Visual afferents from an eye in the terrestrial slug Limax valentianus.

Terrestrial gastropods have a lens-bearing eye on the tip of their tentacles. There are two morphologically distinct photoreceptors, called Type-I and Type-II photoreceptors, in the retina. Type-I photoreceptors are equipped with highly developed photoreceptive microvilli in their outer rhabdomeric segment, whereas Type-II photoreceptors have short and fewer microvilli. Although both types of photoreceptors send afferent projections directly to the brain, their destinations in the brain, called optic neuropiles, have not been sufficiently investigated. Our recent studies revealed that there are commissural fibers in the cerebral ganglia that transmit photic information acquired by bilateral eyes. Moreover, some of the retinal photoreceptors are connected by gap junctions to the photosensitive brain neurons, suggesting the functional interaction of the photic information between the eye and brain photoreceptors, as well as between bilateral eyes. However, it has not been clarified which type of retinal photoreceptors send commissural projections to the contralateral hemiganglion nor interact with the brain photoreceptors. In the present study, we demonstrated by molecular histological analyses and tracer injections that (1) Type-I and Type-II photoreceptors send glutamatergic afferent projections to the medial and lateral lobes of the ipsilateral optic neuropile, respectively, (2) direct synaptic interaction between bilateral optic nerves occurs in the medial lobe of the optic neuropile, and (3) brain photosensory neurons form gap junctions with the medial lobe of the contralateral optic neuropile. These results reveal an ordered pattern of afferent projections from the retina and provide insight into the different functional roles of retinal photoreceptors.

Functional characterization of four opsins and two G alpha subtypes co-expressed in the molluscan rhabdomeric photoreceptor.

BACKGROUND: Rhabdomeric photoreceptors of eyes in the terrestrial slug Limax are the typical invertebrate-type but unique in that three visual opsins (Gq-coupled rhodopsin, xenopsin, Opn5A) and one retinochrome, all belonging to different groups, are co-expressed. However, molecular properties including spectral sensitivity and G protein selectivity of any of them are not determined, which prevents us from understanding an advantage of multiplicity of opsin properties in a single rhabdomeric photoreceptor. To gain insight into the functional role of the co-expression of multiple opsin species in a photoreceptor, we investigated the molecular properties of the visual opsins in the present study. RESULTS: First, we found that the fourth member of visual opsins, Opn5B, is also co-expressed in the rhabdomere of the photoreceptor together with previously identified three opsins. The photoreceptors were also demonstrated to express Gq and Go alpha subunits. We then determined the spectral sensitivity of the four visual opsins using biochemical and spectroscopic methods. Gq-coupled rhodopsin and xenopsin exhibit maximum sensitivity at ~ 456 and 475 nm, respectively, and Opn5A and Opn5B exhibit maximum sensitivity at ~ 500 and 470 nm, respectively, with significant UV sensitivity. Notably, in vitro experiments revealed that Go alpha was activated by all four visual opsins, in contrast to the specific activation of Gq alpha by Gq-coupled rhodopsin, suggesting that the eye photoreceptor of Limax uses complex G protein signaling pathways. CONCLUSIONS: The eye photoreceptor in Limax expresses as many as four different visual opsin species belonging to three distinct classes. The combination of opsins with different spectral sensitivities and G protein selectivities may underlie physiological properties of the ocular photoreception, such as a shift in spectral sensitivity between dark- and light-adapted states. This may be allowed by adjustment of the relative contribution of the four opsins without neural networks, enabling a simple strategy for fine-tuning of vision.

Deterioration of postural control due to the increase of similarity between center of pressure and smooth-pursuit eye movements during standing on one leg.

