Department of Pharmacology, Medicat College of Virginia, Richmond, VA 23298, USA. Abstract. Rats were ... chamber (Lehigh Valley Electronics model 1417 or.
Psychopharmacology
Psychopharmacology(1981) 75 : 383 - 387
9 Springer-Verlag 1981
Discriminative Stimulus Properties of Arecoline: A New Approach for Studying Central Muscarinic Receptors Leonard T. Meltzer* and John A. Rosecrans Department of Pharmacology, Medicat College of Virginia, Richmond, VA 23298, USA
Abstract. Rats were trained to discriminate arecoline (l.74mg/kg) from saline in a milk-reinforced (variable interval i2s) two-lever operant paradigm. The discriminative stimulus (DS) effects of arecoline were antagonized by atropine sulfate, but not by atropine rnethylnitrate or mecamylamine. In contrast to the effects on discrimination, atropine did not antagonize the response rate suppressant effects of arecoline. The DS effect of arecoline completely generalized to oxotremorine, partially generalized to pilocarpine, and did not generalize to nicotine. These data demonstrate that the DS effect of arecoline depends on central muscarinic receptors. Key words: Arecotine - Drug discrimination - Muscarinic receptors
Arecoline (methyl 1,2,5,6-tetrahydro-l-methyl-nicotinate) is the most biologically active substance in the A r e c a c a t e c h u Linn, commonly known as the areca or betel nut. Arecoline is predominantly a muscarinic cholinergic agonist (Baker et al, 1971; Herz et aI. 1967) and at high doses produces some nicotinic cholinergic effects (Herz et al. 1967). The CNS effects of arecoline have been assessed by several neuropharmacNogical and psychopharmacological procedures; including effects on the cortical and subcortical electroencephalogram (Herz 1963; Kawamura and Domino 1969; Riehl et al. 1962; Sitaram et al. 1978; Yamamoto and Domino 1967), motor activity (Pradhan and Dutta ]970), operant behavior (Olds and Domino 1969; Pradhan and Dutta 1970), and tremor (Zetler t971). These studies have provided information concerning the role of central muscarinic systems in mediating or affecting different behaviors. However, these experimental procedures are not specific to studying central muscarinic systems or drug-receptor interactions, since the dependent variables in these studies can be similarly affected by nicotinic cholinergic agonists or other classes of drugs (Kawamura and Domino 1969; Olds and Domino i969; Pradhan and Dutta 1971; Seiden and Dykstra 1977; Yamamoto and Domino 1967; Zetler 1971; Meltzer and Rosecrans unpublished data). It is only through the use of agonist-antagonist interactions that one can ascertain the pharmacological specificity of the drug effect. This may not be sufficient in some instances, since Neuropsychopharmacology Research Unit, Department of Psychiatry, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06710, USA Offprint requests to: Leonard T. Meltzer * Present address:
physiological antagonism (interaction of two systems) may mimic pharmacological antagonism (interaction at the receptor). In order to directly study central muscarinic systems and drug-receptor interactions in intact animals, a more specific task is required. The discriminative stimulus (DS) paradigm provides a specific task in which to study the pharmacological properties of drugs which produce CNS effects. The observation that psychoactive drugs can serve as controlling or DS (internal) in a behavioral paradigm (Barry 1974; Overton 1974; Schuster and Balster 1977) indicates that these drugs produce effects that animals can distinguish from the nondrug condition, as well as from the effects produced by drugs that differ pharmacologically. Use of the DS paradigm has allowed the determination of the structure-activity relationships (SAR) of central serotonergic receptors (Glennon et al. 1980) and the SAR (Chance et al. 1978) and stereospedficity of central nicotinic cholinergic receptors (Meltzer et al. 1980). Arecoline has been demonstrated to serve effectively as a DS (Schechter and Rosecrans 1972b). However, a systematic evaluation of the DS properties of arecoline has not been conducted. The present experiments were designed to characterize the pharmacological specificity of the DS produced by this drug. Materials and Methods Male Sprague-Dawley rats (175-200g) with no previous drug or experimental experience were purchased from Flow Research Animals, Dublin, Virginia, and used in all experiments. These rats were individually housed in a temperaturecontrolled environment under a 12-h light-dark cycle. Initially, food (Purina Rodent Chow) and water were continuously available. After allowing 2 - 4 w e e k s for acclimation, rats were reduced to 80 % of their expected freefeeding weight by restricted feeding. For the remainder of the study, water was freely available in the home cages and adjusted amounts of chow were offered after each experimental session to maintain the animals at 80 % of their expected free-feeding weight. The experimental space was a standard operant test chamber (Lehigh Valley Electronics model 1417 or Coulbourn model El0-10). One wall of the chamber had two levers with a dipper centered between them for delivery of liquid reinforcement. Except where noted, both levers were always in the chambers. Above the dipper was a white house light that was on for the entire session. The experimental chamber was located in a larger sound-insulated and lightproof isolation cubicle. Solid state and electromechanical 0033-3158/81/0075/0383/$01.00
384 programming equipment were used to control sessions. Data were recorded automatically in the form of response and reinforcement totals. Equal parts of sugar and nonfat powdered milk (Land O'Lakes) mixed in tap water and delivered by the dipper (0.01 ml) was the reinforcer.
