Protein Synthesis Inhibition and Appetitive Memory Reconsolidation: Effects of Cycloheximide on Retrained Odor Discrimination Memory in Long-

Evans RatsEric Gonzalez, Johanna Block and Gretchen Hanson Gotthard

Muhlenberg CollegeAllentown, PA 18104Introduction

There is a vast amount of evidence to support that the inhibition of protein synthesis results in the disruption of memory reconsolidation in appetitive tasks (e.g., Fulton et al., 2005; Malenfant et al., 1991; Robinson & Franklin, 2007; Stollhoff et al., 2008; Wanisch et al., 2008). Rodriguez-Ortiz et al. (2008) suggested that the consolidation of new memories requires protein synthesis, while “updated consolidation” of previously-established, unchanged memories does not. The present study examined concept of updated consolidation by training rats on an odor-guided discrimination digging task to a set criterion (Bunsey & Eichenbaum, 1996), then retraining them with identical additional training trials after injection of a protein synthesis inhibitor (cycloheximide, CHX; 1 mg/kg) or vehicle (distilled water). It was hypothesized that reactivating an unchanged memory would not make it susceptible to the effects of CHX, and therefore, produce no memory impairment.

Results The results of the present study showed that CHX had no amnesia-producing effects on relearned, unchanged memory for a non-spatial odor discrimination task.Acquisition The odor discrimination was learned to an equivalent level in all rats. A one-way analysis of variance (ANOVA) showed no difference between groups during the last training trial in terms of average latency to dig [CHX: M= 3.72, SEM= .84; Control: M= 32.82, SEM= 29.69; F(1,15)=.659, p=.430]. Furthermore, all rats reached the 75% acquisition criterion in eight trials.Retraining A repeated measures ANOVA showed a difference between Retraining Trial 1 (CHX: M= 9.25, SEM=2.98; Control: M= 21.32, SEM= 15.94), Retraining Trial 2 (CHX: M= 4.26, SEM=1.08; Control: M=10.89, SEM= 5.36), and Retraining Trial 3 (CHX: M= 3.65, SEM=1.01; Control: M= 19.81, SEM= 17.84) [F(1,15)= 4.12, p= .039], but no significant interaction between CHX and control groups [F(1,15)= .542, p=.593]. Testing A one-way ANOVA showed no statistically significant differences between CHX (M= 5.74, SEM= 2.03) and control (M= 6.72, SEM= 3.09) on latency to dig [F(1,15)= .057, p= .815] (see Figure 2). A preference index score was computed by dividing the time spent digging in the correct cup by the total time spent digging in both cups (+1 indicated perfect preference for the correct cup and -1 indicated perfect preference for the incorrect cup). All rats showed a strong preference for the correct cup (see Figure 3) with no statistical differences between the groups [CHX: M= .98, SEM= .02; Control: M= .90, SEM= .10; F(2,14)= 1.007, p= .390].

Shaping (Days 1-3)

One, then two cups of unscented sand

(10 trials total)

Training (Day 4)

Trained on one odor discrimination (at least eight trials with 75% correct

total)

Retraining (Day 13)

Injected 30 minutes prior to retraining

on odor discrimination (three

trials)

Testing (Day 14)

One probe trial on odor discrimination (No reward, with

criterion of no digging for 30

consecutive seconds to end trial)

Figure 1

MethodSubjects Twenty-one male Long-Evans rats were reduced to and maintained at 85% of their free-feeding weights two weeks prior to and during experimentation. Water was available ad libitum. The environmental conditions of the colony were held constant (temperature: 68° F; 12 hour light/dark cycle with lights on at 6:00 a.m.).

Apparatus Rats were trained and tested in two shoebox cages, identical to the rats’ home cages, with the exception of the bedding replaced by 1.5 inches of play sand. Plastic Gladware® cups (8 ounce) with Velcro® adhesive were filled with sand and anchored to a Plexiglas® plate (l=7.3 in., w=3.5 in.). Scented sand mixtures were created by mixing play sand (1,000 g) with cocoa (10 g) or cinnamon (10 g). To prevent responding to a learned location rather than a learned scent, cups locations were counter-balanced from trial to trial.

Procedure Rats were shaped to dig for Golden Puffs cereal buried in unscented cups of sand for three days prior to training (10 shaping trials total – see Figure 1 for procedure). On training day 4, rats learned the odor discrimination to at least a 75% correct criterion. For seven days, rats participated in an unrelated spatial task (sand maze; Gotthard, 2006), then returned to the present study. Thirty minutes prior to the retraining session, rats received an injection of cycloheximide (1 mg/kg; n=7) or a control injection (distilled water, 1mg/kg; n=10). Twenty-four hours after retraining, rats received one probe trial to test their memory for the odor discrimination.

SummaryProtein synthesis does not appear to be necessary for the reconsolidation of an active, yet unchanged, odor discrimination memory. This is consistent with the work by Rodriguez-Ortiz et al. (2008), which showed that “updated consolidation”, but not retrained/overlearned information, was reliant on protein synthesis. Additionally, the present study expands the relatively limited literature on protein synthesis and appetitive learning and memory.

References•Bunsey, M. & Eichenbaum, H. (1996). Conservation of hippocampal memory function in rats and humans. Nature, 379, 255-257.•Fulton, D., Kemenes, I., Andrew, R.J., & Benjamin, P.R. (2005). A single time-window for protein synthesis-dependent long-term memory formation after one-trial appetitive conditioning. European Journal of Neuroscience, 21, 1347-1358.•Gotthard, G.H. (2006). The sand maze. In M.J. Anderson (Ed.), Tasks and techniques: A sampling of the methodologies for the investigation of animal learning, behavior, and cognition (pp. 87-95). New York: Nova Science Publishers.•Malenfant, S.A., Barry, M., & Fleming, A.S. (1991). Effects of cycloheximide on the retention of olfactory learning and maternal experience effects in postpartum rats. Psychology & Behavior, 49, 289-294.•Robinson, M.J.F., & Franklin, K.B.J. (2007). Effects of anisomycin on consolidation and reconsolidation on a morphine-conditioned place preference. Behavioral Brain Research, 178, 146-153.•Rodriguez-Ortiz, C.J., Garcia-DeLaTorre, P., Benavidez, E., Ballesteros, M. A., Bermudez-Rattoni, F. (2008). Intrahippocampal anisomycin infusions disrupt previously consolidated spatial memory only when memory is updated. Neurobiology of Learning and Memory, 89, 352-359. •Stollhoff, N., Menzel, R., & Eisenhardt, D. (2008). One retrieval trial induces reconsolidation in an appetitive learning paradigm in honeybees (Apis mellifera). Neurobiology of Learning and Memory, 89, 419-425.•Wanisch, K., Wotjak, C.T., & Englemann, M. (2008). Long lasting second stage of recognition memory consolidation in mice. Behavioral Brain Research, 186, 191-196.

Spatial Task (Days 5-12)

Trained on an unrelated spatial

task (i.e., the sand maze) for seven days

Figure 2: Latency to Dig on Test

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Figure 3: Preference Index Score

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AcknowledgmentsThis research was supported in part by grants from the Sentience Foundation (to Eric Gonzalez) and the Provost’s Office at Muhlenberg College (to Johanna Block).