The cAMP Responsive Element Binding protein (CREB) regulates the transcription of other genes and has a well-known role in the stability of synaptic potentiation and memory (1
). A recent series of papers (3
) provided compelling evidence that there are molecular and cellular processes that determine which cells are recruited to store information within a neural circuit. In particular, this work suggested the hypothesis that CREB activated during learning triggers changes in the cell (i.e. increase in excitability) that then affect whether that cell participates in subsequent memories. This idea was tested by artificially increasing the levels of CREB in amygdala neurons using a replication defective Herpes viral vector. The initial results showed that higher CREB levels increase the probability (~3 fold) that amygdala neurons participate in memory for tone fear conditioning (3
). In this form of Pavlovian conditioning, animals learn to associate a tone with an aversive event, such as a mild foot shock. The amygdala is a subcortical brain structure with a well-known role in emotional memories such as fear memory (6
). ARC (Activity-Regulated Cytoskeletal-associated protein) RNA, a gene required for synaptic function and memory, was used to identify the neurons encoding a tone conditioning memory. ARC has been extensively used to determine which neuronal populations are activated by specific behavioral stimuli (8
). Immediately after a memory test, amygdala cells transfected with the viral CREB (identified through its fluorescent tag) were three times more likely to express ARC RNA (i.e., be involved in memory) than neighboring cells (3
). Thus, CREB levels seem to bias which neurons encoded tone conditioning in the amygdala. It is conceivable that an interaction between the effects of the viral vectors used and the genes transfected could have affected the results of these early studies. To address this and other possible confounding interpretations of the results, cell lesion and inactivation strategies were also used to probe the role of CREB in memory allocation (4
A targeted cell lesion strategy was used to explore further the possibility that CREB biases the allocation of memory for tone conditioning in the amygdala (4
). The authors took advantage of a transgenic mouse with a silenced diphtheria toxin receptor to specifically kill the cells with the virally-delivered CREB in mice. A replication defective Herpes viral vector carried CREB and a recombinase that could activate the silenced diphtheria receptor gene by deleting a RNA translation STOP sequence. CREB biased memory allocation since killing the cells transfected with the viral CREB disrupted memory for tone conditioning, while ablating the cells transfected with the control virus had no effect. A series of elegant control experiments showed that ablating the CREB cells did not prevent the animals from making new amygdala-dependent memories, and nor did it affect memories acquired before viral infection. Instead, killing the cells with the viral CREB only affected the memory for tone conditioning acquired after viral transfection.
Targeted killing of nearly 20% of cells in a neural circuit may have had unintended effects that could confound the interpretation of behavioral experiments. However, this does not seem to have been the case since similar results (5
) to those just described were obtained with another approach (10
) that allows for reversible neuronal inactivation. This strategy takes advantage of a Drosophila receptor (the allatostatin receptor) that can be functionally linked to potassium channels in mouse neurons. When activated by the allatostatin receptor, these channels silence neurons (keep them from firing action potentials). Since mice lack the ligand for the allatostatin receptor, only neurons that have the exogenously expressed receptor can be inactivated by treatments with the allatostatin peptide. The results (5
) show that inactivating amygdala cells expressing a virally provided CREB, results in a pronounced amnesia for tone conditioning. This amnesia could be readily reversed simply by retesting the mice a day later in the absence of allatostatin, demonstrating both the reversibility of the allatostatin effects, and the link between activity in the CREB cells and recall. As before, if CREB was replaced with a control gene in the viral vectors, then inactivation of the transfected cells had no impact on memory. Further, the authors showed that CREB manipulations could also affect the allocation of memory for conditioned taste aversion (CTA) (5
), another form of memory that engages the amygdala. Animals that experience intestinal malaise (i.e. nausea) after eating or drinking a novel food learn to avoid that food since they associate it with the subsequent malaise. Inactivating the neurons transfected with the CREB virus had a very specific impact on memory, since inactivation affected a CTA memory acquired after viral transfection, but did not disrupt in the same animals a tone conditioning memory acquired before viral transfection.
Each of the strategies used to study the role of CREB in memory allocation have their own limitations. For example, the expression of the allatostatin receptor could interfere with other receptors in amygdala neurons and thus alter the behavioral results described above. Nevertheless, the convergence of findings with three different strategies (ARC cell tagging, diphtheria and allatostatin inactivation) confirms that CREB has a role in the allocation of memory in the amygdala.
One of the byproducts of the inactivation experiments described above(4
) is the finding that deleting or inactivating 15–20% of cells in an amygdala memory trace does not seem to affect that memory, while deleting 50–60% of those cells does cause a substantial amnesia. This resiliency could be a mechanism for preserving memories in the face of neuronal death or other events that would otherwise erase memory traces.
The studies described above also suggest that there is a competition process that keeps the number of neurons encoding a given memory constant (3
) (but see (11
)). Despite 60–70% of CREB transfected cells being ARC positive and hence engaged in memory (versus 15–20% normally), the overall number of ARC positive cells in the amygdala did not increase. Therefore, it is possible that there are memory allocation mechanisms that control the size of memory traces so as to economize storage space. For example, changes in synaptic inhibition in response to changes in overall levels of circuit excitability could provide a dynamic way to control the overall number of neurons involved in a specific memory, thus modulating the storage space allocated to any one memory.