play an important role in the modulation of emotional state and fear that is exerted by anxiolytic and anxiogenic drugs as well as by environmental stimuli in rats (1). Anxiolytic and anxiogenic ligands of central benzodiazepine recognition sites as well as stress have been shown to modulate the function of GABA, receptors in rat brain (1); anxiogenic ll-carbolines and stress reduce the ability of GABA to enhance the receptor-associated Cl-channel conductance, whereas anxiolytic benzodiazepines elicit an opposite effect. Recently, we showed that brief inhalation of carbon dioxide (CO?), a treatment known to induce anxiety in healthy subjects and anxious patients as well as panic attacks in individuals with panic disorder (2), results in a rapid inhibition of the function of GABA, receptor-coupled Cl- channels (3,4). This action is similar to the effect elicited by various anxiogenic ligands of benzodiazepine recognition sites, which is antagonized by the previous administration of anxiolytic benzodiazepines ( 1) . These observations prompted us to investigate further the pharmacology of the anxiogenic effect of CO, inhalation. Thus, we evaluated the effect of this treatment on the emotional state of rats, as assessed by its ability to facilitate the suppression of drinking induced by punishment (proconflict effect).
PL-322
Proconflict
Effect of CO, Inhalation
Vol. 56, No. 16, 1995
Methods Male Sprague-Dawley rats (Charles River, Como, Italy) with body masses of 200 to 250 g were housed under conditions of controlled temperature and lighting (light period: 0800 - 2000) and were given free access to food and water. Test groups of 15 to 20 rats that had been habituated for 4 to 5 days to the handling maneuvers that precede the experiment were deprived of water for 24 h before the conflict session. The conflict test was performed as previously described (5). Briefly, rats were placed in a clear Plexiglas box (20 by 28 by 20 cm) with a stainless steel grid floor, and the box was enclosed in a sound-attenuated ventilated chamber (Lafayette Instruments, Lafayette, IN, USA). Water was provided through a stainless steel drinking tube that extended 1 cm into the box, 3 cm above the floor. The drinking tube and the grid floor were connected to a constant-current shock generator and to a “drinkometer”. The shock generator delivered one shock (0.4 mA), lasting 0.5 s, for a cumulative period of 15 licks. This period of cumulative drinking was termed the “licking period”. Experiments were performed between 1400 and 1900. Animals were exposed for 1 min to a mixture of 35 % CO, and 65 % O2 that was delivered to the hermetically closed box at the rate of 4 liters/min; CO, content was monitored with a commercial analyzer. Drugs were suspended in distilled water with one drop of Tween 80 per 5 ml and injected intraperitoneally. Each rat was allowed to habituate to the new environment for 15 min, after which the drinking tube was inserted into the cage and the animal was allowed to lick for three licking periods (training period) before the start of the test. Data were analyzed by analysis of variance (ANOVA) followed by Scheffe’s test.
Results Exposure of rats for 1 min to CO2 elicited a proconflict effect. According to previous biochemical studies (4), the animals were tested 10 min after CO, exposure. This treatment decreased by - 40% the number of licking periods during punishment (Table l), without having any effect on unpunished drinking periods (data not shown). This effect of CO, was similar to that exerted by the anxiogenic benzodiazepine receptor ligand FG 7142; as expected (6), at a dose of 10 mg per kilogram of body mass (i.p.), this compound decreased by - 50 % the number of licking periods during punishment (Table 1). The effect of FG 7142, but not that of CQ, was completely prevented by pretreatment with the benzodiazepine recognition site antagonist flumazenil (Table 2), which itself failed to affect punished behavior (Table 1).
TABLE 1 Effect of CO, and the anxiogenic B-carboline FG 7142 on proconflict behavior in rats.
Treatment
Shocks/3 min
No. of animals
Control
31 + 3 (100%)
50
CO,
19 f
l* (61%)
72
FG 7142
16 ) 2* (52%)
16
Flumazenil
32 +_ 1 (103%)
16
Alprazolam
49 + 2*(158%)
16
Rats were exposed to CO, for 1 min and tested 10 min later. FG 7142 (10 mg per kilogram of body mass), alprazolam (0.5 mg/kg), and flumazenil (5 mg/kg) were administered intraperitoneally 30 or 35 min before the start of experiments. Values are means + S.E.M. of two to five experiments with eight to 12 rats per group. *p < 0.01 vs. control group (ANOVA followed by Scheffe’s test).
