Attention, impulsivity and cognitive flexibility in adult male rats exposed to ethanol binge during adolescence as measured in the 5-choice serial reaction time task: the effects of task and ethanol challenges

Svetlana Semenova

Department of Psychiatry, University of California San Diego, La Jolla, CA 92093, USA

Running title: Adolescent ethanol binge, attention, and impulsivity

Svetlana, Semenova, Ph.D., Department of Psychiatry, School of Medicine, University of California San Diego, 9500 Gilman Drive, M/C 0603, La Jolla, California 92093-0603, USA; tel: (858) 534-1528; fax: (858) 858-534-9917; e-mail: ssemenova@ucsd.edu

SUPPLEMENTARY MATERIALS

5-CSRTT procedure and measures
	All testing was conducted in a set of 12 five-hole standard test boxes located within light- and sound-attenuated chambers (Med Associates, St. Albans, VT). Briefly, in this task, after an inter trial interval (ITI), a light stimulus was presented in one of the five response apertures. A nosepoke in the lit aperture was considered as a correct response and resulted in the delivery of a food pellet into the magazine. Nosepokes in non-lit aperture were considered “incorrect responses,” which were followed by a 5 s timeout period and no delivery of food reward. Failure to respond within the limited-hold (LH) period (i.e., an “omission”), additional nosepokes in any aperture after the initial response prior to head entry into the magazine (i.e., perseverative responses), and nosepokes made during the ITI (i.e., before the presentation of the target stimulus; premature responses) resulted in a timeout period and no food reward. Each session lasted 30 min or until 100 trials were completed, whichever occurred first. It takes approximately 40-50 sessions to train rats in the 5-CSRTT.
Accuracy (i.e., the ratio of correct responses to total correct + incorrect responses) is the main measure of attentional performance in the 5-CSRTT. Performance accuracy is a conservative measure because it is independent of omissions. Consequently, accuracy is minimally confounded by factors that nonspecifically suppress responding, such as sedation, locomotor disruption, and reduced motivation. Additionally, a decrease in omissions, typically accompanied by an increase in total correct responses, can reflect improved attention, even if accuracy is unaltered (Amitai and Markou 2010). Premature responses are the primary measure of impulsivity in this task because they represent a failure to inhibit inappropriate responses (i.e., poor inhibitory control). Perseverative responses constitute persistence in an initially rewarded behavior although it is no longer rewarded and are considered a measure of cognitive inflexibility (Dalley et al. 2002). Timeout responses may represent an additional measure of compulsivity related to cognitive inflexibility (Amitai and Markou 2010). Increases or decreases in correct response latency (i.e., the time from the onset of the light stimulus to the performance of a correct nosepoke response) may indicate slower or faster cognitive processing speed, respectively, or they may reflect alterations in the animal’s locomotor behavior or motivation for the reward. However, changes in correct response latency that are not accompanied by altered latencies to retrieve the reward likely reflect true changes in processing speed.

Supplementary Figure 1. Body weight (A) and body weight gain (B) of rats during and after AIE exposure. Data are expressed as mean ± SEM of body weights in grams (A) or as the percentage of body weight before exposure to ethanol/vehicle on P33 (B). *p < 0.05, **p < 0.01, ***p < 0.001, comparisons between AIE-exposed and control rats (Newman-Keuls post hoc test).

Supplementary Table 1. The number of sessions required to complete each training schedule during 5-CSRTT acquisition in rats exposed to ethanol or vehicle during adolescence and trained during adulthood.

Exposure	SD30LH60	SD20LH30	SD10LH15	SD5LH10	SD2.5LH5	SD1.5LH5	SD1LH5	Total

Vehicle		7.33 ± 1.15	1.0 ± 0.00	1.0 ± 0.00	1.17 ± 0.12	1.67 ± 0.35	2.58 ± 0.62	3.42-0.97	18.17 ± 1.43
Ethanol		9.17 ± 1.37	1.08 ± 0.09	1.00 ± 0.0	1.00 ± 0.00	2.5 ± 0.51	2.00 ± 0.50	4.67-1.22	21.42 ± 2.22

SD, stimulus duration (s); LH, limited hold (s). n = 12 per group.

Supplementary Table 2. Speed of responding of adult rats in the 5-CSRTT under baseline and task challenge conditions.

Measure				AIE-exposed rats		Vehicle-exposed rats

A: Baseline performance
Correct response latency (s)		0.72 ± 0.02			0.76 ± 0.02
Incorrect response latency (s)		1.50 ± 0.08			1.6 ± 0.10
Reward latency	(s)			1.91 ± 0.15			1.78 ± 0.07

B: Challenge: 0.5 s stimulus duration
Correct response latency (s)		0.66 ± 0.02 			0.74 ± 0.04
Incorrect response latency (s)		1.40 ± 0.08 			1.61 ± 0.10
Reward latency	(s)			1.79 ± 0.13			2.24 ± 0.62

C: Challenge: 0.25 s stimulus duration
Correct response latency (s)		0.71 ± 0.06			0.74 ± 0.06
Incorrect response latency (s)		1.55 ± 0.05			1.67 ± 0.09
Reward latency	(s)			1.74 ± 0.13			2.06 ± 0.15

D: Challenge: 15 s intertrial interval
Correct response latency (s)		0.92 ± 0.04			1.00 ± 0.07
Incorrect response latency (s)		1.84 ± 0.41			1.52 ± 0.25
Reward latency	(s)			1.96 ± 0.59			1.36 ± 0.04

Supplementary Table 3. ANOVA results for the effects of task challenge with a short stimulus duration.