Procedure Initial Training: One Lever in Chamber. Six 60-90-day old male Sprague-Dawley rats, reduced to approximately 80 % of their normal body weight by restricted feeding, were trained to press one lever in a two-lever operant chamber for milk reinforcement. This lever was designated as the saline lever. After 3 - 4 days of responding on a continuous reinforcement schedule (CRF), rats were trained to respond on the second (drug) lever. Rats were injected with 1.74 mg/kg arecoline and were placed 5 min later in the operant chamber, with only the drug lever present. Rats usually initiated responding spontaneously on the lever, though some were trained if necessary. Session durations were 15rain. After 2 - 3 d a y s of CRF responding on the drug lever, training under saline and drug conditions were alternated. Saline was administered for 2 consecutive days, arecoline for 2 - 4 consecutive days, with only the state-appropriate lever in the chamber. At this time, a variable interval (VI) schedule of reinforcement was instated. The schedule was slowly increased from VI-3 s to VI-12 s. The schedule was increased in 3-s increments on each bar, individually. The criteria for advancement was 40 responses in the first 5 rain for 2 cumulative days in each state. When a rat attained a VI-12 s on both levers, discrimination training began. For half of the rats, the left lever was the saline-correct lever and the right lever was the arecoline-correct lever. The conditions were reversed for the remaining rats. The use of the 1.74 mg/kg training dose was based on pilot studies, which suggested that this dose was effective as a DS. Discrimination Training." Both Levers in Chamber. Rats were injected with arecoline or saline 5 rain before being placed in the operant chamber. Both levers were in the chamber. Responses on the correct lever were reinforced on a VI-12 s schedule. Responses on the incorrect lever had no programmed consequence. Saline and arecoline were administered in a double-alternation procedure (arecoline, arecoline, saline, saline, etc.). Responses on each lever, as well as total reinforcements received, were automatically recorded. Discrimination learning was assessed during a 2-rain nonreinforced period on day1 of each alternation. The data collected during this nonreinforced period are presented as percent drug-bar responding (%DBR), which are the responses on the drug correct lever/total responses. The overall rate of responding (responses per min, RPM) on both levers during this 2-rain period and during training sessions is a measure of drug-induced behavioral disruption. If a rat did not emit at least 5 responses during the 2-rain non-reinforced period, this session was extended until the rat emitted 5 responses. The total time required was recorded and used to calculate the response rate. Agonist and Antagonist Testing. Animals continued to receive 1.74 mg/kg of arecoline and saline according to the doublealternation sequence. The effects of the following experimental manipulations were assessed in these rats: (1) generalization of different doses of arecoline and antagonism of their
effects of atropine; (2) the time-course of the discriminative effects of the training dose (l.74mg/kg) and 1.16mg/kg arecoline; (3) the effects of selective cholinergic antagonists on the training dose; and (4) the generalization of the DS effect ofarecoline to cholinergic agonists. Tests were conducted in a 2-min nonreinforced session following day 2 of an alternation (arecoline, arecoline, test, saline, saline, test, etc.). This procedure did not disrupt the baseline discrimination. Animals were removed from the chambers after 2 min or after 5 responses were emitted, if animals took longer than 2 min to respond. Sessions were conducted for 6 consecutive days. At least 4 training days (one double-alternation) separated antagonist tests. Testing of drugs and doses was randomized so that approximately half of the test trials followed an arecoline-training day, and the rest followed a saline-training day. EDs0 was derived from linear regression analysis and represent the derived dose that produced 50 % DBR.
