The Role of Rac1 in Synaptic Plasticity, Learning and Memory
During the process of learning and memory, neuronal synapses undergo changes involving gene expression, protein synthesis, and cytoarchitectural remodeling, referred to as synaptic plasticity. Changes in the morphology of synapses are necessary for learning and memory storage, and require the rearrangement of the actin cytoskeleton at dendrites and actin rich dendritic spines which are the loci excitatory of synaptic transmission in the central nervous system. Rac1, a protein of the Rho subfamily of GTP binding proteins (GTPases), is largely known for its involvement in cytoskeleton remodeling, and has been implicated in neuronal development, participating in the morphological changes required for migration of newborn neurons to characteristic locations, extension of axons and dendrites into proper target regions, and formation of synapses with appropriate partners. However, the functional role of Rac1 in adult neuronal signaling has been relatively unclear. Our laboratory previously demonstrated that that Rac1 is highly expressed in the adult mouse hippocampus, a part of the brain, which is very crucial for memory acquisition. In vitro studies in hippocampal slices indicated that activation of the hippocampal N-methyl-D-aspartate (NMDA) receptor, the receptor that was modified to produce the “smart mouse” results in membrane translocation and activation of Rac1. Moreover, our laboratory has observed that translocation and activation of Rac1 are associated with fear learning in the adult mice. The purpose of this study was to examine whether Rac1 is required for regulation of the cytoskeletal dynamics leading to morphological plasticity observed at neuronal synapses during hippocampal learning and memory. To evaluate the importance of Rac1 in plasticity and learning we generated a mouse bearing a conditional inactivation of the rac1 gene in the hippocampus (referred herein as Rac1 knockouts [KO] or mutants) using the Cre/LoxP system. We showed that the disappearance of Rac1 in the Rac1 knock out (KO) is time dependent, with a 16% significant decrease at two months and 90% at six months as compared to their wild type (WT) littermates. No differences in protein levels were observed between the wild type and heterozygous littermates. Next, we studied neuronal morphology in hippocampal slices using the rapid Golgi-Cox technique to determine whether Rac1 was necessary for the neuroanatomical and cytoarchitectural changes associated with synaptic plasticity in the adult mice. We showed that loss of Rac1 leads to a significant reduction in spine density in Rac1 mutant mice as compared to the wild type controls. In an attempt to correlate this effect with an alteration in the actin dynamics pathway, we performed Western blot analysis, which showed in the Rac1 mutant significant reduction in p-PAK-1, p-LIMK-1 and p-Cofilin-1, all proteins downstream of Rac1 and essential for cytoskeletal rearrangements leading to dendritic spine development. In order to evaluate the involvement of Rac1 in long term synaptic plasticity (LTP and LTD), hippocampal slices from age matched wild type and Rac1 mutant were given high frequency stimulation (HFS) to generate LTP and low frequency stimulation (LFS) to induce LTD. Rac1 deficient mice showed impaired LTP and LTD as compared to their littermate controls. Furthermore, to assess whether Rac1 is associated with, and necessary for hippocampus-dependent learning and memory in the intact animal, Rac1 mutant mice and their littermate controls were examined in a battery of behavioral tests beginning with six control tests namely, the Open Field Activity, Rotating Rod, Pre-pulse Inhibition of Startle Response, Hot Plate test, Light-dark, Elevated Plus Maze, and ending with two learning and memory tests, the Morris Water Maze and Fear conditioning paradigm. Rac1 deficient mice showed the same performance as the wild type and the heterozygotes in control tests, however, they showed impaired learning in the learning and memory tests. Collectively, these data suggest that Rac1 is an important protein required for proper dendritic spine morphogenesis, long-term synaptic plasticity, as well as learning and memory. These phenomena are abnormal in some autistic disorders, making Rac1 an interesting target in the study of these diseases.