Neuropeptides confer a unique role in the central nervous system (CNS). Their tripartite functionality of facilitating the release of neurotransmitters, remaining bioactive for extended periods of time and their high affinity for G protein-coupled receptors (GPCRs) allows them to drive key intracellular signaling cascades and modulate essential elements of neuronal network processes. The neuropeptide cholecystokinin (CCK) is widely distributed throughout the CNS. Research has shown that high concentrations of CCK exist within the caudate nucleus, the entorhinal cortex, and the limbic system, to name but a few. This allows it to regulate a broad spectrum of fundamental neuronal processes; among which are its modulation of pain, anxiety, hunger, the brain’s reward systems, and learning and memory. Seminal studies exploring the mechanisms of CCK found that CCK was mediated by two variant G protein-coupled receptors – CCK-1 receptor (CCK1R) and CCK-2 receptor (CCK2R). Conducted in the digestive system, these initial studies revealed that CCK mediated processes were diverse, but such results lacked specifics pertaining to its operational functions in the brain.

Long-term potentiation (LTP), one of the prime correlates of learning and memory, has been and still remains predominantly conducted in the hippocampus, an important brain region for exploring such phenomenon. Work stemming from our lab has shown that CCK is a cardinal component of neocortical plasticity, is requisite associative memory formation and facilitates the establishment of visuoauditory associations. In addition, evidence shows it is crucial for certain types of LTP formation. Together, this implies that it is integral to synaptic plasticity, and thus modulates plastic changes in the brain from the synapse to neuronal circuitry. LTP research has demonstrated that its formation is dependent upon numerous protein-specific targets and that LTP is kinase-dependent. Such research has evinced that various kinases and transcription factors (i.e., protein kinase C (PKC), cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), mitogen-activated protein kinase (MAPK), and cAMP response element-binding protein (CREB), respectively) are involved in different stages of LTP formation/induction. However, how CCK facilitates LTP has yet to be fully elucidated.

Preliminary work in in vitro hippocampal neurons examining the potential phosphorylation of cAMP response element-binding protein (CREB), an established regulator of LTP, showed a pronounced difference in phosphorylation between CCK and control. Further exploratory experiments using a phospho-CREB microarray and mass spectrometry of the phosphoproteome revealed an additional array of signal transduction targets. Results showed that in in vitro hippocampal neurons cholecystokinin sulfated octapeptide (CCK-8s) triggers the phosphorylation of kinases associated with the MAPK pathway; the likes of which include: Src proto-oncogene tyrosine-protein kinase (Src), Ras proto-oncogene, GTPase (Ras), BRaf proto-oncogene, serine/threonine kinase (BRaf) and its substrate Mitogen-activated protein kinase kinase 2 (MEK2), along with MAPK, and the downstream phosphorylation of CREB. This strongly suggests that CCK-8s activation of CCK receptors is mediated through the Ras-Raf-MEK-MAPK pathway.

To probe further into the potential involvement of this pathway in CCK-dependent LTP, microelectrode arrays were employed on SD rat hippocampal slice preparations to evaluate field excitatory postsynaptic potentials (fEPSPs). Low frequency stimulation (LFS) coupled with the preapplication of CCK-8s markedly induced LTP formation compared to baseline. The MEK1/2 selective inhibitor U0126 was then applied prior to the paired CCK-8s/LFS LTP induction paradigm, wherein results demonstrated that application of U0126 completely blocks the induction of LTP leading to a significant decrease in fEPSP amplitude compared to baseline and CCK-8s-induced LTP. This strongly suggests that within the hippocampus CCK receptors signal through the Ras-Raf-MEK-MAPK pathway driving the induction of LTP. In parallel, this establishes that the mechanisms of CCK-induced synaptic plasticity (i.e., LTP) are driven primarily by the MAPK pathway. Our results have repeatedly shown that the neuroplastic features of CCK are mediated through CCK2R. However, the hippocampus is known to be rich in CCK1R and studies have shown that it also plays a significant role in hippocampal associated memory. Thus, we aimed to establish which receptor subtype was predominant in mediating the activation of the MAPK pathway. Application of CCK1R antagonist devazepide and CCK2R antagonist netazepide (YF476) prior to CCK-8s stimulation of the MAPK pathway revealed that CCK2R mediates the phosphorylation in this pathway.

The role of classical ionotropic receptors N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) in synaptic plasticity is well-established and known to modulate various key elements of LTP. To evaluate the potential convergence of fast acting ionic transmission on activating the revealed biochemical cascade, we investigated how these receptors might affect the activation of the Ras-Raf-MEK-MAPK pathway. Our findings show that neither tetrodotoxin (TTX), cyanquixaline (CNQX), nor (2R)-amino-5-phosphonopentanoate (APV), have any markedly comprehensive effects on CCK-induced intracellular signaling of MAPK-pathway kinases. This signifies that CCK activated GPCRs act independently of such receptors in the activation of the Ras-Raf-MEK-MAPK pathway.

The nature of neuroplasticity demands a dynamic cytoskeleton to allow reorganization so as to facilitate axonal and dendritic remodeling. Considering the cardinal role that MAPK plays in dendritic morphogenesis, which is fundamental to LTP formation, we further evaluated our phosphoproteome findings to elucidate the possibility of CCK-8s driven plasticity being linked to the actin cytoskeleton and microtubules. Indeed, we found that CCK triggered phosphorylation of a distinct set of proteins within the hippocampal phosphoproteome that are vital to synaptic- and dendritic morphogenesis. This suggests that binding of CCK-8s to its cognate receptors not only mobilizes the Ras-Raf-MEK-MAPK, but also appears to activate proteins known to alter cellular mechanics and shape the cytoskeleton, the likes of which are directly linked to MAPK and activation of the MAPK pathway. These findings imply that CCK-induced LTP results from a coordinated mobilization of key protein targets in the MAPK pathway and subsequent cytoskeletal reorganization.

In conclusion, CCK-8 elicits an extensive array of effects on neurophysiology and plays a crucial role in LTP induction and memory formation. Our results strongly suggest that activation of CCK2R triggers the Ras-Raf-MEK-MAPK signal transduction cascade. Additional experiments elucidated that CCK-dependent LTP can be inhibited through blockade of the MAPK pathway, suggesting that in the hippocampus CCK-induced LTP is MAPK-dependent. Given the possibility of CCK facilitating cytoskeletal remodeling pertinent to synaptic- and dendritic morphogenesis, CCK thus drives brain plasticity through post-translational modifications (PTMs) of proteins that range from those that comprise signal transduction to that requisite to the reorganization of the cytoskeleton and subsequent LTP formation. As demonstrated through an array of congruent results, it is evident that CCK acts as a molecular switch driving plastic changes that lead to the induction of LTP and other correlates of memory formation in the brain.