Function of insulin in snail brain in associative learning.

Insulin is well known as a hormone regulating glucose homeostasis across phyla. Although there are insulin-independent mechanisms for glucose uptake in the mammalian brain, which had contributed to a perception of the brain as an insulin-insensitive organ for decades, the finding of insulin and its receptors in the brain revolutionized the concept of insulin signaling in the brain. However, insulin's role in brain functions, such as cognition, attention, and memory, remains unknown. Studies using invertebrates with their open blood-vascular system have the promise of promoting a better understanding of the role played by insulin in mediating/modulating cognitive functions. In this review, the relationship between insulin and its impact on long-term memory (LTM) is discussed particularly in snails. The pond snail Lymnaea stagnalis has the ability to undergo conditioned taste aversion (CTA), that is, it associatively learns and forms LTM not to respond with a feeding response to a food that normally elicits a robust feeding response. We show that molluscan insulin-related peptides are up-regulated in snails exhibiting CTA-LTM and play a key role in the causal neural basis of CTA-LTM. We also survey the relevant literature of the roles played by insulin in learning and memory in other phyla.

Repetitive noxious stimulus altered the shadow-induced withdrawal behavior in Lymnaea.

Stress alters adaptive behaviors including vigilance behaviors. In Lymnaea one of these vigilance behavior is a heightened withdrawal response to a shadow. The shadow withdrawal response (SWR) is mediated by dermal photoreceptors located primarily on the foot, mantle cavity, and skin around the pneumostome area. Here we asked whether we could obtain a neural correlate of the heightened SWR and other essential behaviors following traumatic stress. We measured the electrophysiological properties of 'Right Pedal Dorsal 11 (RPeD11)', the interneuron that plays a major role in mediating the whole-body withdrawal response. In traumatized snails 24 hours after the trauma they responded not only to a shadow stimulus with an augmented withdrawal response, but suppressed in locomotive, feeding and respiratory behavior. Their behavioral change lasted at least one week. Accompanying the behavioral change in these 'traumatized' preparations there are a number of significant changes in the neuronal properties of RPeD11 compared to naïve preparations. For example, RPeD11 is significantly more depolarized (∼10 mV) has significantly larger input resistance, and the duration of the response elicited by the shadow persists longer. All these changes result in an increased RPeD11 response and seem to raise their defensive alert level.

In vitro aversion conditioning in Lymnaea. Short communication.

In an in vitro semi-intact Lymnaea preparation we were successful in using a training procedure to bring about Conditioning Taste Aversion (CTA ). Following paired presentation of the CS (sucrose) and US (tactile), the CS no longer elicits feeding. We can use sucrose to the lips in the semi-intact preparation as the CS; while we use direct current injection to depolarize RPeD11 as the US. Following pairing of these stimuli, the CS no longer elicits fictive feeding. We can determine the changes in synaptic input to neurons that play key roles in controlling feeding behavior.

Predator detection enables juvenile Lymnaea to form long-term memory.

Learning and memory provide the flexibility an organism requires to respond to changing social and ecological conditions. Juvenile Lymnaea have previously been shown to have a diminished capacity to form long-term memory (LTM) following operant conditioning of aerial respiratory behavior. Juvenile Lymnaea, however, can form LTM following classical conditioning of appetitive behaviors. Here, we demonstrate that laboratory-reared juvenile Lymnaea have the ability to detect the presence of a sympatric predator (i.e. crayfish) and respond to the predator by altering their aerial respiratory behavior. In addition to increasing their total breathing time, predator detection confers on juvenile Lymnaea an enhanced capability to form LTM following operant conditioning of aerial respiratory behavior. That is, these juveniles now have the ability to form long-lasting memory. These data support the hypothesis that biologically relevant levels of stress associated with predator detection induce behavioral phenotypic alterations (i.e. enhanced LTM formation) in juveniles, which may increase their fitness. These data also support the notion that learning and memory formation in conjunction with predator detection is a form of inducible defense.

Differences in LTM-forming capability between geographically different strains of Alberta Lymnaea stagnalis are maintained whether they are trained in the lab or in the wild.

