Retinoic acid reduces the formation of, and acutely modulates, invertebrate electrical synapses.

Communication between cells in the nervous system is dependent on both chemical and electrical synapses. Factors that can affect chemical synapses have been well studied, but less is known about factors that influence electrical synapses. Retinoic acid, the vitamin A metabolite, is a known regulator of chemical synapses, but few studies have examined its capacity to regulate electrical synapses. In this study, we determine that retinoic acid is capable of rapidly altering the strength of electrical synapses in an isomer- and cell-dependent manner. Furthermore, we provide evidence that this acute effect might be independent of either the retinoid receptors or the activation of a protein kinase. In addition to the rapid modulatory effects of retinoic acid, we provide data to suggest that retinoic acid is also capable of regulating the formation of electrical synapses. Long-term exposure to both all-trans-retinoic acid or 9-cis-retinoic acid reduced the proportion of cell pairs forming electrical synapses, as well as reduced the strength of electrical synapses that did form. In summary, this study provides insights into the role that retinoids might play in both the formation and modulation of electrical synapses in the central nervous system.NEW & NOTEWORTHY Retinoids are known modulators of chemical synapses and mediate synaptic plasticity in the nervous system, but little is known of their effects on electrical synapses. Here, we show that retinoids selectively reduce electrical synapses in a cell- and isomer-dependent manner. This modulatory action on existing electrical synapses was rapid and nongenomic in nature. We also showed for the first time that longer retinoid exposures inhibit the formation of electrical synapses.

Activity-dependent modulation of neuronal KV channels by retinoic acid enhances CaV channel activity.

The metabolite of vitamin A, retinoic acid (RA), is known to affect synaptic plasticity in the nervous system and to play an important role in learning and memory. A ubiquitous mechanism by which neuronal plasticity develops in the nervous system is through modulation of voltage-gated Ca2+ (CaV) and voltage-gated K+ channels. However, how retinoids might regulate the activity of these channels has not been determined. Here, we show that RA modulates neuronal firing by inducing spike broadening and complex spiking in a dose-dependent manner in peptidergic and dopaminergic cell types. Using patch-clamp electrophysiology, we show that RA-induced complex spiking is activity dependent and involves enhanced inactivation of delayed rectifier voltage-gated K+ channels. The prolonged depolarizations observed during RA-modulated spiking lead to an increase in Ca2+ influx through CaV channels, though we also show an opposing effect of RA on the same neurons to inhibit Ca2+ influx. At physiological levels of Ca2+, this inhibition is specific to CaV2 (not CaV1) channels. Examining the interaction between the spike-modulating effects of RA and its inhibition of CaV channels, we found that inhibition of CaV2 channels limits the Ca2+ influx resulting from spike modulation. Our data thus provide novel evidence to suggest that retinoid signaling affects both delayed rectifier K+ channels and CaV channels to fine-tune Ca2+ influx through CaV2 channels. As these channels play important roles in synaptic function, we propose that these modulatory effects of retinoids likely contribute to synaptic plasticity in the nervous system.

Disruptions in network plasticity precede deficits in memory following inhibition of retinoid signaling.

