Protein Synthesis is Not Required for Extinction of Paired-Associate Odor DiscriminationsSarah Linderman, Elizabeth Nguyen, and Gretchen Hanson Gotthard

Randolph-Macon Woman’s CollegeLynchburg, VA 24503

Introduction

Much research has shown that the administration of protein synthesis inhibitors blocks the formation of new fear memories (e.g., Nader, Schafe, & LeDoux, 2000). In fact, most of the research in this area has focused on the acquisition of fear responses or used tasks that required animals to respond under aversive conditions (e.g., Morris water maze; Meiri & Rosenblum, 1998).

Additionally, most studies examining the effects of blocked protein synthesis have examined acquisition of a completely new response, rather than extinction of an already-established response (i.e., learning to stop making responses that no longer work). A small number of studies have begun to examine the effects of protein synthesis inhibition on extinction (Lattal & Abel, 2001; Suzuki, et al., 2004); however, the results have been mixed and have been conducted with aversive tasks only (e.g., fear conditioning and the water maze).

The present studied used an appetitive paired-associate digging task (Bunsey & Eichenbaum, 1996) to examine the effects of a protein synthesis inhibitor (cycloheximide) on extinction in rats. Considering extinction is similar to acquisition in that it also involves new learning, it was hypothesized that protein synthesis inhibition would block extinction and produce continued high levels of responding during testing.

Results

AcquisitionAll rats included in the present analyses met acquisition criteria (i.e., two out of the last four training trials correct and at least 75% correct overall during training).

ExtinctionA one-way ANOVA revealed no differences in preference scores for the Cycloheximide (M=.58, SD=.45) and Vehicle (M=.35, SD=.39) groups on the extinction trial, F(12)=.913, p>.05 (see Figure 3).

Additionally, a one-sample t-test showed that the Cycloheximide group had a preference for the correct cup, t(6)=3.38, p=.015, while the Vehicle group showed a marginal preference for the correct cup, t(5)=2.24, p=.07 (see Figure 3).

RetentionA one-way ANOVA revealed no differences in preference scores for the Cycloheximide group (Test 1: M=.43, SD=.79; Test 2: M=.09, SD=.62; Test 3: M=-.37, SD=.75) and the Vehicle group (Test 1: M=.11, SD=.93; Test 2: M=.13, SD=.74; Test 3: M=.07, SD=.82) on Test 1: F(12)=.441, p>.05, Test 2: F(12)=.01, p>.05, or Test 3: F(12)=.99, p>.05 (see Figure 3).

Additionally, a one-sample t-test revealed that the Cycloheximide group [Test 1: t(6)=1.44, p>.05; Test 2: t(6)=.37, p>.05; Test 3: t(6)=-1.29, p>.05] and the Vehicle group [Test 1: t(5)=.29, p>.05; Test 2: t(5)=.43, p>.05; Test 3: t(5)=.2, p>.05] did not differ from chance on all test trials (see Figure 3).

Shaping (Day 1)

One cup of unscented sand (two trials)

Shaping (Day 2)

Two cups of unscented sand (two trials)

Shaping (Day 3)

Two cups of unscented sand (six trials)

Training (Day 4)

Trained on two paired-associate odor discriminations (six trials per discrimination)

Extinction (Day 5)

Extinction trial for one odor discrimination (one trial)

Testing (Day 6)

Test Trial 1: Extinguished Odor

Test Trial 2: Non-Extinguished Odor

Test Trial 3: Extinguished Odor (Reminder Effect)

Figure 1

Method

SubjectsNinety-day old, male Long-Evans rats (N=13) were reduced to and maintained at 85% of their free-feeding weights one week prior to and during experimentation. Water was available ad libitum. Rats were maintained on a 12-hour light/dark cycle.

ApparatusAll shaping, training, and testing took place in the rats’ home cages. Plastic Nalgene® cups (125 ml size) were used for the paired-associate discriminations and were mounted using Velcro® onto rectangular Plexiglas® bases. Odor discriminations were created by mixing play sand (148.5 grams) with different dried spices (i.e., 1.5 grams of oregano, garlic, cinnamon, or nutmeg).

ProcedureRats were shaped to dig in unscented cups of sand for three days prior to training (10 shaping trials total – see Figure 1 for shaping, training, and testing procedure). Rats received one day of training during which they learned two paired-associate discriminations (see Figure 2). One day following training, rats received one extinction trial for one of the odor pairs they learned with a 1 mg/kg cycloheximide injection (n=7) or a vehicle injection (n=6). Twenty-four hours later, rats received three test trials.

Discussion

•Rats learned to dig in the correct odor and showed a statistically significant preference for it in the Cycloheximide group and a marginally significant preference for it in the Vehicle group during their first preference test (i.e., the extinction trial).

•Administration of a protein synthesis inhibitor (cycloheximide) during extinction did not block memory for extinction (as indicated by low preference scores on Tests 1, 2, and 3).

•Results suggest that protein synthesis may not be critical for the new learning associated with extinction in an appetitive paired-associate odor discrimination task.

References

Bunsey, M. & Eichenbaum, H. (1996). Conservation of hippocampal memory function in rats and humans. Nature, 379, 255-257.

Lattal, K. M. & Abel, T. (2001). Different requirements for protein synthesis in acquisition and extinction of spatial preferences and context-evoked fear. The Journal of Neuroscience, 21(15), 5773-5780.

Meiri, N. & Rosenblum, K. (1998). Lateral ventricle injection of the protein synthesis inhibitor anisomycin impairs long-term memory in a spatial memory task. Brain Research, 789, 48-55.

Nader, K., Schafe, G. E., & LeDoux, J. E. (2000). Fear memories require protein synthesis in the amygdala for reconsolidation after retrieval. Nature, 406, 722-726.

Suzuki, A., Josselyn, S. A., Frankland, P. W., Masushige, S., Silva, A. J., & Kida, S. (2004). Memory reconsolidation and extinction have distinct temporal and biochemical signatures. The Journal of Neuroscience, 24(20), 4787-4795.

Figure 2

Oregano Garlic

Cinnamon Nutmeg Cinnamon Nutmeg

Retention

-1

-0.8

-0.6

-0.4

-0.2

0

0.2

0.4

0.6

0.8

1

Pre

fere

nce

Inde

x

Cycloheximide Vehicle

Figure 3

Extinction Test 1 Test 2 Test 3