We found strain differences in the ability of wild Alberta Lymnaea stagnalis to form long-term memory (LTM) following operant conditioning when L. stagnalis were collected from the wild and trained in the laboratory. Lymnaea stagnalis obtained from the Belly River watershed had an enhanced ability to form LTM compared with those from an isolated pond (referred to as Jackson snails). We therefore asked whether the differences in cognitive ability were an epiphenomenon as a result of training in the laboratory. To answer this question we trained each specific strain (Belly and Jackson) in both the laboratory and the field (i.e. in their home pond and in the pond where the other strain resided - referred to as the visitor pond). We found that within each strain there was no difference in the LTM phenotype whether they were trained in the lab or in either their home or visitor pond. That is, the strain differences in the ability to form LTM were still present. Interestingly, we found no strain differences in the ability to learn or the ability to form intermediate-term memory (ITM).

Speed of back-swimming of Lymnaea.

The pond snail, Lymnaea stagnalis, can locomote on its back utilizing the surface tension of the water. We have called this form of movement 'back-swimming'. In order to perform this behavior, the snail must flip itself over on its back so that its foot is visible from above. Little is known about the mechanism of this back-swimming. As a first step for the elucidation of this mechanism, we measured the speed of back-swimming of Lymnaea at the different times of the day. They back-swam significantly faster in the morning than just before dark. These data are consistent with our earlier findings on circadian-timed activity pattern in Lymnaea. Lymnaea appear to secrete a thin membrane-like substance from their foot that may allow them to back-swim. To confirm the existence of this substance and to examine whether this substance is hydrophobic or hydrophilic, we applied a detergent onto the foot during back-swimming. A single drop of 1% Tween 20 drifted Lymnaea away that were still kept at the water surface. These results suggest that Lymnaea secrete a hydrophobic substance from their foot that floats to the water surface allowing Lymnaea to back-swim.

Ketamine inhibits long-term, but not intermediate-term memory formation in Lymnaea stagnalis.

We investigated the effects of the drug ketamine on procedural intermediate- and long-term memory formation in a well-established operant learning and memory model system, Lymnaea stagnalis. Animals were administered ketamine at discrete time points, ranging from 2 h pre-one-trial training (1TT) to 23 h post-1TT. Our results demonstrated that ketamine causes impairment of procedural memory formation, and that ketamine acts differentially, inhibiting only long-term memory (LTM) formation while having no effect on intermediate-term memory (ITM) formation. Ketamine's ability to inhibit LTM was found not to be due to state dependent learning implying that ketamine's effects are therefore specific to the molecular process involved in procedural LTM formation. Given past data from our laboratory, this suggests that ketamine may be exerting its differential effects by altering the gene transcription processes necessary and specific for LTM formation. Additionally, ketamine was found to have no effect on retrieval when administered 1 h before testing. However, ketamine was able to disrupt LTM formation when administered immediately before 1TT and up to 2 h after 1TT. Our findings suggest a longer period of consolidation after 1TT than previously demonstrated in Lymnaea, during which the procedural long-term memory remains labile and is vulnerable to disruption via amnestic agents, such as ketamine.

Hypoxia-induced respiratory patterned activity in Lymnaea originates at the periphery.

Respiration in Lymnaea is a hypoxia-driven rhythmic behavior, which is controlled by an identified network of central pattern generating (CPG) neurons. However, the precise site(s) (i.e., central or peripheral) at which hypoxia acts and the cellular mechanisms by which the respiratory chemosensory drive is conveyed to the CPG were previously unknown. Using semi-intact and isolated ganglionic preparations, we provide the first direct evidence that the hypoxia-induced respiratory drive originates at the periphery (not within the central ring ganglia) and that it is conveyed to the CPG neurons via the right pedal dorsal neuron 1 (RPeD1). The respiratory discharge frequency increased when the periphery, but not the CNS, was made hypoxic. We found that in the semi-intact preparations, the frequency of spontaneously occurring respiratory bursts was significantly lower than in isolated ganglionic preparations. Thus the periphery exerts a suppressive regulatory control on respiratory discharges in the intact animal. Moreover, both anoxia (0% O(2)) and hypercapnia (10% CO(2)) produce a reduction in respiratory discharges in semi-intact, but not isolated preparations. However, the effects of CO(2) may be mediated through pH changes of the perfusate. Finally, we demonstrate that chronic exposure of the animals to hypoxia (90% N(2)), prior to intracellular recordings, significantly enhanced the rate of spontaneously occurring respiratory discharges in semi-intact preparations, even if they were maintained in normoxic saline for several hours. Moreover, we demonstrate that the peripherally originated hypoxia signal is likely conveyed to the CPG neurons via RPeD1. In summary, the data presented in this study demonstrate the important role played by the periphery and the RPeD1 neuron in regulating respiration in response to hypoxia in Lymnaea.

