Protocol for Stress Response Experiment Per Melaney
Protocol for stress response experiment per Melaney
Animals:
Female Sprague-Dawley and male Long Evans rats were purchased from Charles Rivers. The animals were kept under constant temperature, 12 hr light:dark cycle (Lights ON 0600 to 1800) and given food and water at libitum. All animals were housed two per cage for two weeks prior to mating. A trio-mating protocol was used for this purpose. Identification of sperm from vaginal lavage confirmed the conception day. Two to three days before giving birth, the females are separated into individual housing. Upon birth, pups will be culled to six males, six females. Each litter will be assigned to a group. The groups are as follows:
Groups:
- Wean 21d-grouphouse (3 rats/cage)*
- Wean 21d- isolation
- Wean 16d-grouphouse (3 rats/cage)
- Wean 16d-isolation
*Mar’s papers state that housing was 2-4 animals/cage.
Isolated animals will not be allowed to see, touch, or be touched by any other animals.
Care:
Lab will do all care. Cages will be changed weekly once pups are born and they will be feed ad libitum. Early weaned (16d) pups will be fed ground pellets and cow’s milk ad libitum, until they have fully developed incisors to feed normally (21d).
Mar’s Comment: This is very important!!!. I always made sure that at the time of early weaning the milk and powdered pellets were EASILY REACHABLE by the pups. I used to place a petri dish with the milk inside the cage and dip the pup’s mouth on it to “prime” it. I checked these “isolates” every day and changed the petri dish with milk every day without handling them.
Within Litter Design:
Within each litter, one male and one female pup will be assigned to one of six experimental subgroups. They are as follows:
- Basal 75d
- Restraint 15min at 75d
- Circadian 75d
- Basal 30d
- Restraint 15 min at 30d
- Circadian 30d
1-Restraint 15 min at 75d-
2 hrs after lights off, the animal will be restrained for a period of 15 min. Blood will be collected through tail-nick at the following times post-restraint
15’’ 8:15 PM---2015
30’’8:30 PM---2030
120’’10 PM-----2200
Mar suggests doing circadian and stress response on the same animal (see below). In this scenario, two animals would be assigned to both circadian and stress so that alternating times could be obtained from each. This would limit the number of tailnicks and the likelihood of an influential effect on subsequent CORT, ACTH release.
My own opinion is that we should do all the stress time points in the same animal being careful about the technique. Gig has done this successfully and we have too. Others in the field as well. The cleanest way is cath but we already decided against this since the original model did not have cathed animals.
Mar’s Comment- [I guess the multiple tail nicks will affect the subsequent ACTH and CORT release. Is it possible to assign different animals to each time point????. I guess not; in this case, I just wanted to suggest to use the same animal for circadian tail nick and then do the restraint stress on it, getting only one time point from it by decapitation, and getting the brains, and pituitaries too. I guess that my question is: can you assign the same animal for circadian 75d and for one of the stress time points (numbers 2 and 3 of each litter would be only one animal in this way). Just asking!!!!
-Basal at 75d-
Time 2000- Animal will be decapitated at the same time as the Restraint group is starting their restraint time. Brains, pituitaries, thymus and trunk blood will be collected.
This serves as one of the circadian points and pre-stress.
3-Circadian at 75d-
Animal will be tail-nicked at 1600 –start of the rise in CORT, also circadian ACTH peak
2200- nadir of CORT once lights OFF has started, ACTH is stable at about 100 pg from 1800 to 0600
0400- lowest CORT is 4 hrs prior to lights OFF
1000- low CORT
The animal assigned to circadian time point can have the one point collected through trunk blood. We can alternate the time of decapitation in each litter so that one clean one is obtained. This would mean that all circadian points would be obtained on consecutive days (only one nick per day).
Mar’s Comment: I also would like to suggest to get each blood sample point by tail nick in different days, to let the animals recover from the stress of handling, and tail nick [just a reminder: the early weaned/isolated animals are aggressive, jumpy and reactive to handling.
**In order to have a developmental perspective on stress response on these animals, the next three sets of animals within each litter will have procedures performed on 30d and again on 75d (except for 5!).
4-Restraint 15 min at 30d-
5-Basal at 30d- decapitation
6-Circadian at 30d-
**One could argue that these animals could be used for circadian points by decapitation, but we would like to try this to have data that would be part of the student’s thesis work.
Mar’s Comment: (O.K…..it is a nice longitudinal question)
Behavior Test: (Mar’s Protocol Locomotion was done very late- 10 mo and also around 75 d)
Novelty Exploration: Locomotor Activity 90 minutes
2 days prior to restraintPND 28 day old
PND 73
Mar’s Comment: Open Field was done in her protocol.
Millie’s Comment: I suggest Novelty Exploration just because that is the equipment I have available.