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# Savings Protocol # **This protocol was first posted on 5/28/2018 (see version history), prior to data collection. At this stage, the protocol has been formalized in a [registered-report proposal][1].** **The remainder of this page is preserved to document the original posting. There are no substantive modifications from the original plan except that it didn't fully describe the forgotten-memory control group. As of 8/1/2019 the registered-report manuscript is controlling.** ## Protocol History ## - This protocol was developed from Philips et al., 2006, but adapted to work with a within-subjects unilateral sensitization paradigm. - This protocol was then refined and perfected; it was the basis of the work reported in [Perez et al., 2018][2] that was posted on the OSF: - This adaptation is for an experiment to track the transcriptional changes that occur 1 day after savings memory is evoked. ## Overview ## Animals will receive unilateral long-term sensitization. Animals will be tested for savings 1 week after training, a time point at which the behavioral expression of the memory has largely faded. To evoke savings, a reminder shock will be applied on the tail midline. T-SWR durations will be monitored on both sides before training, 1 day after training (to show normal memory), 1 week after training (to show forgetting), 10 minutes after the reminder (to look for short-term changes due to the reminder), and 1 day after the reminder (to look for long-term changes due to the reminder). To compare with a new memory, a 1-day comparison group will follow the same protocol but initially receive sham training. Then, when the original group receives the reminder, this control group will receive normal LTS training. That way, they will be sacrificed 1 day later with a newly expressed memory and fully controlled for the duration of protocol, amount of testing, etc. ## Behavioral Methods ## ### Training ### Animals will receive standard unilateral long-term sensitization training exactly as described in Herdegen et al., 2014. Briefly, training will consist of 4 rounds of noxious shock applied at 30 minute intervals to one side of the body with a hand-held electrode. Each round of shock will consist of 10 pulses of 500ms duration at a rate of 1hz and an amplitude of 90mA. During the course of each shock, the stimulating electrode will be slowly moved from anterior (just behind neck) to posterior (just in front of tail) and back to cover nearly the entire surface of that side of the body. Side of training will be counterbalanced. One of the advantages of this training protocol is that both the induction and expression of sensitization is restricted to one side of the body (Antzoulatos and Byrne 2007; Antzoulatos et al. 2006; Cleary et al. 1998; Scholz and Byrne 1987; Wainwright et al. 2002), enabling each animal to serve as its own control (trained vs. untrained sides). In our paradigm, we typically observe complete forgetting by 7 days after training. For this protocol this will be defined as < 25% change from baseline at the 7 day test. Any animal showing more than this change (on either side) will be excluded from analysis. ### T-SWR Measurement ### As a behavioral outcome, we will measure the duration of the tail-elicited siphon-withdrawal reflex (T-SWR) (see (Walters and Erickson 1986)). The reflex will be evoked by applying a weak shock to one side of the tail using a hand-held stimulator (60Hz biphasic DC pulse for 500ms at 2ma of constant current). T-SWR behavior will be measured as the duration of withdrawal from the moment of stimulation to the first sign of siphon relaxation. For each phase of testing (pre-test, 1-day post, 7-day post, short-term reminder, long-term reminder), responsiveness will be characterized by a series of 8 responses evoked on alternating sides of the body at a 10-min ISI. Scores will be split by side of stimulation (trained vs. untrained) and averaged (4 responses/side for each time point characterized). For the short-term changes after the reminder, it will also be possible to examine each measurement individually, rather than averaged, to explore the time-course of change. Behavioral measurements will be made blind to side of training. ### Reminder ### Folowing Philips et al. (2006), the reminder shock will consist of 2 weak shocks delivered at a 15 min ISI (2s train, 15mA DC pulses at 50Hz). ## Measurement of Gene Expression ## Pleural ganglia will be harvested after the 1 day savings tests (in the 1-day comparison group this is 1 day after standard training). These ganglia contain the VC nociceptors thought to mediate much of LTS memory as well as several interneurons in the T-SWR circuit. Dissection and isolation will take place exactly as described in Herdegen et al., 2014b. Briefly, animals will be anesthetized. Plueral ganglia will then be rapidly excised, placed on ice, and homogenized. After homogenization, RNA will be extracted and reverse-transcribed for microarray and/or qPCR analysis. ## Sample Size Plan ## For microarray, we have found 8 samples to be sufficient to detect strong changes in gene expression. We also follow up microarry with qPCR in independent samples, with 8-10 usually being sufficient. Therefore, we plan for collecting 16-18 savings samples. We have already assessed the transcriptional mechanisms of memory 1 hour after training, so for the 1-day comparison group we will collect only 6-8 samples. Each sample for transcriptional analysis combines tissue from one left-trained animal and one right-trained animal. Therefore, we will need 32-36 aniamals in the savings group and 12-16 in the 1-day control. ## Quality Controls ## ### Behavioral Controls ### * Animals which do not show robust but unilateral LTS at the 1 day test will be excluded from further analsis. This is defined as at least a 30% increase in T-SWR duration relative to baseline at the 1-day post tests on the trained side and less than a 30% change relative to baseline on the untrained side * Animals must experience forgetting to be tested for latent memory. Any animal with >25% increase in T-SWR duration on the trained side prior to the reminder will be excluded from futher analysis. * **Animals should not show habituation on the untrained side. Any animal with <30% decrease in T-SWR on the untrained side at the test prior to savings will be excluded. * Animals in the 1-day comparison group should also show >30% increase in the trained side and no more than a 30% change in the untrained side 1 day after training.* ### Transcriptional Controls ### * Animals in the 1-day comparison must show increased BiP expression on the trained side. * For the savings group, we expect to see increased FMRF expression on the previously trained side, but are somewhat unsure if this will persist with the reminder. Before the expense of microarray, we will use qPCR to measure FMRF. If there seems to be a consistent up-regulation we will use this as an additional criterion for selecting samples. * To be used for transcriptional analysis, animals must show savings memory. [1]: [2]:
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