Drugs. The following drugs were used in these experiments: arecoline hydrobromide (Chemical Dynamics, Plainfield, NJ); atropine methylnitrate, atropine sulfate, and oxotremorine sesquifumarate (Sigma Chemical, St. Louis, MO); mecamylamine HC1 (Merck, Sharp, and Dol~me, West Point, PA); pilocarpine nitrate (Nutritional Biochemical, Cleveland, OH); and optically pure (-)nicotine-di-l-tartrate (synthesized and kindly supplied by Dr. Everette L. May), were obtained as the salt. These drugs were dissolved in 0.9 % saline in a concentration that resulted in an injection volume of 0.1 ml/100g body weight. All injections were SC with a 26 gauge, three-eights inch (1 cm) needle attached to a i ml syringe. In all experiments, drug dosage is expressed as the salt. Results
Acquisition of Arecoline Discrimination. Due to the disruption of responding produced by 1.74mg/kg arecoline, the rats in this group were advanced slowly on the VI schedule. The total number of days (mean _+ SEM) under saline and drug conditions in the initial one lever training segment of the experiment was 44.3 _+ 3.5. Rats learned to discriminate arecoline from saline (84 % DBR and 26 % DBR, respectively) by the second doublealternation of four sessions each. With continued drug and saline administration, the drug- and saline-appropriate responding increased. The mean %DBR responding reached a plateau at approximately 9 5 - 1 0 0 % after 1.74mg/kg arecoline and 5 - 0 % after saline by the tenth double-alternation (total of 40 sessions). Compared to the saline data, arecoline administration produced a decrease in the response rates under both testing and training conditions, and this relationship remained constant throughout the experiment. The test period response rate under the drug state was approximately 25 % of saline values (for example, on the tenth double-alternation the saline and arecoline test session response rates were 21.9 _+ 4.~ and 4.5 + 1.3, respectively). The response rates were higher during the reinforced training period, compared to the nonreinforced test period. The response rate during the training period under the drug state was approximately 50 % of the saline response rate: For example, on the tenth double-alternation the saline and arecoline training session response rates were 35.5 _+ 6.9 and 14.1 _+ 2.6, respectively; in contrast reinforcements received
385 ir--e SALINE-ARECOLINE(B=6) e,-,oATROPINE(4mg;kgl - AREI~OLIIIE(n=6} 120
120
so
/
g c~
I00
9
I0O
60
40
I
q
20-
SAL ATR O~SB 0 ' 8 7 I.'16 SAL
I)4 2*32 SAL ATR 0.58 SAL
0.87 LIB
L?4 P-~2
DOSE OF ARECOUNE {m(:J/kg}
Fig, 1. Dose-eft?ct relationship for DS effects (A) and response rate suppression (B) for arecolinealone and with atropine pretreatment. Each point is the group mean • SEM of one administration in each rat 1oo
o---o
1,16m g / k g (n=5)
-
_
C
;.