Retinoic acid, the active metabolite of vitamin A, is important for vertebrate cognition and hippocampal plasticity, but few studies have examined its role in invertebrate learning and memory, and its actions in the invertebrate central nervous system are currently unknown. Using the mollusc Lymnaea stagnalis, we examined operant conditioning of the respiratory behavior, controlled by a well-defined central pattern generator (CPG), and used citral to inhibit retinoic acid signaling. Both citral- and vehicle-treated animals showed normal learning, but citral-treated animals failed to exhibit long-term memory at 24 h. Cohorts of citral- or vehicle-treated animals were dissected into semi-intact preparations, either 1 h after training, or after the memory test 24 h later. Simultaneous electrophysiological recordings from the CPG pacemaker cell (right pedal dorsal 1; RPeD1) and an identified motorneuron (VI) were made while monitoring respiratory activity (pneumostome opening). Activity of the CPG pneumostome opener interneuron (input 3 interneuron; IP3) was also monitored indirectly. Vehicle-treated conditioned preparations showed significant changes in network parameters immediately after learning, such as reduced motorneuron bursting activity (from IP3 input), delayed pneumostome opening, and decoupling of coincident IP3 input within the network. However, citral-treated preparations failed to exhibit these network changes and more closely resembled naïve preparations. Importantly, these citral-induced differences were manifested immediately after training and before any overt changes in the behavioral response (memory impairment). These studies shed light on where and when retinoid signaling might affect a central pattern-generating network to promote memory formation during conditioning of a homeostatic behavior.NEW & NOTEWORTHY We provide novel evidence for how conditioning-induced changes in a CPG network are disrupted when retinoid signaling is inhibited. Inhibition of retinoic acid signaling prevents long-term memory formation following operant conditioning, but has no effect on learning. Simultaneous electrophysiological and behavioral analyses indicate network changes immediately following learning, but these changes are prevented with inhibition of retinoid signaling, before any overt changes in behavior. These data suggest sites for retinoid actions during memory formation.

Context-Dependent Role of miR-124 in Retinoic Acid-Induced Growth Cone Attraction of Regenerating Motorneurons.

During development and regeneration, growth cones at the tips of extending axons navigate through a complex environment to establish accurate connections with appropriate targets. Growth cones can respond rapidly to classical and non-classical guidance cues in their environment, often requiring local protein synthesis. In vertebrate growth cones, local protein synthesis in response to classical cues can require regulation by microRNAs (miRNAs), a class of small, conserved, non-coding RNAs that post-transcriptionally regulate gene expression. However, less is known of how miRNAs mediate growth cone responses to non-classical cues (such as retinoic acid (RA)), specifically in invertebrates. Here, we utilized adult regenerating invertebrate motorneurons to study miRNA regulation of growth cone attraction to RA, shown to require local protein synthesis. In situ hybridization revealed the presence of miR-124 in growth cones of regenerating ciliary motorneurons of the mollusc Lymnaea stagnalis. Changes in the spatiotemporal distribution of miR-124 occurred following application of RA, and dysregulation of miR-124 (with mimic injection), disrupted RA-induced growth cone turning in a time-dependent manner. This behavioural regulation by miR-124 was altered when the neurite was transected, and the growth cone completely separated from the soma. miR-124 did not, however, appear to be involved in growth cone attraction to serotonin, a response independent of local protein synthesis. Finally, we provide evidence that a downstream effector of RhoGTPases, ROCK, is a potential target of miR-124 during RA-induced growth cone responses. These data advance our current understanding of how microRNAs might mediate cue- and context-dependent behaviours during axon guidance.

Retinoid receptor-based signaling plays a role in voltage-dependent inhibition of invertebrate voltage-gated Ca2+ channels.

The retinoic acid receptor (RAR) and retinoid X receptor (RXR) mediate the cellular effects of retinoids (derivatives of vitamin A). Both RAR and RXR signaling events are implicated in hippocampal synaptic plasticity. Furthermore, retinoids can interact with calcium signaling during homeostatic plasticity. We recently provided evidence that retinoids attenuate calcium current (ICa) through neuronal voltage-gated calcium channels (VGCCs). We now examined the possibility that constitutive activity of neuronal RXR and/or RAR alters calcium influx via the VGCCs. We found that in neurons of the mollusk Lymnaea stagnalis, two different RXR antagonists (PA452 and HX531) had independent and opposing effects on ICa that were also time-dependent; whereas the RXR pan-antagonist PA452 enhanced ICa, HX531 reduced ICa Interestingly, this effect of HX531 occurred through voltage-dependent inhibition of VGCCs, a phenomenon known to influence neurotransmitter release from neurons. This inhibition appeared to be independent of G proteins and was largely restricted to Cav2 Ca2+ channels. Of note, an RAR pan-antagonist, LE540, also inhibited ICa but produced G protein-dependent, voltage-dependent inhibition of VGCCs. These findings provide evidence that retinoid receptors interact with G proteins in neurons and suggest mechanisms by which retinoids might affect synaptic calcium signaling.

