Psychopharmacologia (Berl.) 42, 263- 269 (1975) 9 by Springer-Verlag 1975
Effects of Scopolamine, Physostigmine and Chlordiazepoxide on Punished and Extinguished Water Consumption in Rats KLAUS A. MICZEK and PETER LAU Department of Psychology, Carnegie-Mellon University, Pittsburgh, Pennsylvania Received September 12, 1974; Final Version March 7, 1975
Abstract. It has been postulated that behavioral inhibition due to punishment or extinction may be mediated by brain acetylcholine, and drugs which have disinhibitory action are thought to interact with this system. This notion was tested by comparing the effects of scopolamine, physostigmine and chlordiazepoxide on punished and extinguished water consumption. Scopolamine hydrobromide (0.3, 0.5 mg/kg, i.p.), a centrally and peripherally acting antimuscarinic agent and physostigmine sulfate, (0.3 mg/kg, i.p.), a centrally and peripherally acting acetylcholinesterase inhibitor, lowered both non-punished and punishment suppressed water intake and lick rate, whereas their quaternary analogs which primarily act in the periphery, had no significant effect at comparable
dose levels. Scopolamine and physostigmine suppressed punished water consumption at lower dose levels than nonpunished intake. In contrast, chlordiazepoxide (5.0, 10.0, 20.0 mg/kg, i.p.) enhanced punished as well as non-punished water intake. In a further experiment comparing punishment and extinction suppression, scopolamine and physostigmine did not affect punished or extinguished water intake; chlordiazepoxide (5.0, 10.0, 20.0 mg/kg) reliably increased punished, but not extinguished licking on the water nozzle. These results suggest (1) that scopolamine and chlordiazepoxide do not act via a common mechanism, and (2) that punishment and extinction suppression are not a pharmacological entity.
Key words: Scopolamine Hydrobromide - Physostigmine - Chlordiazepoxide - Punishment - Extinction - Passive Avoidance - Behavioral Inhibition - Licking - Water Consumption.
Cholinergic activity in the central nervous system is thought to mediate behavioral inhibition due to nonreinforcement and punishment. This concept was introduced and developed by Carlton (1963, 1968, 1969) and later expanded by Stein (1969) and Margules and Margules (1973). The major evidence for this theory comes from repeated demonstrations that anticholinergic drugs such as atropine or scopolamine disinhibit punishment suppressed responses in "passive avoidance" or "go-no go" situations (e.g., Bignami, 1967; Bursova etal., 1964; Meyers, 1965) as well as interfere with extinction and habituation processes (e.g., Carleton and Vogel, 1965; Hanson et al., 1967; Heise et al., 1970; Warburton, 1969). There is, however, evidence which limits the generality of the notion that brain acetylcholine exerts an inhibitory role in processes mediating the suppression of behavior. For example, the disinhibitory action of anticholinergics seems to be detectable only in certain classes of behavior. After systemic administration of anticholinergic agents "passive avoidance
deficits" are consistently observed when the punished behavior is a consummatory response (e.g., eating, drinking) or a locomotor response (e.g., shuttling, step-down movement). However, anticholinergic drugs fail to increase the rate of punished operant lever pressing in monkeys or rats (Hanson etal., 1967; Miczek, 1973a). On the other hand, administration of anticholinergic drugs directly to hypothalamic sites disinhibits operant lever pressing, reinforced by food, sweet water, or milk and simultaneously punished by electric shock; but free consumption of milk, food, or water is suppressed by the same treatment (Margules and Stein, 1969; Miczek and Grossman, 1972). Furthermore, the parameters of the various punishment and extinction procedures leading to behavioral suppression appear to be major determinants of scopolamine's disinhibitory effect (e.g., Hanson et al., 1967; Heise et al., 1970; Miczek and Grossman, 1972). The present experiments were undertaken to delineate further the kind of behavioral processes which are mediated by brain acetylcholine. We selected
264
Psychopharmacologia (Berl.), Vol. 42, Fasc. 3 (1975)
punishment and extinction procedures which suppressed behavior on a long-term basis. Since brain acetylcholine has also been linked to thirst (e.g., Fisher and Coury, 1962; Grossman, 1962; Stein and Seifter, 1962), water consumption appeared to be a particularly suitable behavior to study. It was our objective to examine the effects of anticholinergic and anticholinesterase agents on punished and extinguished water consumption. We were also interested in comparing the effects of these drugs with those of chlordiazepoxide. It has been suggested that the benzodiazepines produce their well-known punishment attenuating effect via action on a central cholinergic inhibitory system (Margules and Stein, 1967).