9
=
80r
60
m
~
40 2o
5 15 TIME (rain) POST INdECTION
25
Fig. 2. Time-course of DS effect of arecoIine. Each point is the group mean +_SEM of one administration in each rat were only 10 % less in arecoline than saline sessions, i.e., 43.3 +_ 2.8 and 47.9 + 2.4, respectively, on the tenth doublealternation. Throughout the study, rats injected with arecoline exhibited signs of peripheral muscarinic stimulation (salivation, urination, and defecation),
Pharmacological Specificity of the DS Effect of Arecoline. The DS effect of arecoline was dose-dependent (Fig. 1A). In contrast, the arecoline-induced decrease in response rates did not appear to be dose-dependent (Fig. t B). The training dose of arecoline, which produced approximately 92 % DBR, decreased RPM to 30 % of saline response rates. Decreasing the dose to 0.87 and 0.58 mg/kg decreased the discrimination level to approximately 40 % and 19 % DBR, respectively, the latter value being similar to saline. After 0.87 and 0.58 mg/kg arecoline, the response rates were still decreased to 35 ~s and 50% of the saline response rate, respectively. These data demonstrate a separation of the response-suppressant and discriminative-stimulus effects of arecoline. The time-effect relationship on discrimination for two doses of arecoline is presented in Fig. 2. When tested 5 min after injections, both the training dose (t.74mg/kg) and a dose two-thirds the training dose (1.16mg/kg) produced approximately 95 % DBR. However, the two doses demonstrated different time-effect curves. At 15min postinjection, 55 % DBR was observed after 1.74 mg/kg, while salinelike responding (5 % DBR) was recorded after 1.16 mg/kg. At 25 min after injection of the training dose (1.74 mg/kg), salinelike responding (2 % DBR) was observed. For both doses,
when %DBR had returned to saline levels, the response rates had increased above saline levels (27.9 _+ 10.8 RPM for 1.16mg/kg at 15min, 24.8 _+ 6.5 RPM for 1.74mg/kg at 25 min, compared to 14.8 + 2.8 RPM for saline at 25 min). Table 1 presents the effects of different antagonists on the %DBR produced by the training dose of 1.74mg/kg. Atropine methylnitrate (2 and 4 mg/kg) and mecamylamine (1 and 2.35mg/kg) did not antagonize the DS effect of arecoline. Atropine sulfate in a dose of 2mg/kg did not antagonize the DS effect of 1.74 mg/kg arecoline, but 4 mg/kg decreased the %DBR from a control value of 92.2 • 3.8 to 36,0 _+ 13.1. Atropine sulfate (4mg/kg), administered alone, did not alter response rates or %DBR from saline levels. Pretreatment with 4 mg/kg atropine sulfate produced a onelog shift in the arecoline dose-effect relationship (Fig. 1). The EDso of arecoline alone was 0.83mg/kg, compared to 1.70 mg/kg after atropine pretreatment. However, there was no antagonism of the arecoline-induced suppression of response rates by atropine. Based on gross observations of the subjects, the doses of atropine sulfate and atropine methylnitrate used prevented the peripheral parasympathetic stimulation produced by arecoline. This data again demonstrated a separation of the DS and rate-suppressant effects of arecoline. The generalization of the DS effect of arecoline to different cholinergic agonists is presented in Table2. Different doses of oxotremorine at various time points were tested for generalization. %DBR equal to that produced by the training dose of arecoline (indicating complete generalization) was observed with 0.1 mg/kg oxotremorine sesquifumarate administered 20m in prior to testing. As was evident with arecoline, the suppression of the response rates induced by oxotremorine administration was unrelated to %DBR. Arecoline partially generalized to pilocarpine nitrate administered at different doses and various time points. Between 30 ~ to 55 % DBR was produced by the various time and dose combinations. Due to the peripheral parasympathetic stimulation produced by pilocarpine, it was necessary to pretreat rats with 2 mg/kg atropine methylnitrate. This dose of the peripheral antagonist completely blocked all observable signs of peripheral muscarinic stimulation (salivation, urination, and defecation). Increasing doses of pilocarpine completely disrupted behavior before complete generalization with arecoline could be attained. Arecoline did not generalize to optically pure ( - ) n i c otine-di-l-tartrate, Discussion