Upright postural control is regulated by afferent and efferent/reafferent visual mechanisms. There are two types of efferent and conjugate eye movements: saccades and smooth pursuits. Although postural control is improved by saccades, the effects of smooth pursuits on postural control are still debated, because the difficulties of postural and visual tasks differ in the previous research. Additionally, the mechanisms that interfere with postural control and smooth pursuit are not fully understood. To address these issues, we examined the effects of different patterns of smooth-pursuit eye movement on the path length of the center of pressure (COP) displacement under bipedal and unipedal standing conditions. The relative frequency and amplitude of the COP displacement were remarkably increased when uniform linear visual targets were presented during unipedal standing. In addition, dynamic time warping analysis demonstrated that the similarity between the displacement of the COP and eye movements was increased by the presentation of uniform linear visual targets with orientation selectivity during unipedal standing but not during bipedal standing. In contrast, the attenuation of similarity between the displacement of the COP and eye movements significantly decreased the path length, relative frequency, and amplitude of the COP displacement. Our results indicate that postural stability is deteriorated by the increase of similarity between the displacement of the COP and smooth-pursuit eye movements under unstable conditions.

Compensation mechanism for membrane potential against hypoosmotic stress in the Onchidium neuron.

The effect of acute hypoosmotic stress on the neural response was investigated using the neurons identified in the abdominal ganglion of the amphibious mollusk Onchidium. The membrane potential of an identified neuron (Ip-1/2) was not significantly altered in 50% hypoosmotic artificial sea water. In isotonic 50% artificial seawater (ASW) with osmolarity that was compensated for using glycerol or urea, the membrane potentials of Ip-1/2 were also not altered compared to those in 50% hypoosmotic ASW. However, hyperpolarization was induced in isotonic 50% ASW when osmolarity was compensated for using sucrose or mannose. In the presence of volume-regulated anion channel (VRAC) inhibitors (niflumic acid and glibenclamide), the Ip-1/2 membrane potentials were hyperpolarized in 50% hypoosmotic ASW. These results suggest that there is a compensatory mechanism involving aquaglyceroporin and VRAC-like channels that maintains membrane potential under hypoosmotic conditions. Here, we detected the expression of aquaglyceroporin mRNA in neural tissues of Onchidium.

Regional expression of neuropeptides in the retina of the terrestrial slug Limax valentianus (Gastropoda, Stylommatophora, Limacidae).

Gastropods use lens-bearing eyes to detect ambient light. The retina contains photoreceptors that directly project to the brain. Here, we identified the neurotransmitters that the retinal cells use for projection to the brain in the terrestrial slug Limax. We identified 12 genes encoding neuropeptides as well as a novel vesicular glutamate transporter, a marker of glutamatergic neuron, expressed in the retinal cells. Spatial expression profiles of the neuropeptide genes were determined by in situ hybridization. WWamide/MIP1/Pedal peptide2 were coexpressed in the neurons of the accessory retina. In the main retina, prohormone-4 was expressed in the ventrolateral region. Clionin was expressed in the ventromedial region. Pedal peptide was expressed in the anterior region of the main retina and in the accessory retina. Enterin was expressed in many neurons, including the accessory retina, but not in the dorsal region. FxRIamide1 and 2 were coexpressed in the posterior region. Prohormone-4 variant was uniformly expressed in many neurons but scarcely in the accessory retina. MIP2 was widely expressed throughout the dorsoventral axis in the posteriolateral region of the main retina. Myo1 was expressed in many neurons of the main retina but predominantly in the dorsal region. These expression patterns were confirmed by immunohistochemistry with specific antibodies against the neuropeptides. Projections of these peptidergic retinal neurons were confirmed by immunostaining of the optic nerve. Our present study revealed regional differentiation of the retina with respect to the neurotransmitters that the retinal cells use.

Opsins in the Cephalic and Extracephalic Photoreceptors in the Marine Gastropod Onchidium verruculatum.