Experiment I Scopolamine-treated animals emit more punished behavior than non-treated animals. Since this socalled passive avoidance deficit is typically seen in discrete-trial paradigms, we examined the generality of the p h e n o m e n o n in a situation which involved long-term punishment suppression of water intake to about 50 ~ of non-punished consumption. In contrast to the conventional drinkometer, we used a photocell arrangement for detecting the tongue movements involved in water consumption (Martonyi and Valenstein, 1971).
Method Subjects. Twenty-seven adult male rats of the SpragueDawley strain (Zivic-Miller, Pittsburgh, Pa.), weighing 300- 350 g at the beginning of the experiment, were housed individually in wire-mesh cages in a colony with controlled humidity, temperature, and light cycle (12 hrs light, 12 hrs dark). Throughout the experiment, the rats had free access to food (Purina Laboratory Chow) and were deprived of water for 23 hrs per day. Apparatus. The experiments were conducted in two identical chambers, each housed in a separately ventilated and illuminated (by a 28 W light bulb) enclosure. Each experimental chamber was constructed of Plexiglas walls and ceiling measuring 31 x 28 x 27 cm. The floor consisted of copper rods (0.5 cm in diameter), spaced 2 cm apart. Licking and water consumption were measured by an apparatus similar to one described by Martonyi and Valenstein (1971). Briefly, an elongated slot (2.5 x 0.5 cm) in the center of the front wall, 3 cm above the floor, permitted access to the nozzle of a drinking bottle. An inverted 50 ml centrifuge vial with a stainless steel nozzle was mounted to the back of the front panel. The distance between the tip of the drinking nozzle (0.8 cm in diameter, with an 0.2 cm diameter opening) and the inside of the front wall was about 1 cm, so that each rat lapped water by extending its tongue through the opening in the front wall. Just behind the front wall a light beam was directed at a photocell, detecting each tongue movement toward the drinking nozzle. Licks were registered on a Sodeco counter (model 1425). Water consumption was read to the nearest 0.25 ml. An electric shock generator (Beede Electrical Inst.
Co., Inc., Penacook, N. H., model 228 stimulator) could also be connected between the drinking nozzle and the grid floor. When the rat licked the nozzle, it completed the circuit between floor and nozzle and received an instantaneous electric shock to the tongue. Drugs. The following drugs and dose levels were used; 0.05, 0.1, 0.3, and 0.5 mg/kg scopolamine hydrobromide; 0.025, 0.05, 0.1, 0.3 mg/kg physostigmine sulfate; 0.05, 0.1 mg/kg neostigmine sulfate; 0.1, 0.5mg/kg scopolamine methyl nitrate, all obtained from Sigma Chemical Co., St. Louis, Mo.; 5.0, 10.0, 20.0 mg/kg chlordiazepoxide hydrochloride supplied by Hoffmann-La Roche Co., Nutley, N.J. Each drug solution was prepared in 0.9 ~ saline at frequent intervals; chlordiazepoxide was dissolved immediately before each administration. The drugs were injected intraperitoneally 30 min before the experimental session in a volume of I ml/kg body weight. Procedure. The animals were assigned to two groups according to experimentel conditions: punishment (n = 12) and nonpunishment (n = 13). Each animal was run at the same time each day, 7 days a week, during the light phase of the photocycle. All animals were deprived of water and trained to lick water from the nozzle in the experimental chambers until their daily intake stabilized after about 3 weeks. If an animal's intake (in cc) was within _+ 10~ of five consecutive daily sessions, experimental manipulations were initiated. Each rat was subjected to all drug conditions at all dose levels according to a randomized design, with 7 rats actually completing the entire procedure in the non-punishment group. Only the data