Pharmacological evidence for the role of RAR in axon guidance and embryonic development of a protostome species.

Retinoic acid (RA), the active metabolite of vitamin A, functions through nuclear receptors, one of which is the retinoic acid receptor (RAR). Though the RAR is essential for various aspects of vertebrate development, little is known about the role of RAR in nonchordate invertebrates. Here, we examined the potential role of an invertebrate RAR in mediating chemotropic effects of retinoic acid. The RAR of the protostome Lymnaea stagnalis is present in the growth cones of regenerating cultured motorneurons, and a synthetic RAR agonist (EC23), was able to mimic the effects of retinoic acid in inducing growth cone turning. We also examined the ability of the natural retinoids, all-trans RA and 9-cis RA, as well as the synthetic RAR agonists, to disrupt embryonic development in Lymnaea. Developmental defects included delays in embryo hatching, arrested eye, and shell development, as well as more severe abnormalities such as halted development. Developmental defects induced by some (but not all) synthetic RAR agonists were found to mimic those induced by addition of high concentrations of the natural retinoid isomers. These pharmacological data support a possible physiological role for the RAR in axon guidance and embryonic development of an invertebrate protostome species.

The effect of rearing environment on memory formation.

Lymnaea stagnalis is a well-studied model system for determining how changes in the environment influence associative learning and memory formation. For example, some wild strains of L. stagnalis, collected from separate geographic locations, show superior memory-forming abilities compared with others. Here, we studied memory formation in two laboratory-bred L. stagnalis strains, derived from the same original population in The Netherlands. The two strains were reared in two different laboratories at the University of Calgary (C-strain) and at Brock University (B-strain) for many years and we found that they differed in their memory-forming ability. Specifically, the C-strain required only two training sessions to form long-term memory (LTM) whereas the B-strain required four sessions to form LTM. Additionally, the LTM formed by the B-strain persisted for a shorter amount of time than the memory formed by the C-strain. Thus, despite being derived from the same original population, the C- and B-strains have developed different memory-forming abilities. Next, we raised the two strains from embryos away from home (i.e. in the other laboratory) over two generations and assessed their memory-forming abilities. The B-strain reared and maintained at the University of Calgary demonstrated improved memory-forming ability within a single generation, while the C-strain reared at Brock University retained their normal LTM-forming ability across two subsequent generations. This suggests that local environmental factors may contribute to the behavioural divergence observed between these two laboratory-bred strains.

Identification and Characterization of microRNAs during Retinoic Acid-Induced Regeneration of a Molluscan Central Nervous System.

Retinoic acid (RA) is the biologically active metabolite of vitamin A and has become a well-established factor that induces neurite outgrowth and regeneration in both vertebrates and invertebrates. However, the underlying regulatory mechanisms that may mediate RA-induced neurite sprouting remain unclear. In the past decade, microRNAs have emerged as important regulators of nervous system development and regeneration, and have been shown to contribute to processes such as neurite sprouting. However, few studies have demonstrated the role of miRNAs in RA-induced neurite sprouting. By miRNA sequencing analysis, we identify 482 miRNAs in the regenerating central nervous system (CNS) of the mollusc Lymnaeastagnalis, 219 of which represent potentially novel miRNAs. Of the remaining conserved miRNAs, 38 show a statistically significant up- or downregulation in regenerating CNS as a result of RA treatment. We further characterized the expression of one neuronally-enriched miRNA upregulated by RA, miR-124. We demonstrate, for the first time, that miR-124 is expressed within the cell bodies and neurites of regenerating motorneurons. Moreover, we identify miR-124 expression within the growth cones of cultured ciliary motorneurons (pedal A), whereas expression in the growth cones of another class of respiratory motorneurons (right parietal A) was absent in vitro. These findings support our hypothesis that miRNAs are important regulators of retinoic acid-induced neuronal outgrowth and regeneration in regeneration-competent species.

