This protocol describes a method of examining social hierarchy in a rat model. All the rats perform a complex diving for food task in which they form a distinct hierarchy according to the willingness to dive under water and swim to obtain a food pellet. This method is used to understand decision-making and social relationship among highly social animals in small groups.
Select adult male Sprague Dawley rats weighing 300 to 350 gram. Provide rat chow and water ad libitum. Perform all experiments between 6:00 am and 12:00 pm.
Maintain rats at room temperature of 22 Celsius degrees with 12 hour light and 12 hour dark cycles. A timeline of the experimental protocol. Randomly select 120 rats into one of three experimental groups.
Group one, N equals 60, will be used as a control group. Group two, N equals 30, will be induced with depression. Group three, N equals 30, will also be induced with depression and will subsequently be treated with antidepressant drugs.
Some cross preference tests performed prior and after induction of chronic unpredictable stress and after complex diving for food task. The following stressors are introduced in random order. Group housing by placing six rats per cage for 18 hours.
Tilted cage placement for three hours 45 degrees along the vertical axis. Food deprivation for 18 hours. Water depravation for 18 hours followed by immediate exposure to an empty water bottle.
Soft cage placement for eight hours consisting of 300 milliliters of water spilled into bedding. Continuous lighting and reverse light dark cycle for 48 hours per week And five minutes hot environment at 40 degrees Celsius. Rats are exposed to two of the seven stressors daily in a random order.
One in the daytime, and the second at night for five sequential weeks. Rats from group three are administered antidepressant treatment with amicrom hydrofloride, tricyclic antidepressant 20 milligrams per kilo intraperitoneally once per day for three weeks. Group two of the control group is administered 0.9%saline placebo intraperitoneally once per day for three weeks at the same volume as the antidepressant treatment group.
Prepare apparatus and acclimate rats. Connect two cages 50 on 50 on 50 centimeters to an aquarium 130 on 35 on 50 centimeters via tunnels. Place tubes with food pellets, one food pellet in each tube at one end of the aquarium.
Mark all six rats with different colored marker at the beginning of the experiment to allow individual identification. At any given moment, six rats participated in the experiments simultaneously. Three of them served as control group one and the other three were either depressive group two, or depressive after treatment, group three.
On day one of the experiment, introduce each group of rats to experimental apparatus without water for three hours session. Return rats to standard cage after session. Video-record rats in the apparatus continuously during the three hours session.
Repeat these sessions for days two and three. On days four til 17, add water progressively until maximum water level is reached. On day 17 til 21, maintain maximum water level.
Observe rats diving to access pellets. Record the following parameters. Frequency of entries into tunnel, diving for food, food obtained by attack, food obtained by carrying, and time spent in separate cages.
During the dark cycle place the rats in individual cages in the same room as the housing. Place a bottle of 100 milliliter 1%weight volumes sucrose solution in each cage for 24 hours and allow for adaptation. Remove the bottles and deprive the rats of food and water for 12 hours.
Place two bottles, one containing 100 milliliter of sucrose solution, 1%weight volume and the other 100 milliliter of tap water in each case for four hours. Record the volume in milliliters of both the consumed sucrose solution and water and calculate their affinity to sucrose preference as follows. Sucrose preference in percentage equals sucrose consumption in milliliters, divided by sucrose consumption and milliliters plus water consumption in milliliters.
All times 100%Body weight changes A one way Nova test did not show any differences in changes in body weight between experimental groups for the 21 days of the complex diving for food task. From days to two to 21, there were changes in body weight for all three groups, p-value was less than 0.01. Rats with depressive like symptoms were more active and aggressive than control rats.
Rats induced with the aggressive light behavior showed an increase of entries into the tunnel 130%plus minus 3.7 p value less than 0.01. Diving for food 141%plus minus seven, p value less than 0.01. Food obtained by a pairing, 168%plus minus 12, p value less than 0.01.
Time spent in separate cages 123%plus minus 7.9, p value less than 0.01, and food obtained by attack, 232%plus minus 26, p value less than 0.01. Compared to depressive rats that received antidepressant therapy, 67%plus minus 3.4, 54%plus minus five, 53%plus minus six, 55%plus minus 4.7, 44%plus minus seven respectively. The differences the rats induced with depressive like symptoms and depressed rats treated with antidepressant were statistically greater than the differences between the rats induced with depressive like symptoms and the control group in all five parameters of the complex diving for food tests, p value less than 0.05.
There was no difference in sucrose consumption at the beginning of the experiment. By day 35 of the experiment, the depression group p-value less than 0.01. And the depression group treated with antidepressant therapy, p value less than 0.01 had a significantly lower percent sucrose preference than the control group.
On day 62, the rats induced with depressive like symptoms had a lower percents across preference compared to both the control and the depression group treated with antidepressant treatment, p valued less than 0.05.Conclusion. The complex diving for food task can allow us to assess the relationship between the wide range of mental illness and social organization as well as to study the effectiveness of therapy and social dysfunction. Thank you very much for your attention.