Context learning and the effect of context on memory retrieval in Lymnaea.

Aerial respiratory behavior in Lymnaea was operantly conditioned so that the animals perform aerial respiration significantly less often. Using the standard training procedure (pond water made hypoxic by bubbling N2 through it) both food-deprived and fed animals learned and exhibited long-term memory (LTM). However, food-deprived animals exhibited neither learning nor memory when trained under a condition in which the hypoxic pond water also contained a food odorant (carrot, the food-odorant procedure). Fed animals, however, learned and exhibited LTM with the food-odorant procedure. Thus, the presence of the food odorant per se did not prevent learning or the establishment of LTM. Further experimentation, however, revealed that the ability of the snails to have recall (i.e., memory) for the learned behavior was dependent on the context in which memory was tested. That is, if animals were trained with the food-odorant procedure they could only exhibit recall if tested in the food-odorant context and vice versa with the standard training procedure. Thus, although fed animals could learn and show LTM with either training and testing procedure, LTM could only be seen when they were tested in the context in which they were trained.

Transmitter-receptor interactions between growth cones of identified Lymnaea neurons determine target cell selection in vitro.

In addition to their involvement in transsynaptic communication in the adult nervous system, neurotransmitters also participate in many developmental events, such as neurite initiation and outgrowth. Although growth cones can release transmitters and are themselves sensitive to exogenously applied neurotransmitters, a direct causal relationship between the release of transmitter from one growth cone and its effect on another has not yet been demonstrated. In this study, we provide evidence that dopamine release from the growth cones of an identified Lymnaea neuron, right pedal dorsal 1 (RPeD1), differentially regulates the growth cone behavior of its in vivo target and nontarget neurons in vitro. In coculture, RPeD1 growth cones enhanced the rate of growth cone advance from target cells and synaptic connections developed immediately after contact. In contrast, RPeD1 growth cones not only inhibited the rate of growth cone advance from nontarget cells but they also induced growth cone collapse. Using a "sniffer cell" approach, we demonstrated that both RPeD1 growth cones and somata released dopamine, which can be detected at a distance of several hundred micrometers. RPeD1 somata were used to demonstrate that spontaneous release of dopamine also acted as a chemoattractant for target growth cones but as a chemorepellent for nontarget growth cones. These effects were mimicked by exogenous dopamine application, and both RPeD1 growth cone and soma-induced effects were also blocked in the presence of dopamine receptor antagonists. This study emphasizes the importance of transmitter-receptor interactions between growth cones in target cell selection.

The respiratory central pattern generator of Lymnaea: a model, measured and malleable.

Great progress has been made, and continues to be made in our understanding of the neuronal mechanisms underlying respiration in a wide variety of model systems. The central pattern generator (CPG) controlling aerial respiration in the pond-snail Lymnaea is a particularly well-studied model. Using in vitro and semi-intact preparations, the neural circuitry that controls aerial respiration has been characterized as consisting of three identified interneurons. Furthermore, insight has been gained into the behavioural, cellular and synaptic mechanisms by which this circuit controls respiratory rhythmogenesis. It has also been demonstrated that aerial respiratory behaviour can be modified both by experience and by environmental factors. Studies have shown that, in a behavioural hierarchy, respiration is subservient to the whole-body withdrawal response that respiratory behaviour can be modified through operant conditioning, and that respiratory behaviour is altered by hypoxia. Through research on the Lymnaea respiratory CPG we are coming to a better understanding of the construction and malleability of a CPG network. The malleability of this CPG is of particular interest. No longer can neuronal networks underlying respiratory behaviour be considered hard-wired; they have inherent plasticity.