The role of retinoic acid in the formation and modulation of invertebrate central synapses.

Trophic factors can influence many aspects of nervous system function, such as neurite outgrowth, synapse formation, and synapse modulation. The vitamin A metabolite, retinoic acid, can exert trophic effects to promote neuronal survival and outgrowth in many species and is also known to modulate vertebrate hippocampal synapses. However, its role in synaptogenesis has not been well studied, and whether it can modulate existing invertebrate synapses is also not known. In this study, we first examined a potential trophic effect of retinoic acid on the formation of excitatory synapses, independently of its role in neurite outgrowth, using cultured neurons of the mollusc Lymnaea stagnalis We also investigated its role in modulating both chemical and electrical synapses between various Lymnaea neurons in cell culture. Although we found no evidence to suggest retinoic acid affected short-term synaptic plasticity in the form of post-tetanic potentiation, we did find a significant cell type-specific modulation of electrical synapses. Given the prevalence of electrical synapses in invertebrate nervous systems, these findings highlight the potential for retinoic acid to modulate network function in the central nervous system of at least some invertebrates. NEW & NOTEWORTHY: This study performed the first electrophysiological analysis of the ability of the vitamin A metabolite, retinoic acid, to exert trophic influences during synaptogenesis independently of its effects in supporting neurite outgrowth. It was also the first study to examine the ability of retinoic acid to modify both chemical and electrical synapses in any invertebrate, nonchordate species. We provide evidence that all-trans retinoic acid can modify invertebrate electrical synapses of central neurons in a cell-specific manner.

Extending the duration of long-term memories: Interactions between environmental darkness and retinoid signaling.

Retinoid signaling plays an important role in hippocampal-dependent vertebrate memories. However, we have previously demonstrated that retinoids are also involved in the formation of long-term implicit memory following operant conditioning of the invertebrate mollusc Lymnaea stagnalis. Furthermore, we have discovered an interaction between environmental light/dark conditions and retinoid signaling and the ability of both to convert intermediate-term memory into long-term memory. In this study, we extend these findings to show that retinoid receptor agonists and environmental darkness can both also extend the duration of long-term memory. Interestingly, exposure to constant environmental darkness significantly increased the expression of retinoid receptors in the adult central nervous system, as well as induced specific changes in a key neuron mediating the conditioned behaviour. These studies not only shed more light on how retinoids influence memory formation, but also further link environmental light conditions to the retinoid signaling pathway.

Retinoic acid affects calcium signaling in adult molluscan neurons.

Retinoic acid, the active metabolite of vitamin A, is important for nervous system development, regeneration, as well as cognitive functions of the adult central nervous system. These central nervous system functions are all highly dependent on neuronal activity. Retinoic acid has previously been shown to induce changes in the firing properties and action potential waveforms of adult molluscan neurons in a dose- and isomer-dependent manner. In this study, we aimed to determine the cellular pathways by which retinoic acid might exert such effects, by testing the involvement of pathways previously shown to be affected by retinoic acid. We demonstrated that the ability of all-trans retinoic acid (atRA) to induce electrophysiological changes in cultured molluscan neurons was not prevented by inhibitors of protein synthesis, protein kinase A or phospholipase C. However, we showed that atRA was capable of rapidly reducing intracellular calcium levels in the same dose- and isomer-dependent manner as shown previously for changes in neuronal firing. Moreover, we also demonstrated that the transmembrane ion flux through voltage-gated calcium channels was rapidly modulated by retinoic acid. In particular, the peak current density was reduced and the inactivation rate was increased in the presence of atRA, over a similar time course as the changes in cell firing and reductions in intracellular calcium. These studies provide further evidence for the ability of atRA to induce rapid effects in mature neurons.

