Molecular evidence for two-stage learning and partial laterality in eyeblink conditioning of mice

Laboratories for *Behavioral Genetics and
^‡Mathematical Neuroscience, RIKEN Brain Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan;
^†Department of Molecular Immunology, Graduate School of Agricultural and Life Science, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan; and
^§Neuroscience Program, University of Southern California, Los Angeles, CA 90089-2520

Contributed by Richard F. Thompson, February 13, 2006

Abstract

The anterior interpositus nucleus (AIN) is the proposed site of memory formation of eyeblink conditioning. A large part of the underlying molecular events, however, remain unknown. To elucidate the molecular mechanisms, we examined transcriptional changes in the AIN of mice trained with delay eyeblink conditioning using microarray, quantitative real-time RT-PCR, and in situ hybridization techniques. Microarray analyses suggested that transcriptionally up-regulated gene sets were largely different between early (3-d training) and late (7-d) stages. Quantitative real-time RT-PCR aided by laser microdissection indicated that the expression of representative EARLY genes (Sgk, IkBa, and Plekhf1) peaked at 1-d training in both the paired and unpaired conditioning groups, and was maintained at a higher level in the paired group than in the unpaired group after 3-d training. In situ hybridization revealed increased expression of these genes in broad cerebellar areas, including the AIN, with no hemispheric preferences. In contrast, the expression of representative LATE genes (Vamp1, Camk2d, and Prkcd) was selectively increased in the AIN of the 7-d paired group, with dominance in the ipsilateral AIN. Increased Vamp1 mRNA expression was restricted to the ipsilateral dorsolateral hump, a subregion of the AIN. These expression patterns of two distinct subsets of genes fit well with the two-stage learning theory, which proposes emotional and motor learning phases, and support the notion that AIN has a crucial role in memory formation of eyeblink conditioning.

Footnotes

^¶To whom correspondence may be addressed. E-mail: thompson{at}usc.edu or sitohara{at}brain.riken.jp
Author contributions: J.-S.P., R.F.T., and S.I. designed research; J.-S.P. performed research; S.I. contributed new reagents/analytic tools; J.-S.P. and S.-i.N. analyzed data; and J.-S.P., T.O., R.F.T., and S.I. wrote the paper.
Conflict of interest statement: No conflicts declared.
Abbreviations:

Abbreviations:

AIN,

anterior interpositus nucleus;

CR,

conditioned response;

CS,

conditioned stimulus;

DCN,

deep cerebellar nuclei;

DLH,

dorsolateral hump;

P3,

3-d paired training;

P7,

7-d paired training;

qRT-PCR,

quantitative real-time RT-PCR;

PCG,

plasticity candidate gene;

SHAM,

sham negative control;

U7,

7-d unpaired training;

US,

unconditioned stimulus.
Freely available online through the PNAS open access option.