Different extrinsic trophic factors regulate neurite outgrowth and synapse formation between identified Lymnaea neurons.

The requirement for trophic factors in neurite outgrowth is well established, though their role in synapse formation is yet to be determined. Moreover, the issue of whether the trophic factors mediating neurite outgrowth are also responsible for synapse specification has not yet been resolved. To test whether trophic factors mediating neurite outgrowth and synapse formation between identified neurons are conserved in two molluscan species and whether these developmental processes are differentially regulated by different trophic factors, we used soma-soma and neurite-neurite synapses between identified Lymnaea neurons. We demonstrate here that the trophic factors present in Aplysia hemolymph, although sufficient to induce neurite outgrowth from Lymnaea neurons, do not promote specific synapse formation between excitatory partners. Specifically, the identified presynaptic neuron visceral dorsal 4 (VD4) and postsynaptic neuron left pedal dorsal 1 (LPeD1) were either paired in a soma-soma configuration or plated individually to allow neuritic contacts. Cells were cultured in either Lymnaea brain-conditioned medium (CM) or on poly-L-lysine dishes that were pretreated with Aplysia hemolymph (ApHM), but contained only Lymnaea defined medium (DM; does not promote neurite outgrowth). In ApHM-coated dishes containing DM, Lymnaea neurons exhibited extensive neurite outgrowth, but appropriate excitatory synapses failed to develop between the cells. Instead, inappropriate reciprocal inhibitory synapses formed between VD4 and LPeD1. Similar inappropriate inhibitory synapses were observed in Aplysia hemolymph-pretreated dishes that contained dialyzed Aplysia hemolymph. These inhibitory synapses were novel and inappropriate, because they do not exist in vivo. A receptor tyrosine kinase inhibitor (Lavendustin A) blocked neurite outgrowth induced by both Lymnaea CM and ApHM. However, it did not affect inappropriate inhibitory synapse formation between the neurons. These data demonstrate that neurite outgrowth but not inappropriate inhibitory synapse formation involves receptor tyrosine kinases. Together, our data provide direct evidence that trophic factors required for neurite outgrowth are conserved among two different molluscan species, and that neurite extension and synapse specification between excitatory partners are likely mediated by different trophic factors.

Synthesis and functional integration of a neurotransmitter receptor in isolated invertebrate axons.

Neurotransmitter receptors are considered an important class of membrane proteins that are involved in plasticity-induced changes underlying learning and memory. Recent studies, which demonstrated that the mRNAs encoding for various receptor proteins are localized to specific dendritic domains, allude toward the possibility that these membrane bound molecules may be synthesized locally. However, direct evidence for the local axonal or dendritic synthesis and functional integration of receptor proteins in either vertebrates or invertebrates is still lacking. In this study, using an invertebrate model system we provide the first direct evidence that isolated axons (in the absence of the soma) can intrinsically synthesize and functionally integrate a membrane-bound receptor protein from an axonally injected mRNA. Surgically isolated axons from identified neurons were injected with mRNA encoding a G-protein-coupled conopressin receptor. Immunocytochemical and electrophysiological techniques were used to demonstrate functional integration of the receptor protein into the membrane of the isolated axon. Ultrastructural analysis of axonal compartments revealed polyribosomes, suggesting that some components of the protein synthesizing machinery are indeed present in these extrasomal compartments. Such axonal propensity to locally synthesize and functionally insert transmitter receptors may be instrumental in plasticity induced changes, for instance those that underlie learning and memory.

Operant conditioning in Lymnaea: evidence for intermediate- and long-term memory.

