pCREB in the neonate rat olfactory bulb is selectively and transiently increased by odor preference-conditioned training

Early olfactory preference learning in rat pups occurs when novel odors are paired with tactile stimulation, for example stroking. cAMP-triggered phos phorylation of cAMP response element binding protein (pCREB) has been impli cated as a mediator of learning and memory changes in various animals (Fran k and Greenberg 1994). In the present study we investigate whether CREB is phosphorylated in response to conditioned olfactory training as might be pr edicted given the proposed role of the phosphorylated protein in learning. On postnatal day 6, pups were trained for 10 min using a standard condition ed olfactory learning paradigm in which a conditioned stimulus, Odor, was e ither used alone or paired with an unconditioned stimulus, Stroking (using a fine brush to stroke the pup). In some instances stroking only was used. The pups were sacrificed at 0, 10, 30, or 60 min after the training. Using Western blot analysis, we observed that the majority of olfactory bulbs in conditioned pups (Odor + Stroking) had a greater increase in pCREB activati on at 10 min after training than pups given nonlearning training (Odor only or Stroking only). The phosphorylated protein levels were low at 0 min and at 60 ruin after training. This is in keeping with the slightly delayed an d short-lived activation period for this protein. The localization of pCREB increases within the olfactory bulb as seen by im munocytochemistry. Naive pups were not exposed to odor or training. There w as a significantly higher level of label in mitral cell nuclei within the d orsolateral quadrant of the bulb of pups undergoing odor-stroke pairing. No significant differences were observed among nonlearning groups (Naive, Odo r only, or Stroking only) or among any training groups in the granule or pe riglomerular cells of the dorsolateral region. The localized changes in the nuclear protein are consistent with studies showing localized changes in t he bulb in response to a learned familiar odor. The present study demonstra tes that selective increases in. pCREB occur as an early step following pai ring procedures that normally lead to the development of long-term olfactor y memories in rat pups. These results support the hypothesized Link between pCREB and memory formation.