Table 2.

Methodological considerations for SPS in rats to guide experimental design and methodological reporting.

Experimental Feature	Methodological Recommendations	Additional Considerations
Experimental Conditions	Standardize time of day for the application of all experimental procedures, including SPS	Testing during either the light (inactive) or dark (inactive) phase; discussed in Considerations for Timing of Behavioral Testing
		Treatment of control animals not exposed to SPS; discussed in Control Animals
		Testing personnel; discussed in pop-out “Research personnel as part of an experimental context”
Housing Conditions	Group housing prior to SPS; age- and sex-matched; see Prior to SPS	Housing should be in accordance with the Guide for the Care and Use of Laboratory Animals, 8th edition
Animal Characteristics	SPS was optimized for group sizes of 6–8, age- and sex-matched	Handling frequency discussed in Methodological Considerations: Animal Handling Effects of age discussed in Methodological Considerations: Effects of Age Effects of sex discussed in Methodological Considerations: Effects of Sex Considerations for mice discussed in Methodological Considerations for SPS in Mice
	Handle animals prior to SPS to minimize effects of handling during experimental procedures; discussed in Methodological Considerations: Animal Handling
Restraint (2 h)	Restrainers should be fitted correctly; see Methodological Considerations: Single Prolonged Stress: Restraint Stress	Restrainer type (hard plastic, decapicone, etc.); discussed in Methodological Considerations: Single Prolonged Stress: Restraint Stress
	Separate restrainers should be maintained for different age groups and/or sexes, and cleaned carefully between uses
	Monitor animals to ensure they do not form a C-shape with their spine and become incapable of reorienting themselves as they may asphyxiate; see Methodological Considerations: Single Prolonged Stress: Restraint Stress
	Restrain animals in a procedure room separate from housing rooms to avoid scent transfer
Forced Swim (20 min)	Water temperature between 20 and 24°	Forced swim parameters (i.e., duration) will likely require optimization for females, younger animals, and different animal species/strains; see Methodological Considerations: Single Prolonged Stress: Forced Swim
	Container ~ 68 × 56 × 45 cm
	Closely monitor animals during the forced swim; see Methodological Considerations: Single Prolonged Stress: Forced Swim
	After the forced swim, dry animals as they are removed. Between groups, the swim container should be cleaned and refilled with fresh water
Recovery Period (15 min)	Hyper- and hypo-thermia should be avoided; see Methodological Considerations: Single Prolonged Stress: Forced Swim	A heat source can be used; discussed in Methodological Considerations: Single Prolonged Stress: Forced Swim
Ether exposure (until loss of consciousness, ~5 min)	All procedures with ether, including animal exposure, should occur under a chemical fume hood for safety	Animals can be allowed to regain righting responses under a hood to enable ether evaporating from their fur to dissipate in the hood rather than in the home cage
	Ether should be placed below a vented floor, not in direct contact with animals, and ether vapors should be allowed to fill the ether chamber prior to the addition of animals
	Closely monitor animals during ether exposure; see Methodological Considerations: Single Prolonged Stress: Ether
	After induction of general anesthesia, promptly remove rats from the ether chamber
7 day period following SPS-stressor exposure	Singly house animals for at least 7 days following SPS-stressor exposure; see Post SPS	Considerations for control animals in Control animals., and whether to house control and SPS animals in the same room and considerations for cage cleaning in Post SPS
	Animals should be “undisturbed” for 7 days following SPS-stressor exposure; discussed in Post SPS