Arecoline effectively exerted DS control of behavior at a dose of 1.74mg/kg. An attempt was made to optimize the conditions for discrimination learning by training the rats to respond on the state-appropriate levers from the beginning of the experiment, rather than training the rats to respond on both levers under the nondrug condition first (Overton 1979). During the initial training period, the response requirement for advancement was used to try to control for drug induced disruption of responding, which would interfere with a rat's interaction with the appropriate lever. The response rates under the arecoline condition were approximately 25 ~/o of the saline response rates. This differs from discrimination studies with other drugs (nicotine, Meltzer et al. 1980; LSD and amphetamine, D. Minnema
386 Table I. Effect of antagonists on arecotine cue. Antagonistswere administered SC 25 rain prior to the test session. Arecoline (I.74 mg/kg) and saline were administered SC 5 rain prior to the test session, N indicates the number of subjects completing response requirement/subjects tested. Each vatue is the mean _+SEM Pretreatment
Test
N
RPM
~s
Saline Saline Atropine methylnitrate (2 mg/kg) Atropine methylnitrate (4 mg/kg) Mecamylamine (1 mg/kg) Meeamylamine (2.35 mg/kg) Atropine sulfate (2 mg/kg) Atropine sulfate (4 mg/kg) Atropine sutfate (4 mg/kg)
Saline Ji.74 1.74 1.74 1.74 1.74 1.74 1.74 Saline
6/6 6/6 6/6 6/6 5/5 6/6 6/6 6/6 6/6
17.5 4.3 7,8 5.8 4.6 4.2 5.5 3.7 15.6
3,9 92,2 96.0 97.9 99.1 86,2 93.4 36.0 3.3
+ 3.I 4- 1.2 _+ 3.1 + 1,6 _+ 1.8 • 0.9 _+ 0.9 _+ 0.8 • 3.1
+_ 1.4 _+ 3.8 + 2.7 _+ 2.t • 0.9 _+ 10.0 • 3.8 +_ 13.1 • 3.3
Table2. Generalization of DS efl'ect of arecoline to cholinergic agonists. Agonists were administered SC at times indicated prior to test session, N indicates the number of subjects completing response requirement/number tested. 2 MA indicates 2 mg/kg atropine methylnitrate administered SC I 0-15 rain prior to pilocarpine nitrate. Each value is the mean +_SEM of subjects that responded Drug
Time (rain)
N
RPM
>s
0.20 0.10 0.10 0,10 0.05
5 5 10 20 20
5/5 5/5 4/5 5/5 4/4
3.0 3.8 2.7 6.8 5.1
+_ 2.0 • 1.3 • 0.3 _+ 3.1 • 1.0
60.2 53.1 52.2 87,1 23.3
• t6.7 + i3.5 _+ 16.2 _+ 6.2 +_ 11.3
4.00 8,00 2 M A + 8.00 2MA + 12.00 2MA ~- 16.00 2 MA + 8.00 2MA + t2.00 2 MA + 16.00
10 10 10 10 10 25 25 25
4/5 0/2 5/5 4/5 4/5 5/5 4/4 0/5
3.2 _+ 1.0
44.5 31,5 55,2 32.I 38.0 29.3 -
_+ i6.2
(-)Nicotine bitartrate
0.57 1.14 1.14
I5 15 5
6/6 5/6 5/6
Arecoline HBr
1,74
5 5
Oxotremorine sesquifumarate
Pilocarpine nitrate
Saline
Dose (mg/kg)
OAml/lO0 g
personal communication; phencyclidine, K. Brady personal communication) in which the response rates are similar under both drug and saline conditions. All o f these drugs suppress response rates upon initial administration. However, with repeated administration, tolerance develops to the response rate suppressant effects of nicotine, LSD, amphetamine, a n d phencyctidine, but not to this eft~ct of arecoline. In a separate study, after approximately 30 days of chronic treatment, no tolerance developed to the response rate suppressant effects of arecoline on a VI schedule (Meltzer and Rosecrans manuscript in preparation). However, in the same study, tolerance did develop to the response rate suppressant effects of arecoline on a fixed ratio schedule. It may be argued that if more reinforcements were received under one condition compared to another, that discrimination may be biased towards that lever. Despite the fact that arecoline decreased the response rate in the training condition, the total reinforcements received were similar under both drug and saline states. Thus, the nnmber o f reinforcements received did not affect or bias the discrimination learning. The DS effect of arecoline appears to be mediated through central muscarinic receptors. This was demonstrated by the
9.6 3.2 2,1 1.4 1,8 -
+_ 4.0 + 1.5 • 0.4 +_ 0.5 _+ 0.4
_+ 16.7 _+ 18.8 • t6.0 + 8.0 • I2.4
t8.7 + 3.7 11.4 + 2,4 4.4 _+ 2.7
1.0 +_ 1.0 1.2 + 1.2 17.2 • 8.4
6/6
4.3 + 1.2
92,2 _+ 3.8
6/6
17,5 • 3.1
3.9 +
1.4