of the rats which completed all dosages of one drug and the saline vehicle conditions were analyzed. Drug administrations were scheduled at least 4 days apart. Punishment was introduced for the rats of the second group by presenting electric shock to the tongue for the duration of each lick. The intensity of the shock was adjusted so that the daily intake of each rat was suppressed by about 50 ~ of the preceding punishment-free condition. The shock intensities ranged from 290-470 pA. As soon as the daily intake of each :'at was consistently suppressed to 50 ~o of the non-punished intake, drug treatments were scheduled as described for the non-punishment group. Six rats of the punishment group completed all drug conditions. Following the daily experimental session in the chamber, each rat of both groups was returned to its home cage and given an additional 30 min free access to water. The total number of licks and the water intake during the experimental session as well as the water intake in the home cage were recorded. Statistical Analysis. A two-factor repeated measures design was used, factor one being dosages of drugs and factor two being days of experimental treatment (pre-drug, drug, and post-drug days). In cases of significant overall F-scores, the Newman-Keuls procedure (Winer, 1971) was used to identify differences between individual means.
Results With the presently employed 23 hrs water deprivation regimen all rats achieved a stable daily lick rate and water consumption after about 3 weeks, and maintained these baseline values over several months. Scopolamine hydrobromide decreased non-punished water intake [F(4,28) = 7.9; P < 0.01] and lick rate [ F ( 4 , 2 8 ) = 2.9; P < 0.05] slightly, but signifi-
K. A. Miczek, and P. Lau: Scopolamine, Physostigmine, Chlordiazepoxide and Inhibition ~j
SCOPOLAMINE HYDROBROMIDE
o
~50
SCOPOLAMINE METHYL NITRATE
PHYSOSTIGMINE
NEOSTIGMINE
CHLOR DIAZEPOXIDE
W
Effects of Scopolamine, Physostigmine and Chlordiazepoxide on Punished and Extinguished Water Consumption in Rats KLAUS A. MICZEK and PETER LAU Department of Psychology, Carnegie-Mellon University, Pittsburgh, Pennsylvania Received September 12, 1974; Final Version March 7, 1975
Abstract. It has been postulated that behavioral inhibition due to punishment or extinction may be mediated by brain acetylcholine, and drugs which have disinhibitory action are thought to interact with this system. This notion was tested by comparing the effects of scopolamine, physostigmine and chlordiazepoxide on punished and extinguished water consumption. Scopolamine hydrobromide (0.3, 0.5 mg/kg, i.p.), a centrally and peripherally acting antimuscarinic agent and physostigmine sulfate, (0.3 mg/kg, i.p.), a centrally and peripherally acting acetylcholinesterase inhibitor, lowered both non-punished and punishment suppressed water intake and lick rate, whereas their quaternary analogs which primarily act in the periphery, had no significant effect at comparable
dose levels. Scopolamine and physostigmine suppressed punished water consumption at lower dose levels than nonpunished intake. In contrast, chlordiazepoxide (5.0, 10.0, 20.0 mg/kg, i.p.) enhanced punished as well as non-punished water intake. In a further experiment comparing punishment and extinction suppression, scopolamine and physostigmine did not affect punished or extinguished water intake; chlordiazepoxide (5.0, 10.0, 20.0 mg/kg) reliably increased punished, but not extinguished licking on the water nozzle. These results suggest (1) that scopolamine and chlordiazepoxide do not act via a common mechanism, and (2) that punishment and extinction suppression are not a pharmacological entity.
Key words: Scopolamine Hydrobromide - Physostigmine - Chlordiazepoxide - Punishment - Extinction - Passive Avoidance - Behavioral Inhibition - Licking - Water Consumption.
