Iori Ito

PHYSIOLOGICAL CHARACTERIZATION OF LIP AND TENTACLE NERVES IN LYMNAEA STAGNALIS

The lip and tentacle nerves of the pond snail, Lymnaea stagnalis, were characterized using electrophysiological techniques. When the activity of those nerves was induced in lip-tentacle preparations, aversive taste signals were transmitted through all the lip and tentacle nerves, but appetitive signals could be recorded only through the superior lip nerve. In the CNS immersed in high Mg2+ -high Ca2+ saline, electrical stimuli applied to any of the nerves failed to induce action potentials in one of the regulatory neurons (cerebral giant cell: CGC) involved in feeding responses, implying that the signals are polysynaptically transmitted to the CGC. Intracellular recordings revealed that the CGCs in semi-intact half-body preparations received both appetitive and aversive taste signals not only through the superior lip nerve but also through the median lip nerve. In addition, an osphradium was ruled out as a candidate for appetitive reception. The present results, together with our preceding data arrived at by the histochemical analyses, indicate that the appetitive taste transduction responsible for generating feeding responses is performed through the superior lip nerve with some contribution of the median lip nerve. The data showing that the CGC can receive various taste signals suggests that it may play a crucial role in feeding behavior as demonstrated in the study of conditioned taste-aversion.

The Nitric Oxide/Cyclic GMP Pathway in the Olfactory Processing System of the Terrestrial Slug Limax marginatus

Abstract To examine the distribution of nitric oxide (NO)-generative cells and NO-responsive cells in the tentacles and procerebral lobes (olfactory processing center) of terrestrial slugs, we applied NADPH diaphorase (NADPH-d) histochemistry and NO-induced cyclic GMP (cGMP)-like immunohistochemistry. We found that NADPH-d reactive cells/fibers and cGMP-like immunoreactive cells/fibers were different, but they were localized adjacent to each other, in both the tentacles and the procerebral lobes. Then, we measured the concentration of NO that was generated around the procerebral lobes using an NO sensitive electrode, when the olfactory nerve was electrically stimulated as a replacement for an odorant stimulus. Stimulation of the olfactory nerve evoked an increase in NO concentration at nanomolar levels, suggesting that binding of nanomolar concentrations of NO to the prosthetic heme group activates soluble guanylyl cyclase. Taken together with previously reported physiological data, our results, therefore, showed that the NO/cGMP pathways are involved in slug olfactory processing.

Effects of electrical stimulation of the tentacular digits of a slug upon the frequency of electrical oscillations in the procerebral lobe

To find the primary mechanism for the frequency changes of electrical oscillations in the procerebral (PC) lobe of a slug, we electrically stimulated the tip, middle and basal regions of the digits of the superior and inferior tentacles and recorded the local field potentials from the PC lobe. Stimuli to the middle and basal regions of the digits of the inferior tentacle significantly decreased the frequency of electrical oscillations in the PC lobe, whereas those to the tip regions of the digits of the inferior tentacle and all regions of the digits of the superior tentacle increased it. These findings suggest that the change in the frequency of electrical oscillations in the PC lobe depends on the excited region in the digits, providing the first presentation of the physiological difference in the olfactory function between the superior and inferior tentacles.

Distributions of γ-Aminobutyric Acid Immunoreactive and Acetylcholinesterase-Containing Cells in the Primary Olfactory System in the Terrestrial Slug Limax marginatus

Abstract The tentacular ganglion, the primary olfactory system of terrestrial slugs, exhibits spontaneous oscillations with a spatial coherence. The digit-like extensions (digits) of the tentacular ganglion presumably house the cell bodies of the neurons underlying the oscillations. The present study was designed to identify the anatomical and physiological determinants of these oscillations with a special focus on whether the neurons located in the digits contribute to the coherent oscillations. We recorded field potentials from the spatially separated sites in the digits in the terrestrial slug Limax marginatus. We also simultaneously recorded tentacular nerve to monitor the coherent oscillations. The spatially separated regions in the digits oscillated at the same frequency as the tentacular nerve, indicating a single coherent activity. To study the neural networks underlying the coherent oscillations, we examined the distributions of acetylcholinesterase (AChE)-containing and γ-aminobutyric acid immunoreactive (GABA-ir) neurons. AChE-containing and GABA-ir fibers were found to connect the neurons in a branch of the digits with those in other branches. We also used a vital staining technique with 1,1′-didodecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate to examine the projections of neurons in the digits. Large stained cells were detected in many branches of the digits after placing the dye on one of the cell masses located in right and left sides of the tentacular ganglion. They were detected in the cell masses and in many branches of the digits after placing the dye on a branch of the digits. Our results showed that the slug primary olfactory system has highly interconnected neural networks.

Odor responses and spontaneous oscillatory activity in tentacular nerves of the terrestrial slug, Limax marginatus

We studied the neural oscillatory activity in the peripheral olfactory system of the tentacles in the terrestrial slug, Limax marginatus, by extracellular recording. Recordings from the cut-ends of the inferior tentacular nerves connected to the inferior tentacular ganglia and sensory epithelia showed spontaneous oscillatory activity at frequencies of 0.1-30 Hz. This spontaneous activity was dominated by the 0.6-6 Hz band. Ethanol odor stimulation decreased the amplitude in the 0.6-6 Hz band and increased those in the 6-15 and 15-30 Hz bands. Antagonists of the gamma-aminobutyric acid (GABA) receptor, bicuculline and picrotoxin, resulted in suppression of spontaneous activity and modulated the odor response in the 0.6-6 Hz band. Our results indicate the involvement of GABA-mediated oscillatory activity in the tentacular nerves in the olfactory processing in molluscs.

