The current scheme of a CaMK cascade composed of upstream activating kinases, including CaMKKα and CaMKKβ, and downstream effector kinases, including CaMKI and CaMKIV/Gr, raises questions concerning unique and redundant functions of the respective cascade component and the potential for specialized pairings of upstream and downstream kinases. In vitro studies using purified kinases revealed the two upstream kinases to be similar in their capacity to activate CaMKI and CaMKIV/Gr (2
). Similarly, experiments on cell lines transfected with the respective upstream kinase revealed redundant capacity of CaMKKα and CaMKKβ to activate downstream CaMKIV/Gr- and CREB-dependent transcription (2
). In contrast, a more complex picture has emerged from studies on genetic mouse models deficient in individual components of the CaMK cascade, including CaMKIV/Gr, CaMKKβ, and now CaMKKα. CaMKIV/Gr deficiency was associated with a profound deficit in LTP and CREB activation in several memory-related forebrain areas and impaired contextual and cued-fear LTM but normal spatial LTM. CaMKIV/Gr deficiency was also associated with defective stress-induced CREB activation and decreased stress-induced anxiety-like behavior (18
). CaMKKβ deficiency impaired CREB activation upon spatial training and delayed the formation of spatial LTM but spared fear memory (38
). In contrast, the present studies demonstrated that CaMKKα was dispensable for spatial memory but was revealed to promote contextual fear memory and CaMKIV/Gr and CREB activation upon fear conditioning. Unlike CaMKIV/Gr- and CaMKKβ-deficient mice, CaMKKα KO mice exhibited normal hippocampal LTP. Collectively, these findings suggest divergent functions of the upstream components of the CaMK cascade in learning and memory.
Genetic deletion of CaMKKα resulted in relatively selective deficits in fear memory for context. The deletion did not disrupt normal levels of anxiety, as demonstrated in the elevated plus maze and open field preparations. Additionally, the deletion did not result in an overall disruption of fear learning per se. Tone testing indicated that the CaMKKα KO mice could acquire and express fear memory, even at remote testing points following conditioning. In contrast, these same KO mice showed significant and persistent deficits in context fear. However, the possibility that CaMKKα deficiency imparts a subtler deficit in cued fear learning that would require testing of larger populations of mice to be uncovered cannot be ruled out. Intriguingly, the CaMKKα KO mice showed context fear deficits at every time point across repeated measures but showed no deficit at all in extinction of the freezing response across tests. That is, the CaMKKα deletion may have weakened the formation or early consolidation of the context fear memory (as demonstrated by the immediate postshock freezing deficit and at the 24 h time point) but may also have left extinction learning mechanisms intact. Overall, the unique dissociations found with these animals include a deficit in fear learning but not in anxiety, a deficit in context fear but not in tone fear, and finally a deficit in acquisition/consolidation of context fear but not in extinction.
One caveat of the above behavioral studies is that they were carried out exclusively on male mice, leaving open the possibility of a sexual bias in the reported findings. There are well-characterized differences between male and female rodents in their acquisition of contextual fear learning. Females are slower to acquire contextual fear than males, although this difference disappears with repeated conditioning trials or longer context exposure prior to conditioning (34
). These differences, some of which may relate to estrous cycle-dependent changes in neurosteroid levels that impact the hippocampus, may reflect on the interaction between the sex of the animal and the genetic mutation(s) under study (55
). Accordingly, the possibility that male CaMKKα KO mice more readily exhibit a deficit in contextual fear conditioning than their female counterparts cannot be ruled out, as this remains to be verified experimentally.
To date, much progress has been made in delineating the putative cellular substrates of learning and memory, specifically in the fear conditioning preparation. Critical roles have been revealed for CREB- and CaMKIV/Gr-related processes in fear acquisition and expression (6
). The findings here together with those of the aforementioned studies outline a linear relay within the CaMK cascade that is composed of CaMKKα, its downstream substrate CaMKIV/Gr, and the CaMKIV/Gr substrate CREB that is operative in fear memory. It should be noted that the deficit in phosphorylated CREB (pCREB) formation was selectively observed upon fear conditioning of CaMKKα KO mice but not at baseline, where the pCREB levels were either comparable to WT mice or even increased (such as in the hippocampus). This is in contrast to the more global deficit in pCREB formation observed in CaMKIV/Gr KO mice both at baseline and upon fear conditioning, reflective of the role of CaMKIV/Gr as a key CREB kinase (18
). These findings may be interpreted as suggesting that the role of the CaMKKα pathway in CREB activation is constrained to specific behavioral responses (e.g., fear memory).
The profound deficit in CREB activation in the hippocampus of CaMKKα KO mice upon fear conditioning is of particular significance given the critical role the hippocampus plays in processing the spatial context and relaying the information to the amygdala in the course of contextual fear conditioning (43
). CREB-responsive element-dependent gene expression driven by CREB and related transcription factors is activated in the hippocampus upon contextual fear conditioning (23
). Global CREB deficiency impairs contextual fear memory consolidation, as does antagonism of CREB-responsive element-dependent transcription in the hippocampus (3
). Contextual fear conditioning up-regulates the expression of a number of genes in the hippocampus, several of which are targets of calcium/CREB signaling that have been implicated in contextual fear memory consolidation (25
). Of particular interest is brain-derived neurotrophic factor, which is induced in a calcium- and CREB-dependent manner and is up-regulated after contextual fear conditioning (15
). Brain-derived neurotrophic factor appears to selectively promote contextual fear memory consolidation; its deficiency or antagonism impairs contextual fear memory consolidation while sparing cued fear (29
). Other targets of calcium/CREB signaling include a number of immediate-early activation genes, such as c-Fos, which is induced by the CaMK cascade and whose deficiency impairs contextual fear conditioning (12
). Further studies would be required to elucidate the contributions of these and other targets of the CaMK cascade to the observed deficit in contextual fear conditioning in CaMKKα KO mice.
Given that all the component kinases of the CaMK cascade share a strongly overlapping expression profile in forebrain tissues, these results can be interpreted to suggest that CaMKKα and CaMKKβ have both unique and redundant functions in activating downstream CaMK and/or other substrates, including CaMKIV/Gr. For example, the impairment of contextual fear memory in both CaMKIV/Gr and CaMKKα KO mice but not CaMKKβ KO mice argues for an upstream role of CaMKKα in regulating CaMKIV/Gr-dependent contextual fear learning. On the other hand, the absence in CaMKKα KO mice of LTP deficits common to CaMKKβ and CaMKIV/Gr KO mice suggests that CaMKKβ is the more relevant kinase in mediating LTP potentiation by CaMKIV/Gr.
In addition to activating downstream CaMKI and CaMKIV/Gr, both CaMKKα and CaMKKβ have been demonstrated to independently activate other signaling pathways. CaMKKα has been demonstrated to act as an Akt kinase that phosphorylates Akt on the regulatory threonine 308 residue, leading to its activation (57
). Both CaMKKα and CaMKKβ have also been shown to phosphorylate and activate AMP kinase (4
). The roles of these alternative pathways in promoting fear memory formation by CaMKKα are currently unclear and are the subject of ongoing investigation.