Expression of a retinoic acid receptor (RAR)-like protein in the embryonic and adult nervous system of a protostome species.

The vitamin A metabolite, retinoic acid, is an important molecule in nervous system development and regeneration in vertebrates. Retinoic acid signaling in vertebrates is mediated by two classes of nuclear receptors, the retinoid X receptors (RXRs) and the retinoic acid receptors (RARs). Recently, evidence has emerged to suggest that many effects of retinoic acid are conserved between vertebrate and invertebrate nervous systems, even though the RARs were previously thought to be a vertebrate innovation and to not exist in non-chordates. We have cloned a full-length putative RAR from the CNS of the mollusc Lymnaea stagnalis (LymRAR). Immunoreactivity for the RAR protein was found in axons of adult neurons in the central nervous system and in growth cones of regenerating neurons in vitro. A vertebrate RAR antagonist blocked growth cone turning induced by exogenous all-trans retinoic acid, possibly suggesting a role for this receptor in axon guidance. We also provide immunostaining evidence for the presence of RAR protein in the developing, embryonic CNS, where it is also found in axonal processes. Using qPCR, we determined that LymRAR mRNA is detectable in the early veliger stage embryo and that mRNA levels increase significantly during embryonic development. Putative disruption of retinoid signaling in Lymnaea embryos using vertebrate RAR antagonists resulted in abnormal eye and shell development and in some instances completely halted development, resembling the effects of all-trans retinoic acid. This study provides evidence for RAR functioning in a protostome species.

Retinoic acid induces changes in electrical properties of adult neurons in a dose- and isomer-dependent manner.

The electrical activity of neurons is known to play a role in neuronal development, as well as repair of adult nervous tissue. For example, the extension of neurites and motility of growth cones can be modulated by changes in the electrical firing of neurons. The vitamin A metabolite retinoic acid also plays a critical role during nervous system development and is also known to elicit regenerative responses, namely the induction, enhancement, and directionality of neurite outgrowth. However, no studies have previously reported the ability of retinoic acid to modify the electrical activity of neurons. In this study, we determined whether retinoic acid might exert effects on the nervous system by altering the electrical properties of neurons. Using cultured adult neurons from Lymnaea stagnalis, we showed that acute application of retinoic acid can rapidly elicit changes in neuronal firing properties. Retinoic acid caused the presence of atypical firing behavior such as rhythmic bursting and altered the shape of action potentials, causing increases in half-amplitude duration and decay time. Retinoic acid also caused cell silencing, whereby neuronal activity was halted within an hour. These effects of retinoic acid were shown to be both dose and isomer dependent. We then showed that the effects of retinoic acid on cell firing (but not silencing) were significantly reduced in the presence of an retinoid X receptor pan-antagonist HX531. This study suggests that some of the effects of retinoic acid during neuronal development or regeneration might possibly occur as a result of changes in electrical activity of neurons.

Retinoid signaling is necessary for, and promotes long-term memory formation following operant conditioning.

Retinoic acid, a metabolite of vitamin A, is proposed to play an important role in vertebrate learning and memory, as well as hippocampal-dependent synaptic plasticity. However, it has not yet been determined whether retinoic acid plays a similar role in learning and memory in invertebrates. In this study, we report that retinoid signaling in the mollusc Lymnaea stagnalis, is required for long-term memory formation following operant conditioning of its aerial respiratory behaviour. Animals were exposed to inhibitors of the RALDH enzyme (which synthesizes retinoic acid), or various retinoid receptor antagonists. Following exposure to these inhibitors, neither learning nor intermediate-term memory (lasting 2 h) was affected, but long-term memory formation (tested at either 24 or 72 h) was inhibited. We next demonstrated that various retinoid receptor agonists promoted long-term memory formation. Using a training paradigm shown only to produce intermediate-term memory (lasting 2 h, but not 24 h) we found that exposure of animals to synthetic retinoids promoted memory formation that lasted up to 30 h. These findings suggest that the role of retinoids in memory formation is ancient in origin, and that retinoid signaling is also important for the formation of implicit memories, in addition to its previously demonstrated role in hippocampal-dependent memories.