Aerial respiration of the pond snail, Lymnaea stagnalis, can be operantly conditioned; however, the parameters necessary to produce long-term (LTM) or intermediate term memory (ITM) have not previously been investigated. We conducted training using procedures that varied in the duration of the training session, the number of training sessions per day or the amount of time between subsequent training sessions (SI). We found that by varying the duration and frequency of the training session learning could be differentially produced. Furthermore, the ability to form LTM was dependent not only on the duration of the training session was also the interval between training sessions, the SI. Thus it was possible to produce ITM, which persists for up to 3 hr, and not form LTM, which persists at least 18 hr. Learning, ITM, and LTM can be differentially produced by altering the SI, the duration of the training session, or the number of training sessions per day. These findings may allow us to begin to elucidate the underlying neural mechanisms of learning, ITM, and LTM.

Target cell contact suppresses neurite outgrowth from soma-soma paired Lymnaea neurons.

Neurite extension from developing and/or regenerating neurons is terminated on contact with their specific synaptic partner cells. However, a direct relationship between the effects of target cell contact on neurite outgrowth suppression and synapse formation has not yet been demonstrated. To determine whether physical/synaptic contacts affect neurite extension from cultured cells, we utilized soma-soma synapses between the identified Lymnaea neurons. A presynaptic cell (right pedal dorsal 1, RPeD1) was paired either with its postsynaptic partner cells (visceral dorsal 4, VD4, and Visceral dorsal 2, VD2) or with a non-target cell (visceral dorsal 1, VD1), and the interactions between their neurite outgrowth patterns and synapse formation were examined. Specifically, when cultured in brain conditioned medium (CM, contains growth-promoting factors), RPeD1, VD4, and VD2 exhibited robust neurite outgrowth within 12-24 h of their isolation. Synapses, similar to those seen in vivo, developed between the neurites of these cells. RPeD1 did not, however, synapse with its non-target cell VD1, despite extensive neuritic overlap between the cells. When placed in a soma-soma configuration (somata juxtaposed against each other), appropriate synapses developed between the somata of RPeD1 and VD4 (inhibitory) and between RPeD1 and VD2 (excitatory). Interestingly, pairing RPeD1 with either of its synaptic partner (VD4 or VD2) resulted in a complete suppression of neurite outgrowth from both pre- and postsynaptic neurons, even though the cells were cultured in CM. A single cell in the same dish, however, extended elaborate neurites. Similarly, a postsynaptic cell (VD4) contact suppressed the rate of neurite extension from a previously sprouted RPeD1. This suppression of the presynaptic growth cone motility was also target cell contact specific. The neurite suppression from soma-soma paired cells was transient, and neuronal sprouting began after a delay of 48-72 h. In contrast, when paired with VD1, both RPeD1 and this non-target cell exhibited robust neurite outgrowth. We demonstrate that this neurite suppression from soma-soma paired cells was target cell contact/synapse specific and Ca(2+) dependent. Specifically, soma-soma pairing in CM containing either lower external Ca(2+) concentration (50% of its control level) or Cd(2+) resulted in robust neurite outgrowth from both cells; however, the incidence of synapse formation between the paired cells was significantly reduced. Taken together, our data show that contact (physical and/or synaptic) between synaptic partners strongly influence neurite outgrowth patterns of both pre- and postsynaptic neurons in a time-dependent and cell-specific manner. Moreover, our data also suggest that neurite outgrowth and synapse formation are differentially regulated by external Ca(2+) concentration.

Sevoflurane induced suppression of inhibitory synaptic transmission between soma-soma paired Lymnaea neurons.

The cellular and synaptic mechanisms by which general anesthetics affect cell-cell communications in the nervous system remain poorly defined. In this study, we sought to determine how clinically relevant concentrations of sevoflurane affected inhibitory synaptic transmission between identified Lymnaea neurons in vitro. Inhibitory synapses were reconstructed in cell culture, between the somata of two functionally well-characterized neurons, right pedal dorsal 1 (RPeD1, the giant dopaminergic neuron) and visceral dorsal 4 (VD4). Clinically relevant concentrations of sevoflurane (1-4%) were tested for their effects on synaptic transmission and the intrinsic membrane properties of soma-soma paired cells. RPeD1- induced inhibitory postsynaptic potentials (IPSPs) in VD4 were completely and reversibly blocked by sevoflurane (4%). Sevoflurane also suppressed action potentials in both RPeD1 and VD4 cells. To determine whether the anesthetic-induced synaptic depression involved postsynaptic transmitter receptors, dopamine was pressure applied to VD4, either in the presence or absence of sevoflurane. Dopamine (10(-]5) M) activated a voltage-insensitive K(+) current in VD4. The same K(+) current was also altered by sevoflurane; however, the effects of two compounds were nonadditive. Because transmitter release from RPeD1 requires Ca(2+) influx through voltage-gated Ca(2+) channels, we next tested whether the anesthetic-induced synaptic depression involved these channels. Individually isolated RPeD1 somata were whole cell voltage clamped, and Ca(2+) currents were analyzed in control and various anesthetic conditions. Clinically relevant concentrations of sevoflurane did not significantly affect voltage-activated Ca(2+) channels in RPeD1. Taken together, this study provides the first direct evidence that sevoflurane-induced synaptic depression involves both pre- and postsynaptic ion channels.

