De Novo synthesis of CREB in a presynaptic neuron is required for synaptic enhancement involved in memory consolidation

Abstract

Interaction between the activator type of cyclic AMP response element binding protein (CREB1) and the repressor type (CREB2) results in determining the emergence of long-lasting synaptic enhancement involved in memory consolidation. However, we still do not know whether the constitutively expressed forms of CREB are enough or the newly synthesized forms are required for the synaptic enhancement. In addition, if the newly synthesized forms are needed, we must determine the time for translation of CREB from its mRNA. We applied the methods of RNA interference and real-time polymerase chain reaction (PCR) to CREB in the cerebral giant cells of Lymnaea. The cerebral giant cells play an important role in associative learning and employ a CREB cascade for the synaptic enhancement to neurons such as the B1 motoneurons. We injected the small interfering RNA (siRNA) of CREB1 or CREB2 into the cerebral giant cells and examined the changes in amplitude of excitatory postsynaptic potential (EPSP) recorded in the B1 motoneurons. The changes in the amounts of CREB1 and CREB2 mRNAs were also examined in the cerebral giant cells. The EPSP amplitude was suppressed 15 min after injection of CREB1 siRNA, whereas that was augmented 60 min after injection of CREB2 siRNA. In the latter case, the decrease in the amount of CREB2 mRNA was confirmed by real-time PCR. Our results showed that the de novo synthesized forms of CREB are required within tens of minutes for the synaptic enhancement in memory consolidation.

One-trial conditioned taste aversion inLymnaea: good and poor performers in long-term memory acquisition

“…It has been hypothesized that the cerebral giant cells and the B2 motor neurons play key roles in mediating the learning and LTM formation of this CTA (Kojima et al, 1997;Kojima et al, 2000;Kojima et al, 2001;Hatakeyama et al, 2004a;Sadamoto et al, 2004b). Consistent with this hypothesis are the data showing that altered gene activity and new protein synthesis must occur in these and other neurons if LTM is to form following the CTA procedure Hatakeyama et al, 2004b;Hatakeyama et al, 2006;Azami et al, 2006;Wagatsuma et al, 2006). In addition, our recent findings showed that snails are both able to discriminate different tastes and continue to eat other foods following the CTA procedure (Sugai et al, 2006).…”

“…the consolidation process) following learning requires both altered gene activity and de novo protein synthesis (Davis and Squire, 1984;Milner et al, 1998;Kandel and Squire, 1999;McGaugh and Izquierdo, 2000;Kandel, 2001;Sangha et al, 2003a;Epstein et al, 2003;Epstein et al, 2004;Ribeiro et al, 2005;Azami et al, 2006;Wagatsuma et al, 2006). An advantage of using a one-trial conditioning procedure that results in LTM formation is that the consolidation process must begin at some time following the single pairing of the stimuli; providing a precise point in time to apply amnesiac agents.…”

“…Before we can formulate a viable, testable hypothesis at the cellular and molecular levels (e.g. the presence of different ratios of two types of cyclic-AMP responsive element binding protein in key neurons for Lymnaea CTA) Wagatsuma et al, 2005;Wagatsuma et al, 2006), we must first examine a number of the important parametric characteristics of this one-trial CTA learning (e.g. the formation, strength and modifiability of LTM).…”

“…However, the advantage that cooling has over other such amnesiac agents is that it can be applied quickly, it is relatively harmless, and it can be reversed quickly. Knowing that the molecular processes that underlie memory formation occur within 10·min after the one-trial conditioning session may allow us to differentiate between causal and correlative molecular events in key neurons that are necessary for behavioral memory Wagatsuma et al, 2006). That is, if a change in a molecular marker does not occur within 10·min of the termination of the one-trial conditioning procedure then it is probably not an early, causal, necessary process for memory formation.…”

Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient

“…Isolation of preand postsynaptic compartments is important for examining signaling and protein synthesis, which occurs in both compartments. [7][8][9][10][11][12][13] The Percoll-sucrose gradient is a crude purification so we saw minor cellular, nuclear and organelle contamination in the SN fraction but the overall separation was good.…”