Novel interactive effects of darkness and retinoid signaling in the ability to form long-term memory following aversive operant conditioning.

The vitamin A metabolite, retinoic acid, is important for memory formation and hippocampal synaptic plasticity in vertebrate species. In our studies in the mollusc Lymnaea stagnalis, we have shown that retinoic acid plays a role in memory formation following operant conditioning of the aerial respiratory behaviour. Inhibition of either retinaldehyde dehydrogenase (RALDH) or the retinoid receptors prevents long-term memory (LTM) formation, whereas synthetic retinoid receptor agonists promote memory formation by converting intermediate-term memory (ITM) into LTM. In this study, animals were exposed to constant darkness in order to test whether light-sensitive retinoic acid would promote memory formation. However, we found that exposure to constant darkness alone (in the absence of retinoic acid) enhanced memory formation. To determine whether the memory-promoting effects of darkness could override the memory-inhibiting effects of the retinoid signaling inhibitors, we exposed snails to RALDH inhibitors or a retinoid receptor antagonist in constant darkness. We found that darkness overcame the inhibitory effects of RALDH inhibition, but did not overcome the inhibitory effects of the retinoid receptor antagonist. We also tested whether constant darkness and training affected the mRNA levels of the retinoid metabolic enzymes RALDH and Cyp26, or the mRNA levels of the retinoid receptors, but found no significant effect. Overall, these data demonstrate an interaction between environmental light conditions and the retinoid signaling pathway, which influence long-term memory formation in a mollusc.

Non-canonical retinoid signaling in neural development, regeneration and synaptic function.

Canonical retinoid signaling via nuclear receptors and gene regulation is critical for the initiation of developmental processes such as cellular differentiation, patterning and neurite outgrowth, but also mediates nerve regeneration and synaptic functions in adult nervous systems. In addition to canonical transcriptional regulation, retinoids also exert rapid effects, and there are now multiple lines of evidence supporting non-canonical retinoid actions outside of the nucleus, including in dendrites and axons. Together, canonical and non-canonical retinoid signaling provide the precise temporal and spatial control necessary to achieve the fine cellular coordination required for proper nervous system function. Here, we examine and discuss the evidence supporting non-canonical actions of retinoids in neural development and regeneration as well as synaptic function, including a review of the proposed molecular mechanisms involved.

Presence of Ribeiroia ondatrae in the developing anuran limb disrupts retinoic acid levels.

The widespread reports of malformed frogs have sparked interest worldwide to try and determine the causes of such malformations. Ribeiroia ondatrae is a digenetic trematode, which has been implicated as one such cause, as this parasite encysts within the developing tadpole hind limb bud and inguinal region causing dramatic limb malformations. Currently, the mechanisms involved in parasite-induced limb deformities remain unclear. We sought to investigate whether the level of retinoic acid (RA), a morphogenetic factor known to play a critical role in limb bud formation, is altered by the presence of R. ondatrae within the infected tadpole. Alteration of RA levels within the limb bud caused by the presence of the parasite may be achieved in three ways. First, metacercariae are actively secreting RA; second, cercariae, upon entering the limb/inguinal region, may release a large amount of RA; finally, the metacercariae may induce either an increase in the synthesis or a decrease in the degradation of the host's endogenous retinoic acid levels. Here, we show through high performance liquid chromatography and mass spectrometry that limb bud tissue of Lithobates sylvaticus, which has been parasitised, contains 70% more RA compared to the unparasitised control. Furthermore, parasites that have encysted within the limb buds appear to contain substantially less RA (56%) than the free swimming cercariae (defined as the infectious stage of the parasite). Taken together, these data illustrate for the first time that encystment of R. ondatrae leads to an increase in RA levels in the tadpole limb bud and may offer insight into the mechanisms involved in parasite-induced limb deformities.

