Morphological and physiological characteristics of dermal photoreceptors in Lymnaea stagnalis.

Dermal photoreceptors located in the mantle of Lymnaea stagnalis were histologically and physiologically characterized. Our previous study demonstrated that the shadow response from dermal photoreceptors induces the whole-body withdrawal response. Through the interneuron, RPeD11, we detected that the light-off response indirectly originated from a dermal photoreceptor. Previous observations, based on behavioral pharmacology, revealed that cyclic guanosine monophosphate acts as a second messenger in the dermal photoreceptor. Furthermore, gastropods possess dermal photoreceptors containing rhodopsin, as a photopigment, and another photo-sensitive protein, arrestin, responsible for terminating the light response. Thus, we chose three antibodies, anti-cGMP, anti-rhodopsin, and anti-ß-arrestin, to identify the dermal photoreceptor molecules in Lymnaea mantle. Extracellular recording, using a suction electrode on the mantle, revealed a light off-response from the right parietal nerve. Overlapping structures, positive against each of the antibodies, were also observed. Numerous round, granular particles of 3-47 µm in diameter with one nucleus were distributed around pneumostome and/or inside the mantle. The cells surrounding the pneumostome area, located 10 µm beneath the surface, tended to have smaller cell soma ranging from 3 to 25 µm in diameter, while cells located in other areas were distributed uniformly inside the mantle, with a larger diameter ranging from 12 to 47 µm. The histological examination using back-filing Lucifer Yellow staining of the right parietal nerve with the three dermal photoreceptor antibodies confirmed that these overlapping-stained structures were dermal photoreceptors in Lymnaea.

The shadow-induced withdrawal response, dermal photoreceptors, and their input to the higher-order interneuron RPeD11 in the pond snail Lymnaea stagnalis.

The shadow-induced withdrawal response in Lymnaea stagnalis is mediated by dermal photoreceptors located on the foot, mantle cavity, and skin around the pneumostome area. Here, we determined whether we could obtain a neural correlate of the withdrawal response elicited by a shadow in a higher-order central neuron that mediates withdrawal behavior. We measured the electrophysiological properties of the higher-order interneuron Right Pedal Dorsal 11 (RPeD11), which has a major role in Lymnaea withdrawal behavior. In semi-intact preparations comprising the circumesophageal ganglia, the mantle cavity and the pneumostome, but not the foot and eyes, a light-on stimulus elicited a small short-lasting hyperpolarization and a light-off stimulus elicited a depolarization of RPeD11. We also determined that dermal photoreceptors make a monosynaptic contact with RPeD11. The dermal photoreceptor afferents course to the circumesophageal ganglia via the anal and genital nerves to the visceral ganglion, and/or via the right internal and external parietal nerves to the parietal ganglion. Finally, in addition to responding to photic stimuli, RPeD11 responds to both mechanical and chemical stimuli delivered to the pneumostome.

Increase in excitability of RPeD11 results in memory enhancement of juvenile and adult Lymnaea stagnalis by predator-induced stress.

Memory consolidation following learning is a dynamic process. Thus, long-term memory (LTM) formation can be modulated by many factors, including stress. We examined how predator-induced stress enhances LTM formation in the pond snail Lymnaea stagnalis at both the behavioral and electrophysiological levels. Training snails in crayfish effluent (CE; i.e., water from an aquarium containing crayfish) significantly enhanced LTM. That is, while memory persists for only 3h in adult control experiments following a single 0.5-h training session in pond water in which the pneumostome receives a contingent tactile stimulus to the pneumostome; when the snails are trained in CE, the memory persists for at least 24h. In juveniles, the data are more dramatic. Juveniles are unable to form LTM in pond water, but form LTM when trained in CE. Here we examined whether juvenile snails form LTM following a one-trial training procedure (1TT). Following the 1TT procedure (a single-trial aversive operant conditioning training procedure), juveniles do not form LTM, unless trained in CE. Concomitantly, we observe changes in the excitability of RPeD11, a key neuron mediating the whole snail withdrawal response, which may be a neural correlate of enhanced memory formation.

Cyclic nucleotide-gated channels are involved in phototransduction of dermal photoreceptors in Lymnaea stagnalis.