Measure						Factors
AIE exposure		Challenge		Interaction

Accuracy (%)				0.006			116.9 ****		1.51
Correct Responses (%)			0.87			55.4 ****		2.9 (p = 0.06)
Incorrect responses (%)			0.09			58.14 ****		0.52
Omissions (%)				1.09			3.95 *			1.71

Premature responses			2.95			0.86			0.06
Perseverative responses			0.52			7.28 **			1.65
Timeout responses			2.37			10.08 ***		0.44

Correct response latency (s)		1.26			0.90			0.43
Incorrect response latency (s)		3.21			0.93			0.33
Reward latency (s)			0.69			0.23			0.73

Performance in a session during challenge with a shorter stimulus duration (SD 0.25 s and 0.5 s) than that used during training was compared with performance under baseline conditions (SD 1 s). Two-way ANOVA F values for main effects of the factors AIE exposure (df = 1,21) and Stimulus duration challenge (df = 2,42) and their interaction (df = 2,42). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Supplementary Table 4. ANOVA results for the effects of task challenge with a long intertrial interval.

Measure						Factors
AIE exposure		Challenge		Interaction

Accuracy (%)				0.01			1.38			0.99
Correct Responses (%)			1.02			72.13 ****		0.25
Incorrect responses (%)			0.04			2.93			1.25
Omissions (%)				0.10			74.25 ****		0.40

Premature responses			0.10			122.7 ***		0.34
Perseverative responses			0.94			43.77****		0.41
Timeout responses			0.78			6.24 *			0.50

Correct response latency (s)		3.3 (p = 0.08)		29.28 ***		0.66
Incorrect response latency (s)		0.20			0.35			1.00
Reward latency (s)			1.48			0.53			0.86

Performance in a session during a task challenge with a longer ITI (ITI 15 s) than that used during training was compared with performance under baseline conditions (ITI 5 s). Two-way ANOVA F values for main effects of the factors AIE exposure (df = 1,21) and ITI challenge (df = 1,21) and their interaction (df = 1,21). ***p < 0.001, ****p < 0.0001.

Supplementary Table 5. ANOVA results for the effects of acute ethanol challenge (vehicle, 1.5, and 3 g/kg) on 5-CSRTT performance in AIE-exposed and control rats during adulthood.

Measure						Factors
AIE exposure		Challenge		Interaction

Trials completed			9.36 **			8.37 ***		8.39 ***
Accuracy (%)				0.77			2.84 (p = 0.06)		2.32
Correct Responses (%)			3.83 (p = 0.06)		8.15 **			3.55 *
Incorrect responses (%)			2.3			0.55			0.35
Omissions (%)				6.25 *			8.87 ***		4.17 *

Premature responses			8.73 **			0.86			1.26
Perseverative responses			1.95			2.66 (p = 0.08)		0.23
Timeout responses			5.87 *			0.99			0.71

Correct response latency (s)		0.36			0.80			0.60
Incorrect response latency (s)		2.4			1.04			0.13
Reward latency (s)			0.34			0.49			0.65

Two-way ANOVA F values for main effects of the factors AIE exposure (two levels; df = 1,21) and Ethanol challenge (three levels; df = 2,42) and their interaction (df = 2,42). *p < 0.05, **p < 0.01, ***p < 0.001.

Supplementary Table 6. Speed of responding of adult rats in the 5-CSRTT after acute ethanol challenge.

Measure			Ethanol dose 		AIE-exposed		Vehicle-exposed
 				(g/kg)

Correct response latency (s)	vehicle			0.72 ± 0.03		0.81 ± 0.03
Incorrect response latency (s)	vehicle			1.51 ± 0.18		1.44 ± 0.19
Reward latency (s)		vehicle			2.51 ± 0.63		2.11 ± 0.22

Correct response latency (s)	1.5			0.82 ± 0.04		0.86 ± 0.05
Incorrect response latency (s)	1.5			1.95 ± 0.15		1.63 ± 0.25
Reward latency (s)		1.5			2.02 ± 0.31		2.03 ± 0.24

Correct response latency (s)	3			0.83 ± 0.05		0.78 ± 0.12
Incorrect response latency (s)	3			1.87 ± 0.16		1.7 ± 0.36
Reward latency (s)		3			2.09 ± 0.25		4.31 ± 2.97