Twenty-two male, Long-Evans Hooded rats (Harlan Sprague-Dawley, Blue Spruce Farms stock, Indianapolis, IN) were received in four groups of 5 or 6 rats each at 6 weeks of age. They were individually housed in clear plastic cages (25.9 × 47.6 × 20.9 cm) with an approximately 3 cm depth of wood shavings, and had free access to food and water. Once training began, they were fed once per day approximately 30-min following any experimental session. Weights were permitted gradually to increase during training, and were maintained at approximately 340 ± 10 g. Room lights were on a 12:12 light/dark cycle (lights on at 6:00 a.m.). One or two non-nutritive rodent enrichment items (Bio-Serve, Inc., Frenchtown, NJ) were introduced to the home cage at various times with no change in behavioral performance during experimental sessions. Housing conditions and animal care and use were in accordance with U. S. Public Health Service Policy.
The six custom-made operant conditioning chambers are described elsewhere (Ator, 1991
). Briefly, two rodent levers were mounted 13 cm apart on one wall with identically colored cue lights mounted one over each lever that provided the only illumination. A food cup, into which 45-mg food pellets (P.J. Noyes, Lancaster, NH, USA) could be delivered, was centered on the chamber wall opposite the levers. The sound of an electromechanical relay operation accompanied each pellet delivery. White noise and a ventilation fan in a larger sound-attenuating enclosure masked extraneous sounds. Experimental events and the collection of behavioral data were controlled by an IBM-compatible computer, solid-state chamber interface cards, and MED-PC® State Notation software (Georgia, VT, USA). The computer program was custom-written for the laboratory, and included collection of data on a pellet-by-pellet basis. Lever presses and feeder operations also were monitored with an event recorder (Esterline-Angus, Indianapolis, IN, USA). Another clear Plexiglas operant chamber with an associated control console (Gerbrands Corp., Model 2150, Arlington, MA, USA) was used for initial training of the lever press for each rat by use of a separate, hand-operated switch that could operate the feeder. In that chamber, a cue light was over the lever, and the food cup was located next to the lever.
TPA023 and TPA023B were generously donated by Merck, Sharp, & Dohme (Harlow, UK). Lorazepam was generously donated by Wyeth Research (Princeton, NJ, USA). Zolpidem hemitartrate was purchased from RBI (Natick, MA, USA). All drugs were given as i.p. injections in a volume of 1 ml/kg, except that TPA023B at 18 and 32 mg/kg were administered as 2 ml/kg. TPA023B and TPA023 doses were dissolved in polyethylene glycol 400, which then was diluted 50% with 0.9% saline, and maintained for no more than 7 days. Lorazepam doses were prepared as a stock solution of 80:20 propylene glycol:polyethylene glycol 400 (maintained for up to 30 days), which then was diluted 50% with 0.9% saline (maintained for up to 7 days) for injection. Zolpidem hemitartrate was dissolved in 0.9% saline in a volume of 20 ml, and maintained until it had been used. Doses are expressed as the form of the drug given above (zolpidem base was 80.4% of the salt).
Drug Discrimination Training
Because of the importance of the outcome of training for the TPA023B group in relation to the other three, the methodology will be described in detail. The groups of rats were trained in the following order: zolpidem (n=5), lorazepam (n=6), TPA023 (n=6), TPA023B (n=5). Each rat first was habituated to the clear Plexiglas chamber and to the sound of the operation of the pellet feeder. Once pellets were being retrieved reliably when the feeder operated, lever press training by reinforcing successive approximations (shaping) began. Once the lever press was acquired, training switched to one of the six experimental chambers to which the rat was assigned.
