The role of retinoic acid in long-term memory formation and synaptic plasticity in the mollusc Lymnaea stagnalis

The active metabolite of vitamin A, retinoic acid (RA), is involved in memory formation and hippocampal plasticity in vertebrates. A similar role for retinoid signaling in learning and memory formation has not previously been examined in an invertebrate species. However, the conservation of retinoid signaling between vertebrates and invertebrates is supported by the presence of retinoid signaling machinery in invertebrates. For example, in the mollusc Lymnaea stagnalis the metabolic enzymes and retinoid receptors have been cloned from the CNS. In this study I demonstrated that impairing retinoid signaling in Lymnaea by either inhibiting RALDH activity or using retinoid receptor antagonists, prevented the formation of long-term memory (LTM). However, learning and intermediate-term memory were not affected. An additional finding was that exposure to constant darkness (due to the light-sensitive nature of RA) itself enhanced memory formation. This memory-promoting effect of darkness was sufficient to overcome the inhibitory effects of RALDH inhibition, but not that of a retinoid receptor antagonist, suggesting that environmental light conditions may influence retinoid signaling. Since RA also influences synaptic plasticity underlying hippocampal-dependent memory formation, I also examined whether RA would act in a trophic manner to influence synapse formation and/or synaptic transmission between invertebrate neurons. However, I found no evidence to support an effect of RA on post-tetanic potentiation of a chemical synapse. Retinoic acid did, however, reduce transmission at electrical synapses in a cell-specific manner. Overall, these studies provide the first evidence for a role of RA in the formation of implicit long-term memories in an invertebrate species and suggest that the role of retinoid signaling in memory formation has an ancient origin.

The role of microRNAs and retinoid signaling during spinal cord regeneration in the adult newt.

The molecular events after spinal cord injury that lead to the establishment of a permissive environment and epimorphic regeneration remain unclear. Two molecular pathway regulators that may converge to create a spinal cord regeneration-permissive environment in the urodele are retinoic acid (RA) and microRNAs (miRNAs). Recent evidence suggests that RARβ-mediated signaling is necessary for tail and caudal spinal cord regeneration in the adult newt. MicroRNAs are attractive candidates as mediators of retinoid signaling during regeneration, as their pleiotropic effects are vital in situations where global changes in gene expression are required. Thus, the overall aim of this thesis was to determine if miRNAs are involved in tail and caudal spinal cord regeneration in the adult newt, and if they act as regulators and/or effectors of retinoid signaling during this process. I have demonstrated here, for the first time, that multiple miRNAs are dysregulated in response to spinal cord injury in the adult newt, as well as in response to inhibition of retinoid signaling. Two of these miRNAs, miR-133a and miR-1, appear to target RARβ2 transcripts both in vivo and in vitro. Inhibition of RA signaling via RARβ with a selective antagonist, LE135, alters the pattern of expression of these miRNAs, which leads to an inhibition of tail regeneration. These data are indicative of a negative feed back loop, albeit potentially an indirect one. I also aimed to examine which miRNAs are affected by inhibiting RA synthesis during regeneration, and provided a long list of miRNAs that are dysregulated. These data provide the foundation for future studies on the putative roles of these miRNAs, as well as their function in retinoid signaling. Overall, these studies provide the first evidence for a role for miRNAs as mediators of retinoid signaling during caudal spinal cord regeneration in any system.