CNS serotonin content mediating food deprivation-enhanced learning is regulated by hemolymph tryptophan concentration and autophagic flux in the pond snail.

Nutritional status affects cognitive function in many types of organisms. In the pond snail Lymnaea stagnalis, 1 day of food deprivation enhances taste aversion learning ability by decreasing the serotonin (5-hydroxytryptamin; 5-HT) content in the central nervous system (CNS). On the other hand, after 5 days of food deprivation, learning ability and the CNS 5-HT concentration return to basal levels. How food deprivation leads to alterations of 5-HT levels in the CNS, however, is unknown. Here, we measured the concentration of the 5-HT precursor tryptophan in the hemolymph and CNS, and demonstrated that the CNS tryptophan concentration was higher in 5-day food-deprived snails than in non-food-deprived or 1-day food-deprived snails, whereas the hemolymph tryptophan concentration was not affected by the duration of food deprivation. This finding suggests the existence of a mediator of the CNS tryptophan concentration independent of food deprivation. To identify the mediator, we investigated autophagic flux in the CNS under different food deprivation conditions. We found that autophagic flux was significantly upregulated by inhibition of the tropomyosin receptor kinase (Trk)-Akt-mechanistic target of rapamycin complex 1 (MTORC1) pathway in the CNS of 5-day food-deprived snails. Moreover, when autophagy was inhibited, the CNS 5-HT content was significantly downregulated in 5-day food-deprived snails. Our results suggest that the hemolymph tryptophan concentration and autophagic flux in the CNS cooperatively regulate learning ability affected by different durations of food deprivation. This mechanism may underlie the selection of behaviors appropriate for animal survival depending on the degree of nutrition.

DNA Instability in Neurons: Lifespan Clock and Driver of Evolution.

In the last ten years, the discovery of neuronal DNA postmitotic instability has changed the theoretical landscape in neuroscience and, more broadly, biology. In 2003, A. M. Olovnikov suggested that neuronal DNA is the "initial substrate of aging". Recent experimental data have significantly increased the likelihood of this hypothesis. How does neuronal DNA accumulate damage and in what genome regions? What factors contribute to this process and how are they associated with aging and lifespan? These questions will be discussed in the review. In the course of Metazoan evolution, the instability of neuronal DNA has been accompanied by searching for the pathways to reduce the biological cost of brain activity. Various processes and activities, such as sleep, evolutionary increase in the number of neurons in the vertebrate brain, adult neurogenesis, distribution of neuronal activity, somatic polyploidy, and RNA editing in cephalopods, can be reconsidered in the light of the trade-off between neuronal plasticity and DNA instability in neurons. This topic is of considerable importance for both fundamental neuroscience and translational medicine.

Effects of 5-HT and insulin on learning and memory formation in food-deprived snails.

The pond snail Lymnaea stagnalis learns conditioned taste aversion (CTA) and consolidates it into long-term memory (LTM). How well they learn and form memory depends on the degree of food deprivation. Serotonin (5-HT) plays an important role in mediating feeding, and insulin enhances the memory consolidation process following CTA training. However, the relationship between these two signaling pathways has not been addressed. We measured the 5-HT content in the central nervous system (CNS) of snails subjected to different durations of food deprivation. One-day food-deprived snails, which exhibit the best learning and memory, had the lowest 5-HT content in the CNS, whereas 5-day food-deprived snails, which do not learn, had a high 5-HT content. Immersing 1-day food-deprived snails in 5-HT impaired learning and memory by causing an increase in 5-HT content, and that the injection of insulin into these snails reversed this impairment. We conclude that insulin rescues the CTA deficit and this may be due to a decrease in the 5-HT content in the CNS of Lymnaea.

Previous motor activity affects the transition from uncertainty to decision making in snails.

One of the most widely accepted benefits of enhanced physical activity is an improvement in the symptoms of depression, including the facilitation of decision making. Up until now, these effects have been shown in rodents and humans only. Little is known about their evolutionary origin or biological basis, and the underlying cellular mechanisms also remain relatively elusive. Here, we demonstrate for the first time that preceding motor activity accelerates decision making in an invertebrate, the pond snail Lymnaea stagnalis To investigate decision making in a novel environment, snails, which normally live in water, were placed on a flat dry surface to simulate the potentially threatening consequence of being in an arid environment. This stimulus initiated two distinct phases in snail behaviour: slow circular movements, followed by intense locomotion in a chosen direction. The first phase was prolonged when the test arena was symmetrically lit, compared with one with an apparent gradient of light. However, forced muscular locomotion for 2 h prior to the test promoted the transition from random circular motions to a directional crawl, accompanied by an increase in crawling speed but with no effect on the choice of direction. Intense locomotion for 2 h also produced a strong excitatory effect on the activity of serotonergic neurons in L. stagnalis Our results suggest that the beneficial effects of physical exercise on cognitive performance in mammals might have deep roots in evolution, granting the opportunity to unravel the origins of such effects at the single-neuron and network levels.