Molluscan RXR Transcriptional Regulation by Retinoids in a Drosophila CNS Organ Culture System.

Retinoic acid, the active metabolite of Vitamin A, is important for the appropriate development of the nervous system (e.g., neurite outgrowth) as well as for cognition (e.g., memory formation) in the adult brain. We have shown that many of the effects of retinoids are conserved in the CNS of the mollusc, Lymnaea stagnalis. RXRs are predominantly nuclear receptors, but the Lymnaea RXR (LymRXR) exhibits a non-nuclear distribution in the adult CNS, where it is also implicated in non-genomic retinoid functions. As such, we developed a CNS Drosophila organ culture-based system to examine the transcriptional activity and ligand-binding properties of LymRXR, in the context of a live invertebrate nervous system. The novel ligand sensor system was capable of reporting both the expression and transcriptional activity of the sensor. Our results indicate that the LymRXR ligand sensor mediated transcription following activation by both 9-cis RA (the high affinity ligand for vertebrate RXRs) as well as the vertebrate RXR synthetic agonist, SR11237. The LymRXR ligand sensor was also activated by all-trans RA, and to a much lesser extent by the vertebrate RAR synthetic agonist, EC23. This sensor also detected endogenous retinoid-like activity in the CNS of developing Drosophila larvae, primarily during the 3rd instar larval stage. These data indicate that the LymRXR sensor can be utilized not only for characterization of ligand activation for studies related to the Lymnaea CNS, but also for future studies of retinoids and their functions in Drosophila development.

Developmental expression of a molluscan RXR and evidence for its novel, nongenomic role in growth cone guidance.

It is well known that the vitamin A metabolite, retinoic acid, plays an important role in vertebrate development and regeneration. We have previously shown that the effects of RA in mediating neurite outgrowth, are conserved between vertebrates and invertebrates (Dmetrichuk et al., 2005, 2006) and that RA can induce growth cone turning in regenerating molluscan neurons (Farrar et al., 2009). In this study, we have cloned a retinoid receptor from the mollusc Lymnaea stagnalis (LymRXR) that shares about 80% amino acid identity with the vertebrate RXRalpha. We demonstrate using Western blot analysis that the LymRXR is present in the developing Lymnaea embryo and that treatment of embryos with the putative RXR ligand, 9-cis RA, or a RXR pan-agonist, PA024, significantly disrupts embryogenesis. We also demonstrate cytoplasmic localization of LymRXR in adult central neurons, with a strong localization in the neuritic (or axonal) domains. Using regenerating cultured motor neurons, we show that LymRXR is also present in the growth cones and that application of a RXR pan-agonist produces growth cone turning in isolated neurites (in the absence of the cell body and nucleus). These data support a role for RXR in growth cone guidance and are the first studies to suggest a nongenomic action for RXR in the nervous system.

A novel, nongenomic mechanism underlies retinoic acid-induced growth cone turning.

The vitamin A metabolite, retinoic acid (RA), is well known for its roles in neural development and regeneration. We have previously shown that RA can induce positive growth cone turning in regenerating neurons in vitro. In this study, we address the subcellular mechanisms underlying this chemo-attractive response, using identified central neurons from the adult mollusc, Lymnaea stagnalis. We show that the RA-induced positive growth cone turning was maintained in the presence of the transcriptional inhibitor, actinomycin D. We also physically transected the neurites from the cell body and showed that isolated growth cones retain the capacity to turn toward a gradient of RA. Moreover, this attractive turning is dependent on de novo local protein synthesis and Ca(2+) influx. Most of RA's actions during neurite outgrowth and regeneration require gene transcription, although these data show for the first time in any species, that the chemotropic action of RA in guiding neurite outgrowth, involves a novel, nongenomic mechanism.