Cholinergic activity in the central nervous system is thought to mediate behavioral inhibition due to nonreinforcement and punishment. This concept was introduced and developed by Carlton (1963, 1968, 1969) and later expanded by Stein (1969) and Margules and Margules (1973). The major evidence for this theory comes from repeated demonstrations that anticholinergic drugs such as atropine or scopolamine disinhibit punishment suppressed responses in "passive avoidance" or "go-no go" situations (e.g., Bignami, 1967; Bursova etal., 1964; Meyers, 1965) as well as interfere with extinction and habituation processes (e.g., Carleton and Vogel, 1965; Hanson et al., 1967; Heise et al., 1970; Warburton, 1969). There is, however, evidence which limits the generality of the notion that brain acetylcholine exerts an inhibitory role in processes mediating the suppression of behavior. For example, the disinhibitory action of anticholinergics seems to be detectable only in certain classes of behavior. After systemic administration of anticholinergic agents "passive avoidance
deficits" are consistently observed when the punished behavior is a consummatory response (e.g., eating, drinking) or a locomotor response (e.g., shuttling, step-down movement). However, anticholinergic drugs fail to increase the rate of punished operant lever pressing in monkeys or rats (Hanson etal., 1967; Miczek, 1973a). On the other hand, administration of anticholinergic drugs directly to hypothalamic sites disinhibits operant lever pressing, reinforced by food, sweet water, or milk and simultaneously punished by electric shock; but free consumption of milk, food, or water is suppressed by the same treatment (Margules and Stein, 1969; Miczek and Grossman, 1972). Furthermore, the parameters of the various punishment and extinction procedures leading to behavioral suppression appear to be major determinants of scopolamine's disinhibitory effect (e.g., Hanson et al., 1967; Heise et al., 1970; Miczek and Grossman, 1972). The present experiments were undertaken to delineate further the kind of behavioral processes which are mediated by brain acetylcholine. We selected
264
Psychopharmacologia (Berl.), Vol. 42, Fasc. 3 (1975)
punishment and extinction procedures which suppressed behavior on a long-term basis. Since brain acetylcholine has also been linked to thirst (e.g., Fisher and Coury, 1962; Grossman, 1962; Stein and Seifter, 1962), water consumption appeared to be a particularly suitable behavior to study. It was our objective to examine the effects of anticholinergic and anticholinesterase agents on punished and extinguished water consumption. We were also interested in comparing the effects of these drugs with those of chlordiazepoxide. It has been suggested that the benzodiazepines produce their well-known punishment attenuating effect via action on a central cholinergic inhibitory system (Margules and Stein, 1967).
Experiment I Scopolamine-treated animals emit more punished behavior than non-treated animals. Since this socalled passive avoidance deficit is typically seen in discrete-trial paradigms, we examined the generality of the p h e n o m e n o n in a situation which involved long-term punishment suppression of water intake to about 50 ~ of non-punished consumption. In contrast to the conventional drinkometer, we used a photocell arrangement for detecting the tongue movements involved in water consumption (Martonyi and Valenstein, 1971).
Method Subjects. Twenty-seven adult male rats of the SpragueDawley strain (Zivic-Miller, Pittsburgh, Pa.), weighing 300- 350 g at the beginning of the experiment, were housed individually in wire-mesh cages in a colony with controlled humidity, temperature, and light cycle (12 hrs light, 12 hrs dark). Throughout the experiment, the rats had free access to food (Purina Laboratory Chow) and were deprived of water for 23 hrs per day. Apparatus. The experiments were conducted in two identical chambers, each housed in a separately ventilated and illuminated (by a 28 W light bulb) enclosure. Each experimental chamber was constructed of Plexiglas walls and ceiling measuring 31 x 28 x 27 cm. The floor consisted of copper rods (0.5 cm in diameter), spaced 2 cm apart. Licking and water consumption were measured by an apparatus similar to one described by Martonyi and Valenstein (1971). Briefly, an elongated slot (2.5 x 0.5 cm) in the center of the front wall, 3 cm above the floor, permitted access to the nozzle of a drinking bottle. An inverted 50 ml centrifuge vial with a stainless steel nozzle was mounted to the back of the front panel. The distance between the tip of the drinking nozzle (0.8 cm in diameter, with an 0.2 cm diameter opening) and the inside of the front wall was about 1 cm, so that each rat lapped water by extending its tongue through the opening in the front wall. Just behind the front wall a light beam was directed at a photocell, detecting each tongue movement toward the drinking nozzle. Licks were registered on a Sodeco counter (model 1425). Water consumption was read to the nearest 0.25 ml. An electric shock generator (Beede Electrical Inst.