The activity of isolated neurons and the modulatory state of an isolated nervous system represent a recent behavioural state.

Behavioural/motivational state is known to influence nearly all aspects of physiology and behaviour. The cellular basis of behavioural state control is only partially understood. Our investigation, performed on the pond snail Lymnaea stagnalis whose nervous system is useful for work on completely isolated neurons, provided several results related to this problem. First, we demonstrated that the behavioural state can produce long-term changes in individual neurons that persist even after neuron isolation from the nervous system. Specifically, we found that pedal serotonergic neurons that control locomotion show higher activity and lower membrane potential after being isolated from the nervous systems of hungry animals. Second, we showed that the modulatory state (the chemical neuroactive microenvironment of the central ganglia) changes in accordance with the nutritional state of an animal and produces predicted changes in single isolated locomotor neurons. Third, we report that observed hunger-induced effects can be explained by the increased synthesis of serotonin in pedal serotonergic neurons, which has an impact on the electrical activity of isolated serotonergic neurons and the intensity of extrasynaptic serotonin release from the pedal ganglia.

Multistable coordination of feeding motor rhythms in semi-intact preparation of Lymnaea stagnalis. Short communication.

It is well known that most rhythm-generating neuronal ensembles are multifunctional and can generate different motor rhythms in different contexts. This implies that coordination of multifunctional networks must also be flexible or multistable. Coordination of radula movements and gut contractions was studied in semi-intact preparations of L. stagnalis using video registration and an event recorder. Several context-dependent stable variants of radula-gut coordination were detected. Our data suggest that this preparation is a promising model for studying mechanisms of multistable motor rhythm coordination.

Insulin and Memory in Invertebrates.

Insulin and insulin-like peptides (ILP) help to maintain glucose homeostasis, whereas insulin-like growth factor (IGF) promotes the growth and differentiation of cells in both vertebrates and invertebrates. It is sometimes difficult to distinguish between ILP and IGF in invertebrates, however, because in some cases ILP has the same function as IGF. In the present review, therefore, we refer to these peptides as ILP/IGF signaling (IIS) in invertebrates, and discuss the role of IIS in memory formation after classical conditioning in invertebrates. In the arthropod Drosophila melanogaster, IIS is involved in aversive olfactory memory, and in the nematode Caenorhabditis elegans, IIS controls appetitive/aversive response to NaCl depending on the duration of starvation. In the mollusk Lymnaea stagnalis, IIS has a critical role in conditioned taste aversion. Insulin in mammals is also known to play an important role in cognitive function, and many studies in humans have focused on insulin as a potential treatment for Alzheimer's disease. Although analyses of tissue and cellular levels have progressed in mammals, the molecular mechanisms, such as transcriptional and translational levels, of IIS function in cognition have been far advanced in studies using invertebrates. We anticipate that the present review will help to pave the way for studying the effects of insulin, ILPs, and IGFs in cognitive function across phyla.

Coordination of rhythm-generating units via NO and extrasynaptic neurotransmitter release.

The buccal ganglia of the mollusc, Lymnaea stagnalis, contain two distinct but interacting rhythm-generating units: the central pattern generator for the buccal rhythm and nitrergic B2 neurons controlling gut motility. Nitric oxide (NO) has previously been demonstrated to be involved in the activation of the buccal rhythm. Here, we found that NO-generating substances (SNP and SNAP) activated the buccal rhythm while slowing the endogenous rhythm of B2 bursters. The inhibitor of NO-synthase, L-NNA, the NO scavenger PTIO, or the inhibitor of soluble guanylyl cyclase, ODQ, each produced opposite, depolarising effects on the B2 neuron. In isolated B2 cells, only depolarising effects of substances interfering with NO production or function (PTIO, L-NNA and ODQ) were detected, whereas the NO donors had no hyperpolarising effects. However, when an isolated B2 cell was placed close to its initial position in the ganglion, hyperpolarising effects could be obtained with NO donors. This indicates that extrasynaptic release of some unidentified factor(s) mediates the hyperpolarising effects of NO donors on the B2 bursters. The results suggest that NO is involved in coordination between the radula and foregut movements and that the effects of NO are partially mediated by the volume chemical neurotransmission of as yet unknown origin.

Another Example of Conditioned Taste Aversion: Case of Snails.

Conditioned taste aversion (CTA) in mammals has several specific characteristics: (1) emergence of a negative symptom in subjects due to selective association with a taste-related stimulus, (2) robust long-term memory that is resistant to extinction induced by repeated presentation of the conditioned stimulus (CS), (3) a very-long-delay presentation of the unconditioned stimulus (US), and (4) single-trial learning. The pond snail, Lymnaea stagnalis, can also form a CTA. Although the negative symptoms, like nausea, in humans cannot be easily observed in invertebrate animal models of CTA, all the other characteristics of CTA seem to be present in snails. Selective associability was confirmed using a sweet sucrose solution and a bitter KCl solution. Once snails form a CTA, repeated presentation of the CS does not extinguish the CTA. A long interstimulus interval between the CS and US, like in trace conditioning, still results in the formation of a CTA in snails. Lastly, even single-trial learning has been demonstrated with a certain probability. In the present review, we compare, in detail, CTA in mammals and snails, and discuss the possible molecular events in CTA.