Dermal photoreceptors in the pond snail Lymnaea stagnalis mediate the whole-body withdrawal response, including pneumostome closure, elicited by a shadow passing over the pneumostome area. The pneumostome closure response is part of the defense reaction in Lymnaea. The shadow or 'light-off' stimulus elicits activity in a higher order interneuron, RPeD11, which has a major role in mediating defensive withdrawal behavior elicited by noxious or threatening stimuli. Here, we tested our hypothesis that cyclic nucleotide-gated (CNG) channels are involved in the dermal photoreceptor-mediated transduction of the shadow stimulus. The response to the shadow stimulus recorded in RPeD11 was abolished by 500 µM cis-diltiazem, which blocks cGMP-activated conductance of CNG channels. On the other hand, the shadow response elicited in RPeD11 was not blocked by 2-amino ethyldiphenyl borate (2-APB), a transient receptor potential (TRP) channel blocker. Consistent with the electrophysiologic data, cis-diltiazem blocked the shadow-evoked withdrawal response, whereas 2-APB did not block the withdrawal response evoked by the shadow stimulus in intact freely behaving Lymnaea. Together, these findings support the hypothesis that the second messenger in dermal photoreceptors involves CNG and not TRP channels.

Potassium currents in isolated statocyst neurons and RPeD1 in the pond snail, Lymnaea stagnalis.

To begin to determine the underlying neural mechanisms of memory formation, we studied two different cell types that play important roles in different forms of associative learning in Lymnaea. Statocyst neurons (hair cells) mediate classical conditioning, whereas RPeD1 is a site of memory formation induced by operant conditioning of aerial respiration. Because potassium (K(+)) channels play a critical role in neuronal excitability, we initiated studies on these channels in the aforementioned neurons. Three distinct K(+) currents are expressed in the soma of both the hair cells and RPeD1. In hair cells and RPeD1, there is a fast activating and rapidly inactivating 4-aminopyridine (4-AP)-sensitive A current (I(A)), a tetraethyl ammonium (TEA)-sensitive delayed rectifying current, which exhibits slow inactivation kinetics (I(KV)), and a TEA- and 4-AP-insensitive Ca(2+)-dependent current (I(Ca-K)). In hair cells, the activation voltage of I(A); its half-maximal steady-state activation voltage and its half-maximal steady-state inactivation were at more depolarized levels than in RPeD1. The time constant of recovery from I(A) inactivation was slightly faster in hair cells. I(A) in hair cells is also smaller in amplitude than in RPeD1 and is activated at more depolarized potentials. In like manner, I(KV) is smaller in hair cells and is activated at more depolarized potentials than in RPeD1.

Electrophysiological responses to light of neurons in the eye and statocyst of Lymnaea stagnalis.

Lymnaea can be classically conditioned by pairing photic stimulation with a rotational stimulus. The electrophysiological properties of the Lymnaea photoreceptors and statocyst neurons are incompletely known. There are 2 types of ocular photoreceptors and 3 types of statocyst "hair cells." Type A photoreceptors had a response latency from 200 to 400 ms, with a graded depolarizing response having maximum action spectra at 480-500 nm, corresponding to the beta(max) of rhodopsin. Additionally they extend their axons in the direction of the other type of photoreceptor neuron, the type T cell. These neurons have a 2-component response to light: a response reversibly reduced in Ca(2+)-free saline, and a component persisting in Ca(2+)-free saline. Type T cells send processes into the cerebral ganglion and terminate close to the ending of the statocyst hair cells. Hair cells send their terminal branches to the cerebral ganglia close to the terminations of the type T cells. Caudal hair cells respond to a light flash with a depolarization, whereas the rostral cells respond with a hyperpolarization. The response latency in all hair cells was dependent on the stimulus intensity; the brightest light tested had a latency of 200 ms. The photo-induced response was abolished in Ca(2+)-free saline, whereas it was still present in high Ca(2+)-high Mg(2+) saline, consistent with the hypothesis that the connection between the photoreceptors and hair cells is monosynaptic. Thus the sensory information necessary for forming an association between photic and rotational stimuli converges on the statocyst neurons.

Conditioned taste aversion with sucrose and tactile stimuli in the pond snail Lymnaea stagnalis.

A new form of taste aversion conditioning was established in the pond snail Lymnaea stagnalis. An associative memory, lasting 24h, was produced in the pond snail with 20 pairings of 100 mM sucrose as the conditioned stimulus (CS) and mechanical stimulation to the head as the unconditioned stimulus (UCS). Animals exposed to reverse pairings of the CS and UCS failed to learn the association. The learning was characterized by a shift in the response to the UCS from a whole-body withdrawal response to the cessation of feeding behavior.

