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.

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.

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.

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.

Retinoic acid inhibits neuronal voltage-gated calcium channels.

Retinoic acid is the active metabolite of vitamin A and regulates several important cellular processes by activating retinoic acid receptors (RAR) and retinoid X receptors (RXR). These receptors generally act as transcription factors, though non-genomic actions of both retinoic acid and the receptors have also been reported. One such nongenomic effect includes the modulation of Ca2+ levels during homeostatic synaptic plasticity in the hippocampus. Retinoic acid can thus affect Ca2+ signaling and can potentially control both synaptic plasticity and neuronal firing. However, whether retinoic acid can regulate voltage-gated Ca2+ channels (either via genomic or nongenomic actions), which are fundamental to these processes, has not yet been studied in detail. Here we demonstrate the effects of retinoic acid on the biophysical properties of voltage-gated Ca2+ channels in cultured invertebrate motorneurons. Overnight exposure to physiological concentrations of retinoic acid significantly inhibited the voltage-gated Ca2+ current (ICa) in an isomer-dependent manner. Specifically, all-trans retinoic acid (atRA), but not 9-cis RA (9cRA), depolarized the voltage of half-maximal activation of ICa. AtRA also reduced the rate of channel activation and delayed recovery from inactivation. We provide evidence that both L-type and non-L-type voltage-gated Ca2+ channels are affected by atRA, as both nifedipine-sensitive and nifedipine-resistant ICa were inhibited in these neurons. These effects of retinoic acid are thought to be at least partially mediated by the retinoid receptors, as treatment of the neurons with synthetic RAR and RXR agonists produced a similar inhibition of ICa.

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.