Vol. 56, No. 16, 1995
PL-323
Proconflict Effect of CO, Inhalation
To investigate further the proconflict action of CO2 inhalation, we examined the effect of prior administration of the anxiolytic and antipanic drug alpmzolam (0.5 mg/kg, i.p.) (2,7). Alprazolam completely prevented the proconflict effect elicited by CO2 (Table 2).
TABLE 2 Effects of alurazolam and flumazenil on the orocontlict Treatment Control
Shocks/3 min
effect of CO, inhalation No. of antmals
31 &- 3 (100%)
50
Alprazolam
+ CO,
41 k 2*(132%)
30
Flumazenil
+ CO,
I8 + 2% (58%)
20
Flumazenil
+ FG 7142
29 + 2 (94%)
I6
Rats were exposed to CO, for I min and tested IO min later. FG 7142 (10 mgikg), alprazolam (0.5 mg/kg), and flumazenil (5 mg/kg) were administered intraperitoneally 30 or 35 min before the start of experiments. Value are means + S.E.M. of two to five experiments with eight to 12 rats per group. *p < 0.01 vs control group (ANOVA followed by Scheffe’s test).
Discussion We have shown that a brief exposure to CO, has a proconflict effect in rats. To our knowledge, this observation constitutes the first experimental evidence that CO1 inhalation alters the emotional state of laboratory animals. This effect was similar to that elicited by the anxiogenic B-carboline derivative FG 7142 and was abolished by prior administration of alprazolam. Given that the enhancement of shock-induced suppression of drinking in rats is predictive of an anxiogenic action (6), our results are consistent with the clinical evidence that CO, inhalation induces panic attacks in patients with anxiety disorders and that this effect is sensitive to the action of benzodiazepines (2,7). Although our data do not clarify the mechanism of the panic-inducing action of COz inhalation, the observations that prior administration of anxiolytic benzodiazepines prevent the CO*-induced proconflict effect in rats (Table 2), the CO,-induced reduction in GABA, receptor function in rat brain (3,4), as well as the CO,-induced anxiety and panic attacks in humans (2,7) may support the idea that central GABA-mediated transmission is involved in the pharmacology of CO, inhalation. However, the finding that CO,-induced proconflict effect is attenuated by alprazolam does not exclude the possibility that different neurotransmitters other than GABA may be involved in the behavioural effect elicited by CO, inhalation. Accordingly, alprazolam is able to attenuate panic attack induced by agents acting through a variety of pharmacologic mechanisms (8). Experiments are now in progress to evaluate whether serotoninergic anxiolytics mimick the action of alprazolam. Finally, the present results, together with previous biochemical data (3,4) suggest that rats exposed to different concentrations of CO, may be a new useful model with which to study the neurochemistry and pharmacology of changes in emotional state as well as the efficacy of new anxiolytic drugs. Acknowledgments Supported by grant 92.00022.PF41 from the National Research Council (CNR) (Targeted Project, “Prevention and Control Disease Factors’); Subproject, “Stress”.
PL-324
Proeonflict
Effect of CO, Inhalation
Vol. 56, No. 16, YXk5
References 1. 2. 3. 4.
G. BIGGIO, A. CONCAS, M.G. CORDA, 0. GIORGI, E. SANNA and M. SERRA, Pharmacol. Ther. 48, 121-142 (1990). S.W. WOODS, D.S. CHARNEY, J. LOKE, W.K. GOODMAN, D.E. REDMOND and G.R. HENINGER, Arch. Gen. Psychiatry 43, 900-905 (1986). E. SANNA, T. CUCCHEDDU, M. SERRA, A. CONCAS and G. BIGGIO, Eur. J. Pharmacol. 216, 457-458 (1992). A. CONCAS, E. SANNA, T. CUCCHEDDU, M.P. MASCIA, G. SANTORO, E. MACIOCCO and G. BIGGIO, Prog. Neuro-Psychopharmacol. Biol. Psychiatry 1z, 651-661 (1993). M.G. CORDA, 0. GIORGI, B. LONGONI and G. BIGGIO, Eur. J. Pharmacol. 159, 233238 (1989). M.G. CORDA, W.D. BLAKER, W.B. MENDELSON, A. GUIDOTTI and E. COSTA, Proc. Natl. Acad. Sci. USA, 80, 2072-2076 (1983). H. POLS, J. ZANDBERGEN, C. DE LOOF and E. GRIEZ, Acta Psychiatry Stand. 84, 585-590 (1991). M.R. LIEBOWITZ, A.J. FYER, J.M. GORMAN, R. CAMPEAS, A. LEVIN, S.R. DAVIES, D. GOETZ and D.F. KLEIN. J. Clin. Psychophartnacol., 6, 13-20 (1986).