Experimental sessions were conducted Monday through Friday with each group running within the same hour each morning. The cue lights over both levers were illuminated when the rat was placed in the chamber. Each rat was assigned a no-drug (ND) and drug (D) lever, the side positions of which were counterbalanced within each training group. Lever assignments also were counterbalanced across successive rats assigned to a particular experimental chamber. Shaping the lever press again was necessary due to the location of the food cup on the opposite wall, and shaping began on the ND lever. Pellet delivery was followed by a 1-s timeout, during which the cue lights were off and lever presses were counted, but had no programmed consequences. When a rat began to press the ND lever reliably, the response requirement was increased within and across the 20-min sessions until it was a fixed ratio (FR) of 5 responses per pellet. The criterion for raising the FR value by 1 was receipt of approximately 20 pellets (+/− 3) at a given FR value. Timeout was increased to 5-s at FR 2 and to 10-s at FR 5. A consecutive-response contingency was in effect such that pressing the other lever prior to completing the response requirement on the ND lever reset the count.
Drug discrimination training per se began when a rat reached FR 5 on the ND lever. Injections of zolpidem 2.0 mg/kg, i.p., lorazepam 1.0 mg/kg, i.p., TPA023 1.0 mg/kg, i.p., or TPA023B 3.2 mg/kg, i.p. occurred at pretreatment intervals (min) of 30, 60, 15, and 15, respectively. These intervals and doses were based on successful previous training for zolpidem and lorazepam in our laboratory, on evidence for discriminative effects of TPA023 in rats trained to discriminate flunitrazepam (Ator, unpublished), and on information that parameters for behavioral effects of TPA023B should be similar to TPA023 (Gerard R. Dawson, personal communication). Rats were placed in the experimental chamber for the last (or total) 15-min of the pretreatment interval during which timeout was in effect. At the end of the presession timeout, the cue lights were illuminated and pellet reinforcement depended on meeting the FR contingency on the D lever. Initially, the response requirement was 1, with a timeout of 1-s after each pellet; and shaping again was used to train the rat to press the D lever. After a rat was pressing the D lever, a double-alternation sequence of D and ND training sessions began (i.e., DD NDND DD NDND), all sessions were preceded by a 15-min timeout, and the timeout after each pellet delivery was 10-s. The FR requirement was raised to 10 across sessions, generally according to the following sequence: D FR1-2, D FR 3-4, ND FR 3-4, ND FR 4-5, D FR 4-5, D FR 5-6, ND FR 5-6, ND FR 6-7, etc., but it was customized for individual rats to promote each rat’s completing the sequence quickly and obtaining at least 20-25 pellets (usually more) per session under both D and ND conditions. The within-session increase on D training days occurred after at least 20 pellets had been obtained. Single alternation of D and ND training sessions generally began after the first FR10 session and performance was assessed to determine whether the performance criterion (see below) to be able to begin testing was met.
After a mean of 11.8 alternating D and ND sessions at FR 10 at the 3.2 mg/kg training dose, during which no rat met the criterion for testing, training conditions were individualized based on performance. Rats were given 4 to 6 sessions under any one condition before the next manipulation was made. Initially the approach was to raise the FR to values greater than 10 by 3 or 5 responses all at once, given that the number of pellets per session was >30 for the previous four sessions. It was lowered on an individual basis if a rat failed to obtain at least 20 pellets in a session, and then gradually raised again to at least FR 10. When this manipulation was not proving successful at achieving criterion performance, the pretreatment time was increased to 30 min. Next, the pretreatment time was held constant, but the dose was increased to 10 mg/kg, and a double-alternation sequence of D and ND sessions was used. After 19 sessions, the FR was increased to 15 for an additional 16 sessions, this time with single alternation of D and ND sessions. The dose then was raised to 18 mg/kg, and the pretreatment time was increased to 120-min based on newly obtained pharmacokinetic information for rats (John R. Atack, personal communication). After 20 sessions, the FR was lowered to 10 for 10 additional sessions before increasing it to 15 for 19 sessions. In a final manipulation, which took the long elimination half-life of TPA023B into account, rats were given one day off (O) after a drug session (i.e., one cycle of training consisted of D O ND). This 3-session cycle was repeated for 13 cycles with no trend toward improvement in accuracy.