antagonism by atropine sulfate, and not by atropine methylnitrate or mecamytamine, of the DS effects o f arecotine. Complete generalization to oxotrernorine and the tack o f generalization to nicotine also demonstrates the involvement of muscarinic receptors, It should be a d d e d that the lack of antagonism by atropine methylnitrate and mecamylamine does not appear to be a function o f the dose. Atropine methylnitrate has been demonstrated to be at least tenfold as potent as atropine sulfate in blocking the effects of peripheral muscarinic stimulation by acetylcholine (Morrison et al. 1969). The highest dose of mecamylamine tested was equimolar with 4 mg/kg atropine sulfate. It is also 2.5-times the dose required to completely block the discriminative stimulus effects of 1.14mg/kg nicotine (Meltzer et al. 1980). Arecoline did not generalize to optically pure ( - ) n i c o t i n e . This is similar to the lack o f generalization of nicotine to arecoline in rats trained to discriminative nicotine from saline (Schechter and Rosecrans 1972c). Rats can also discriminative arecoline from nicotine (Schechter and Rosecrans t972a). These studies support the existence of separate muscarinic and nicotinic cholinergic systems. Pilocarpine, in doses that were eightfold the molar concentration of the training dose of arecoline, failed to
387 produce complete generalization to the DS effect of arecoline. One explanation for this is that pilocarpine did not enter the CNS in a high enough concentration to mimic the arecoline effect. In the present study, rats were pretreated with atropine methylnitrate prior to pilocarpine administration. This antagonized all observable signs of parasympathetic stimulation. The fact that responding was completely disrupted by the highest doses of pilocarpine tested, even after blockade of peripheral muscarinic receptors, implies that pilocarpine did indeed reach behaviorally effective levels in the CNS. This data may provide evidence for the existence of two central muscarinic systems, only one mediating the DS effect of arecoline. Thus, the lack of complete generalization by pilocarpine may be due to either a lower receptor affinity or intrinsic activity for the receptors that mediate the DS effect of arecoline. There was a dissociation of the dose-effect relationships of the DS effects and response rate suppressant effects of arecoline. The ~ D B R produced by the lowest dose of arecoline was similar to saline values, but the response rate was 50 % of the saline value. A dissociation between response rates and discriminability has also been observed for amphetamine (Kuhn et al. 1974). In addition, atropine sulfate antagonized the DS effects of arecoline, but not the response rate suppressant effects. These data may also indicate the presence o f two separate muscarinic cholinergic systems in the brain, one that mediates the discriminative stimulus effects of arecoline and a second that mediates rates of responding or m o t o r output. In addition, the sensitivity of the latter system to muscarinic stimulation appears to be greater than the former. Support for the existence of multiple muscarinic receptors comes from studies on peripheral organs (Barlow et al. 1976, 1979) and studies on the CNS (Birdsall et al. 1978, 1980). There are also multiple cholinergic pathways in the brain (McGeer and McGeer 1979). An alternative, but not exclusive, explanation is that the DS effects of arecoline or muscarinic stimulation are very weak and, hence, require a maximum stimulation of the central muscarinic system by arecoline. In contrast, operant behavior response rates are very sensitive to disruption by muscarinic stimulation. Thus, discrimination can only be attained at doses that disrupt behavior (decreased response rates). It may be concluded that the discrimination procedure is specific for muscarinic stimulation, but may not be very sensitive. This differential sensitivity hypothesis (DS effect versus response rate) may be confounded by the lack of tolerance to the response rate suppressant effects of arecoline on a VI schedule.
Aclcnowledgements.This work was supported by the National Institute of Drug Abuse grant DA-07029. References
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Received September 17, 1980; Final version August 28, 198I