Involvement of the ryanodine receptor in morphologic modification of Hermissenda type B photoreceptors after in vitro conditioning.

We examined whether Ca(2+) induced Ca(2+) release through ryanodine receptors is involved in the conditioning of specific morphologic changes at the axon terminals of type B photoreceptors in the isolated circumesophageal ganglion of Hermissenda. Calcium chelation by bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid prevented the conformational change at the terminals after five paired presentations of light and vibration, which produce terminal branch contraction of B photoreceptors. Two ryanodine receptor blockers, dantrolene and micromolar concentrations of ryanodine, depressed the increase in excitability due to in vitro conditioning and the increase in intracellular Ca(2+) in response to membrane depolarization. Although the ability to increase intracellular Ca(2+) was depressed, synaptic transmission was preserved in the normal state from hair cells under dantrolene and ryanodine incubation. Ryanodine receptor blockers also prevented contraction at the B photoreceptor axon terminals. These results suggest that the ryanodine receptor has a crucial role in inducing the in vitro conditioning specific changes both physiologically and morphologically, including "focusing" at the B photoreceptor axon terminal.

Photoresponse from the statocyst hair cell in Lymnaea stagnalis.

Sensory cells for associative learning of light and turbulence were studied in Lymnaea. Intracellular recordings with Lucifer Yellow filled electrodes were made from photoreceptors and statocyst hair cells. Photoreceptors had a long latency, graded depolarizing response to a flash of light; they extended their axon to the cerebral ganglion. The caudal hair cell, one of 12 cells in the statocyst, responded to brief light with a depolarization and superimposed impulse activity. It formed its terminal arborization close to the photoreceptor endings in the cerebral ganglion. Ca(2+)-free saline reversibly abolished the photoresponse in the hair cell, suggesting the information was conveyed via a chemical synapse. These findings demonstrated that sensory information for associative learning was convergent at the statocyst hair cell.

In vitro conditioning induces morphological changes in Hermissenda type B photoreceptor.

Short- and long-term synaptic plasticity are considered to be cellular substrates of learning and memory. The mechanisms underlying synaptic plasticity especially with respect to morphology, however, are not known. In vitro conditioning in molluscan preparations is a well established form of short-term synaptic plasticity. Five paired presentations of light and vestibular stimulation to the isolated nervous system of Hermissenda results in an increase in excitability of the identified neuron, the type B photoreceptor, indicated by 2 measures, an increase in the input resistance and a cumulative depolarization after the cessation of light stimulus recorded from the cell soma. The terminal branches of type B photoreceptors iontophoretically injected with fluorescent dye were analyzed using computer-aided 3-dimensional reconstruction of images obtained using a confocal microscope under 'blind' conditions. The terminal branches contracted along the centro-lateral axis within an hour after conditioning, paralleling the increase in neuronal excitability. These data suggest that in vitro conditioning in Hermissenda is a form of short-term synaptic plasticity that involves changes in macromolecular synthesis.

Associative learning acquisition and retention depends on developmental stage in Lymnaea stagnalis.

Associative learning dependent on visual and vestibular sensory neurons and the underlying cellular mechanisms have been well characterized in Hermissenda but not yet in Lymnaea. Three days of conditioning with paired presentations of a light flash (conditional stimulus: CS) and orbital rotation (unconditional stimulus: UCS) in intact Lymnaea stagnalis results in a whole-body withdrawal response (WBWR) to the CS. In the current study, we examined the optimal stimulus conditions for associative learning, including developmental stage, number of stimuli, interstimulus interval, and intertrial interval. Animals with a shell length longer than 18 mm (sexually mature) acquired and retained the associative memory, while younger ones having a shell length shorter than 15 mm acquired but did not retain the memory to the following day. For mature animals, 10 paired presentations of the CS and UCS presented every 2 min were sufficient for the induction of a WBWR to the CS. Furthermore, animals conditioned with the UCS presented simultaneously with the last 2 s of the CS also exhibited a significant WBWR in response to the CS. Blind animals did not acquire the associative memory, suggesting that ocular photoreceptors, and not dermal photoreceptors, detected the CS. These results show that maturity was key to retention of associative learning.