Induction of LTM following an Insulin Injection.

The pond snail Lymnaea stagnalis learns conditioned taste aversion (CTA) and consolidates it into long-term memory (LTM). One-day food-deprived snails (day 1 snails) show the best CTA learning and memory, whereas more severely food-deprived snails (5 d) do not express good memory. However, previous studies showed that CTA-LTM was indeed formed in 5-d food-deprived snails (day 5 snails), but its recall was prevented by the effects of food deprivation. CTA-LTM recall in day 5 snails was expressed following 7 d of feeding and then 1 d of food deprivation (day 13 snails). In the present study, we thus hypothesized that memory recall occurs because day 13 snails are in an optimal internal state. One day of food deprivation before the memory test in day 13 snails increased the mRNA level of molluscan insulin-related peptide (MIP) in the CNS. Thus, we further hypothesized that an injection of insulin into day 5 snails following seven additional days with access to food (day 12 snails) activates CTA neurons and mimics the food deprivation state before the memory test in day 13 snails. Day 12 snails injected with insulin could recall the memory. In addition, the simultaneous injection of an anti-insulin receptor antibody and insulin into day 12 snails did not allow memory recall. Insulin injection also decreased the hemolymph glucose concentration. Together, the results suggest that an optimal internal state (i.e., a spike in insulin release and specific glucose levels) are necessary for LTM recall following CTA training in snails.

Direct and decarboxylation-dependent effects of neurotransmitter precursors on firing of isolated monoaminergic neurons.

To elucidate mechanisms that underlie the profound physiological effects of the monoamine precursors 5-hydroxy-L-tryptophan (5-HTP) and L-3,4-dihydroxyphenylalanine (L-DOPA), we examined their action on single monoaminergic neurons isolated from the ganglia of the gastropod snail Lymnaea stagnalis. In isolated serotonergic PeA motoneurons, 5-HTP produced excitation. The effect was mimicked by serotonin at 0.5-1 microM, masked by pretreatment with serotonin at higher concentrations, and abolished by the inhibitor of aromatic amino acid decarboxylase (AAAD) m-hydroxybenzylhydrazine (NSD-1015), the inhibitor of the vesicular monoamine transporter reserpine or the serotonin receptor antagonist mianserin. Exposure of the dopaminergic interneurons RPeD1 to L-DOPA caused a biphasic effect composed of a depolarization followed by a hyperpolarization. AAAD inactivation with NSD-1015, as well as the blockade of dopamine receptors with sulpiride, resulted in the enhancement of the excitatory effect, and the abolition of the inhibitory effect. Dopamine caused hyperpolarization and masked the inhibitory phase of L-DOPA action. The results show that precursors affect the rate of firing of isolated monoaminergic neurons and that their effect is completely or partially mediated by the enhanced synthesis of the respective neurotransmitter, followed by extrasynaptic release of the latter and activation of extrasynaptic autoreceptors.

Possible involvement of nitric oxide in coordination of buccal rhythm and gut motility in Lymnaea stagnalis.

NO-generating drugs (SNP, SNAP, NaNO2) activated the standard buccal rhythm and simultaneously slowed down the endogenous rhythm of the NO-synthesizing neuron B2 which enhances gut motility. In the B2, these drugs caused giant hyperpolarizing waves strongly associated with the buccal cycles. Hyperpolarizing waves on the B2 could also be induced by prolonged stimulation of the B2. The inhibitor of NO-synthase L-NNA or the NO scavanger PTIO abolished giant hyperpolarizing waves, depolarised the B2 and activated fast bursting in the B2. The results suggest that NO may be involved in coordination of radula and foregut movements.

A CREB2-targeting microRNA is required for long-term memory after single-trial learning.

Although single-trial induced long-term memories (LTM) have been of major interest in neuroscience, how LTM can form after a single episode of learning remains largely unknown. We hypothesized that the removal of molecular inhibitory constraints by microRNAs (miRNAs) plays an important role in this process. To test this hypothesis, first we constructed small non-coding RNA (sncRNA) cDNA libraries from the CNS of Lymnaea stagnalis subjected to a single conditioning trial. Then, by next generation sequencing of these libraries, we identified a specific pool of miRNAs regulated by training. Of these miRNAs, we focussed on Lym-miR-137 whose seed region shows perfect complementarity to a target sequence in the 3' UTR of the mRNA for CREB2, a well-known memory repressor. We found that Lym-miR-137 was transiently up-regulated 1 h after single-trial conditioning, preceding a down-regulation of Lym-CREB2 mRNA. Furthermore, we discovered that Lym-miR-137 is co-expressed with Lym-CREB2 mRNA in an identified neuron with an established role in LTM. Finally, using an in vivo loss-of-function approach we demonstrated that Lym-miR-137 is required for single-trial induced LTM.