Inhibition of calcineurin extends the duration of early associative olfactory memory

The search for which molecular players are involved in the transfer from short term memory to long term memory has led down several different avenues. The role of protein phosphatases in learning and memory has been one well-studied route of investigation. Specifically, protein phosphatase 2B (calcineurin, CN) has received a significant amount of attention due to its promotion of the dephosphorylation of CREB. Researchers have ascertained that over-expression of CN is associated with memory retention deficits and impaired memory consolidation in mice (Mansuy, Mayford, Jacob, Kandel & Bach, 1998; Foster, Sharrow, Masses, Norris, & Kumar, 2001). In contrast, CN inhibition enhances conditioned place preference (Gerdjikov & Beninger, 2005) and contextual learning (Ikegami & Inokuchi, 2000). -- The present study hypothesized that an infusion of FK506 (a CN inhibitor) bilaterally into the olfactory bulbs would prevent the dephosphorylation of CREB and, prolong the duration of a conditioned odor preference, which normally only lasts 24 h with a single training trial. On post natal day (PND) 6, rat pups received a 2 mg/kg subcutaneous injection of isoproterenol (ISO), a β-adrenoceptor agonist (the unconditioned stimulus, US), and after a 10 minute exposure to peppermint (the conditioned stimulus; CS) were infused with FK506 or vehicle into the olfactory bulbs. Subsequently, preference for peppermint was assessed 24 hrs, 48 hrs, 72 hrs, 96 hrs and 1 week after training. Immunohistochemistry for pCREB revealed that unilateral infusion of FK506 resulted in an amplification of phosphorylated CREB in the olfactory bulb 40 min after training relative to sham side infusions. Additionally, pups infused bilaterally with FK506 maintained a learned preference for peppermint 48, 72 and 96 hrs after training. These results support the hypothesis that prolonging CREB phosphorylation with CN inhibition can extend the duration of conditioned olfactory memory. -- CN inhibition also modified the conventional inverted U curve obtained when ISO is used to replace stroking, as the US. Under normal conditions, 2 mg/kg of systemic ISO is effective, while higher and lower doses are not able to produce learning. When pups were infused with FK506 to inhibit CN, learning occurred with the low dose of 1 mg/kg ISO, and with the higher dose 6 mg/kg. We assumed that high doses of ISO do not elicit olfactory memory due to excessive phosphatase activity, and that low doses do not provide sufficient cAMP activation. CN inhibition blocks phosphatase activity, thus allowing learning to occur at higher doses of ISO. For the low dose effect, we hypothesize that AC9, a positive regulator of cAMP inhibited by CN, is relieved from inhibition and cAMP production is increased to provide sufficient activity to phosphorylate CREB and produce learning following normally insufficient (1 mg/kg ISO) activation. Alternatively, normally subthreshold phosphorylation elicited by small elevations of cAMP via low dose ISO may be raised to suprathreshold levels by phosphatase inhibition. -- Calcineurin inhibitors thus have promise as memory enhancers since they facilitate memory initiation and memory duration from sub- to supra-optimal associative learning conditions