Co., Inc., Penacook, N. H., model 228 stimulator) could also be connected between the drinking nozzle and the grid floor. When the rat licked the nozzle, it completed the circuit between floor and nozzle and received an instantaneous electric shock to the tongue. Drugs. The following drugs and dose levels were used; 0.05, 0.1, 0.3, and 0.5 mg/kg scopolamine hydrobromide; 0.025, 0.05, 0.1, 0.3 mg/kg physostigmine sulfate; 0.05, 0.1 mg/kg neostigmine sulfate; 0.1, 0.5mg/kg scopolamine methyl nitrate, all obtained from Sigma Chemical Co., St. Louis, Mo.; 5.0, 10.0, 20.0 mg/kg chlordiazepoxide hydrochloride supplied by Hoffmann-La Roche Co., Nutley, N.J. Each drug solution was prepared in 0.9 ~ saline at frequent intervals; chlordiazepoxide was dissolved immediately before each administration. The drugs were injected intraperitoneally 30 min before the experimental session in a volume of I ml/kg body weight. Procedure. The animals were assigned to two groups according to experimentel conditions: punishment (n = 12) and nonpunishment (n = 13). Each animal was run at the same time each day, 7 days a week, during the light phase of the photocycle. All animals were deprived of water and trained to lick water from the nozzle in the experimental chambers until their daily intake stabilized after about 3 weeks. If an animal's intake (in cc) was within _+ 10~ of five consecutive daily sessions, experimental manipulations were initiated. Each rat was subjected to all drug conditions at all dose levels according to a randomized design, with 7 rats actually completing the entire procedure in the non-punishment group. Only the data of the rats which completed all dosages of one drug and the saline vehicle conditions were analyzed. Drug administrations were scheduled at least 4 days apart. Punishment was introduced for the rats of the second group by presenting electric shock to the tongue for the duration of each lick. The intensity of the shock was adjusted so that the daily intake of each rat was suppressed by about 50 ~ of the preceding punishment-free condition. The shock intensities ranged from 290-470 pA. As soon as the daily intake of each :'at was consistently suppressed to 50 ~o of the non-punished intake, drug treatments were scheduled as described for the non-punishment group. Six rats of the punishment group completed all drug conditions. Following the daily experimental session in the chamber, each rat of both groups was returned to its home cage and given an additional 30 min free access to water. The total number of licks and the water intake during the experimental session as well as the water intake in the home cage were recorded. Statistical Analysis. A two-factor repeated measures design was used, factor one being dosages of drugs and factor two being days of experimental treatment (pre-drug, drug, and post-drug days). In cases of significant overall F-scores, the Newman-Keuls procedure (Winer, 1971) was used to identify differences between individual means.
Results With the presently employed 23 hrs water deprivation regimen all rats achieved a stable daily lick rate and water consumption after about 3 weeks, and maintained these baseline values over several months. Scopolamine hydrobromide decreased non-punished water intake [F(4,28) = 7.9; P < 0.01] and lick rate [ F ( 4 , 2 8 ) = 2.9; P < 0.05] slightly, but signifi-
K. A. Miczek, and P. Lau: Scopolamine, Physostigmine, Chlordiazepoxide and Inhibition ~j
SCOPOLAMINE HYDROBROMIDE
o
~50
SCOPOLAMINE METHYL NITRATE
PHYSOSTIGMINE
NEOSTIGMINE
CHLOR DIAZEPOXIDE
W