AbstractThe marine gastropod Onchidium verruculatum has a pair of ocular photoreceptors, the stalk eyes, on the tip of its stalk near the head, as well as several extracephalic photosensory organs. The retinas of the stalk eye consist of two morphologically distinct visual cells, namely, the type I cells equipped with well-developed microvilli and the type II cells with less developed microvilli. The extracephalic photosensors comprise the dorsal eye, dermal photoreceptor, and brain photosensitive neurons. The characteristics of these cephalic and extracephalic photosensory organs have been studied from morphological and electrophysiological perspectives. However, little is known about the visual pigment molecules responsible for light detection in these organs. In the present study, we searched for opsin molecules that are expressed in the neural tissues of Onchidium and identified six putative signaling-competent opsin species, including Xenopsin1, Xenopsin2, Gq-coupled rhodopsin1, Gq-coupled rhodopsin2, Opsin-5B, and Gq-coupled rhodopsin-like. Immunohistochemical staining of four of the six opsins revealed that Xenopsin1, Gq-coupled rhodopsin1, and Gq-coupled rhodopsin2 are expressed in the rhabdomere of the stalk eye and in the dermal photoreceptor. Xenopsin2 was expressed in the type II photoreceptors of the stalk eye and in the ciliary photoreceptors of the dorsal eye. These immunohistochemical data were consistent with the results of the expression analysis, revealed by quantitative reverse transcription polymerase chain reaction. This study clarified the identities of opsins expressed in the extracephalic photosensory organs of Onchidium and the distinct molecular compositions among the photoreceptors.

FxRIamide regulates the oscillatory activity in the olfactory center of the terrestrial slug Limax.

The terrestrial slug Limax acquires odor-aversion memory. The procerebrum is the secondary olfactory center in the brain of Limax, and functions as the locus of the memory formation and storage. The change in the local field potential oscillation in the procerebrum reflects the information processing of the learned odor. However, it is not fully understood what factors, intrinsic or extrinsic in the procerebrum, alter the oscillatory activity and how it is regulated. In the present study, we found that FxRIamide (Phe-x-Arg-Ile-NH2), which was previously identified as a myomodulatory peptide in the gastropod Fusinus ferrugineus, downregulates the oscillatory frequency of the local field potential oscillation in the procerebrum of Limax. FxRIamide peptides were encoded by two distinct transcripts, which exhibit partially overlapping expression patterns in the brain. Immunohistochemical staining revealed a scattered distribution of FxRIamide-expressing neurons in the cell mass layer of the procerebrum, in addition to the ramified innervation of FxRIamidergic neurons in the neuropile layers. Down-regulation of the oscillatory frequency of the local field potential was explained by the inhibitory effects of FxRIamide on the bursting neurons, which are the kernels of the local field potential oscillation in the procerebrum. Our study revealed the previously unidentified role of FxRIamide peptides in the network of interneurons of Limax, and these peptides may play a role in the mnemonic functions of the procerebrum.

Integration of ocular and non-ocular photosensory information in the brain of the terrestrial slug Limax.

In the terrestrial slugs Limax, most of the photosensory information is thought to be acquired by an eye located on the superior tentacles, by which the slugs avoid light. Recent studies, however, suggested that the brain also plays a role as a photosensor in their negative phototaxis behavior. In the present study, we investigated how the photosensory information acquired by the eye and brain is integrated. The visual pathway in the brain was traced by incorporating tracer molecules from the cut end of an optic nerve, and commissural interactions were found in optic neuropiles located in the lateral regions of the cerebral ganglia. A cluster of neuronal cell bodies located near the dorsal surface of the cerebral ganglion had connections with the contralateral optic neuropile via gap junctions. Some of these neuronal cell bodies were Opn5A-immunoreactive, and contained numerous photic vesicle-like structures. Light-induced spikes were recorded extracellularly from the dorsal surface of these neuronal clusters, and they were synchronous with the spikes recorded from the cut end of the cerebral commissure. This study suggests that both the light information from the eye and the contralateral cerebral ganglion are integrated in the optic neuropile.

Detection of circulating tumor DNA in patients of operative colorectal and gastric cancers.

Liquid biopsy is a non-invasive tool to examine the genetic profile of tumors by identification of mutated circulating tumor DNA (ctDNA), which is often analyzed by next generation sequencing (NGS) or droplet digital PCR (ddPCR) assay. We first examined the ctDNA mutation in pre-operative plasma samples obtained from 154 colorectal cancer (CRC) and 46 gastric cancer (GC) patients, using the NGS-based panel assay. The overall detection rate of mutated ctDNA was 72.0% (144 of 200 patients), and the panel-based screening identified 207 and 47 mutations from CRC and GC patients, respectively. The ddPCR analysis was then performed on post-operative samples of 77 patients, and detection of mutated ctDNA was earlier than imaging-based diagnosis in all of 6 patients who showed the tumor recurrences after surgery. Our data also revealed that patients with positive post-operation ctDNA level showed significant shorter recurrence-free survival compared to the patients with negative ctDNA level (HR 14.9; 95% CI, 0.7-313.5; p < 0.0001). These findings suggested that screening of mutated ctDNA by liquid biopsy aids in identifying the patients at high risk of post-operative recurrence, and serial screening of ctDNA would allow to monitor the response after treatment and/or early detection of tumor recurrence.