Generalization and Substitution Tests
The performance criterion was that (a) at least 95% of all responses in a session had to occur on the appropriate lever and (b) the first 10 consecutive lever presses had to be on the appropriate lever. Once this dual criterion was met in four consecutive sessions, a test session with the training drug vehicle and one with the training drug dose were given. Contingencies in test sessions were the same as training sessions except that making 10 consecutive responses on either lever produced a food pellet. These performances were assessed for demonstration of stimulus control by the training conditions. Stimulus control was demonstrated if (1) at least 80% of the total responses in the session were made on the ND lever in the session when vehicle had been injected, (2) at least 80% of the total responses in the session were made on the D lever in the session when the training dose had been injected, and (3) these results occurred in both test sessions sequentially. If the first and second elements of the stimulus-control criterion were not met, at least four D and ND single-alternation training sessions had to meet the performance criterion before both stimulus control tests were repeated.
Once stimulus control was demonstrated for zolpidem, lorazepam, and TPA023, tests could occur every third session, given that the performance criterion was met in the intervening ND and D sessions. Failure to meet criterion in any one training session required that criterion performance be shown in 4 consecutive training sessions in which D and ND alternated before the next test could occur. Dose-effect determinations were made for each group first with its training drug. Before testing with TPA023B, the zolpidem-trained rats were tested with CP730,330 (a partial BZ agonist), L-838,417, and dextofisopam; the lorazepam-trained rats were tested with CP730,330, dextofisopam, and pentobarbital; these data are not presented here. Tests with TPA023B were conducted with a pretreatment interval of 15-min.
Assessment of the Ataxic Effects of TPA023B
Ataxia after TPA023B was assessed in the TPA023B group approximately 5 months after the termination of training, during which the rats were not used in any experiments. The decision to make this assessment was based on a noticeable flaccid body tone after the first administration of 18 mg/kg with a pretreatment of 120 min. The procedure used was described by Melnick et al. (2002)
. Briefly, the frequency of “paw slips,” defined as one paw falling below the floor rods in the experimental chamber and exposing the ankle joint to the observer, was assessed in 5-min sessions for each rat. A baseline measurement of paw slips was made for each rat 120-min after a vehicle injection. Twenty-four hours later, rats were pretreated with 32 mg/kg TPA023B 120-min prior to a second 5-min determination of paw slips.
One-way analysis of variance (ANOVA) was used to determine whether the total number of sessions to meet the performance criterion for initial testing differed among the lorazepam, zolpidem, and TPA023 groups. For drug discrimination test sessions, responding across levers was calculated as the percentage of total responding on the D lever, excluding responses during timeouts. Consistent with the drug discrimination literature, full generalization was concluded if the percentage of D-lever responding was 80% or more. Conversely, 20% or less of D-lever responding was not considered to indicate D-like discriminative stimulus effects. Given the 95% accuracy criterion for training session performance, these thresholds can be considered significantly different from chance for a conditional discrimination (Sidman, 1980
). Response rates were calculated by dividing the total responses on both levers when the cue lights were illuminated by the total session time minus timeouts. ED50
values were determined by drawing a line from the Y-axis at 50% to the group mean curve and from that point to the X-axis. ED50
values are reported to the nearest 1/8 log10
-unit dose where the line met the Y-axis. The ED50
for reduction in rate of responding was determined in an analogous manner by using the mean rate of responding under vehicle test conditions as 100%, and dividing that rate by 2 to determine 50%. The number of sessions to meet criterion after novel doses of the training drug were analyzed separately for each group using a repeated measures ANOVA with the within-subjects factor of Dose (the lowest dose of TPA023 was omitted so that the number of doses would be equal across groups for this analysis). Sessions to meet criterion after novel TPA023B tests in zolpidem-, lorazepam-, and TPA023-trained rats were analyzed using a 3-X-5 mixed ANOVA with the between-subjects variable of Training Drug (lorazepam, zolpidem, TPA023) and the within-subjects variable of TPA023B Dose (0.32, 1.0, 3.2, 10, 32 mg/kg) tested. Ataxia data are expressed as the mean (±SEM) number of paw slips under both conditions. A paired-samples t
-test was used to compare paw slips after vehicle with those after TPA023B. All determinations of statistical significance were made at p
<.05. Statistical analyses were conducted using the Statistical Package for the Social Sciences, Version 13.0.