Excitatory synaptogenesis between identified Lymnaea neurons requires extrinsic trophic factors and is mediated by receptor tyrosine kinases.

Neurotrophic factors have well established roles in neuronal development and adult synaptic plasticity, but their precise role in synapse formation has yet to be determined. This paper provides the first direct evidence that neurotrophic factors in brain conditioned medium (CM) differentially regulate excitatory and inhibitory synapse formation. Somata of identified presynaptic and postsynaptic neurons were isolated from the CNS of Lymnaea and were cultured in a soma-soma configuration in the presence (CM) or absence [defined medium (DM)] of trophic factors. In DM, excitatory synapses did not form. When they were paired in CM or in DM containing Lymnaea epidermal growth factor (EGF); however, all presynaptic neurons reestablished their specific excitatory synapses, which had electrical properties similar to those seen in vivo. CM-induced formation of excitatory synapses required transcription and de novo protein synthesis, as indicated by the observations that synapse formation was blocked by the protein synthesis inhibitor anisomycin and the protein transcription blocker actinomycin D; the CM factor was inactivated by boiling. They were also blocked by receptor tyrosine kinase inhibitors (lavendustin A, genistein, K252a, and KT5926) but not by inactive analogs (genistin and lavendustin B), suggesting that the effect was mediated by receptor tyrosine kinases. These results, together with our previously published data, demonstrate that trophic factors are required for excitatory, but not inhibitory, synapse formation and extends the role of EGF from cell proliferation, neurite outgrowth, and survival to excitatory synapse formation.

Trophic factor-induced plasticity of synaptic connections between identified Lymnaea neurons.

Neurotrophic factors participate in both developmental and adult synaptic plasticity; however, the underlying mechanisms remain unknown. Using soma-soma synapses between the identified Lymnaea neurons, we demonstrate that the brain conditioned medium (CM)-derived trophic factors are required for the formation of excitatory but not the inhibitory synapse. Specifically, identified presynaptic [right pedal dorsal 1 (RPeD1) and visceral dorsal 4 (VD4)] and postsynaptic [visceral dorsal 2/3 (VD2/3) and left pedal dorsal 1 (LPeD1)] neurons were soma-soma paired either in the absence or presence of CM. We show that in defined medium (DM-does not contain extrinsic trophic factors), appropriate excitatory synapses failed to develop between RPeD1 and VD2/3. Instead, inappropriate inhibitory synapses formed between VD2/3 and RPeD1. Similarly, mutual inhibitory synapses developed between VD4 and LPeD1 in DM. These inhibitory synapses were termed novel because they do not exist in the intact brain. To test whether DM-induced, inappropriate inhibitory synapses could be corrected by the addition of CM, cells were first paired in DM for an initial period of 12 hr. DM was then replaced with CM, and simultaneous intracellular recordings were made from paired cells after 6-12 hr of CM substitution. Not only did CM induce the formation of appropriate excitatory synapses between both cell pairs, but it also reduced the incidence of inappropriate inhibitory synapse formation. The CM-induced plasticity of synaptic connections involved new protein synthesis and transcription and was mediated via receptor tyrosine kinases. Taken together, our data provide the first direct insight into the cellular mechanism underlying trophic factor-induced specificity and plasticity of synaptic connections between soma-soma paired Lymnaea neurons.