Negative relationship between odor-induced spike activity and spontaneous oscillations in the primary olfactory system of the terrestrial slug Limax marginatus.

Abstract Although primary olfactory systems in various animals display spontaneous oscillatory activity, its functional significance in olfactory processing has not been elucidated. The tentacular ganglion, the primary olfactory system of the terrestrial slug Limax marginatus, also displays spontaneous oscillatory activity at 1–2 Hz. In the present study, we examined the relationship between odor-evoked spike activity and spontaneous field potential oscillations in the tentacular nerve, representing the pathway from the primary olfactory system to the olfactory center. Neural activity was recorded from the tentacular nerve before, during and after application of various odors (garlic, carrot, and rat chow) to the sensory epithelium and the changes in firing rate and spontaneous oscillations were analyzed. We detected the baseline amplitude of the oscillations and baseline spike activity before stimulation. Odor stimulations for 20 s or 60 s evoked a transient increase in the firing rate followed by a decrease in the amplitude of spontaneous oscillations. The decrease in the amplitude was larger in the first 8 s of stimulation and subsequently showed recovery during stimulation. The amplitude of the recovered oscillations often fluctuated. Odor-evoked spikes appeared when the amplitude of the recovered oscillations was transiently small. These results suggest that the large oscillations could inhibit spike activity whereas the first transient increase in spike activity was followed by the decrease in the oscillation amplitude. Our results indicate that there is a significant negative correlation between spontaneous oscillations and odor-evoked spike activity, suggesting that the spontaneous oscillations contribute to the olfactory processing in slugs.

Complement receptor 3-like immunoreactivity in the light green cells and the canopy cells of the pond snail, Lymnaea stagnalis

We observed CR3-like immunoreactivity in the central nervous system (CNS) and its surrounding peripheral nerves of the pond snail, Lymnaea stagnalis. In the CNS of L. stagnalis, the immunoreactivity presenting meshwork-like structure was detected in some neurosecretory cells, which are the light green cells (LGCs) and the canopy cells (CCs), both controlling the body growth. The immunoreactivity was also observed along the edges of median lip nerves. The immunoreactive regions in the median lip nerves appeared to form the axonal plates, from which the LGCs and the CCs release molluscan insulin-related peptides (MIPs) into the blood. By contrast, no immunoreactivity was detected in other neurosecretory cells or their release sites, for example the caudodorsal cells and the cerebral commissure, which release ovulation hormones. The present findings, therefore, suggested that CR3 expresses only in the neurosecretory cells releasing MIPs in L. stagnalis.

Complement Receptor 3-Like Immunoreactivity in the Superior and Inferior Tentacles of Terrestrial Slug, Limax marginatus

Abstract Complement receptor 3 (CR3), one of cell adhesion molecules, plays a crucial role in secretion of mammalian neutrophils. To study whether CR3 is also involved in neurosecretion of gastropod molluscs, we examined the CR3-like immunoreactivity in the central nervous system (CNS) and the superior and inferior tentacles of the terrestrial slug, Limax marginatus. In the CNS of L. marginatus, we did not detect the specific immunoreactivity at all. In contrast, the CR3-like immunoreactivity was observed in the cell bodies and processes of collar cells of the superior and inferior tentacles. In particular, granules contained in the cell bodies of collar cells exhibited the CR3-like immunoreactivity. Retrograde labeling of horseradish peroxidase applied on the sensory epithelia (SE) of superior and inferior tentacles showed that the collar cells of these tentacles project their processes to the SE. In the previous study, homogenate of the superior tentacles injected into the body cavity of slugs stimulated spermatogenesis and simultaneously inhibited egg-laying behavior, suggesting that the tentacular hormone acts as gonadotropic hormone. Taken together, CR3 expressed in the collar cells is considered to be involved in the secretion of tentacular hormone in L. marginatus.

Mapping of odor-related neuronal activity using a fluorescent derivative of glucose

Activity labeling was applied to the olfactory systems of the terrestrial slug Limax valentianus using 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG), a fluorescent derivative of glucose. 2-NBDG was incorporated into cultured Limax olfactory interneurons, and this was partially blocked by the presence of a high concentration of glucose in the medium, indicating that a part of the uptake of 2-NBDG is mediated by glucose transporters. Next, in order to map odor-related neuronal activity in the primary olfactory center, tentacular ganglion, we injected 2-NBDG into the body cavities of slugs and exposed them to odors or clean air (control). In the odor-stimulated animals, the cell mass region was strongly stained. The digit-like extensions and the neuropil region were also stained in some animals. The control animals showed no staining. The neurons in the cell mass are thought to be involved in generating oscillating activities in the tentacular ganglion, and their activation may imply modulation of oscillatory activity during odor processing. Our results show that 2-NBDG is useful for mapping neuronal activity in vivo.