Distribution and physiological effect of enterin neuropeptides in the olfactory centers of the terrestrial slug Limax.

In gastropods, the function of neuropeptides has been studied primarily in the peripheral motor systems. Their functional roles in the central nervous system have received less attention. The procerebrum, the secondary olfactory center of the terrestrial slug Limax, consists of several hundred thousand interneurons, and plays a pivotal role in olfactory learning and memory. In the present study, we found that enterin, known as a myoactive peptide functioning in the enteric and vascular system of Aplysia, is expressed in the procerebrum of Limax. These enterin-expressing neurons primarily make projections within the cell mass layer of the procerebrum. The oscillatory frequency of the local field potential in the procerebrum was reduced by an exogenous application of enterin. The local field potential oscillation in the tentacular ganglion, the primary olfactory center, was also modulated by enterin. Whole-cell patch-clamp recordings revealed that the modulatory effect in the procerebrum was due to the inhibitory effect of enterin on the bursting neurons, which function as the kernels determining the oscillatory activity of the procerebrum. Our results revealed the novel role of the myoactive neuropeptide enterin in the higher olfactory function in terrestrial gastropods.

Light avoidance by a non-ocular photosensing system in the terrestrial slug Limax valentianus.

Although the eye is the best-studied photoreceptive organ in animals, the presence of non-ocular photosensing systems has been reported in numerous animal species. However, most of the roles that non-ocular photosensory systems play remain elusive. We found that the terrestrial slug Limax valentianus avoids light and escapes into dark areas even if it is blinded by the removal of the bilateral superior tentacle. The escape behaviour was more evident for short-wavelength light. Illumination to the head with blue but not red light elicited avoidance behaviour in the blinded slugs. Illumination to the tail was ineffective. The light-avoidance behaviour of the blinded slugs was not affected by the removal of the penis, which lies on the brain in the head, suggesting that the penis is dispensable for sensing light in the blinded slug. mRNA of Opn5A, xenopsin, retinochrome and, to a lesser extent, rhodopsin was expressed in the brain according to RT-PCR. Light-evoked neural responses were recorded from the left cerebro-pleural connective of the isolated suboesophageal ganglia of the brain, revealing that the brain is sensitive to short wavelengths of light (400-480 nm). This result is largely consistent with the wavelength dependency of the light-avoidance behaviour of the blinded slugs that we observed in the present study. Our results strongly support that the terrestrial slug L. valentianus detects and avoids light by using its brain as a light-sensing organ in the absence of eyes.

Co-expression of opsins in the eye photoreceptor cells of the terrestrial slug Limax valentianus.

Visual opsins coupled with Gq -type G protein have been considered to be responsible for the vision in mollusks. Recent transcriptomic studies, however, revealed the presence of opsin mRNA belonging to different groups of opsin subfamilies in the eyes of mollusks. In the present study, we found that at least three different opsins, Gq -coupled rhodopsin, opsin5A, and xenopsin, are co-expressed in the rhabdomeric photoreceptor cell in the eyes of the terrestrial slug Limax valentianus. These opsins were all localized to the microvilli of the rhabdomere. Co-expression of rhodopsin and opsin5A mRNA was also demonstrated by dual fluorescence in situ hybridization. Co-expression of multiple opsins in the rhabdomeric photoreceptors cells may explain the previously reported shift in the action spectra of the electroretinogram of eyes of Limax flavus between the light- and dark-adapted states, which was also reproduced in the present study in L. valentianus.

RFamidergic neurons in the olfactory centers of the terrestrial slug Limax.

BACKGROUND: The terrestrial slug Limax has long been used as a model for the study of olfactory information processing and odor learning. Olfactory inputs from the olfactory epithelium are processed in the tentacular ganglion and then in the procerebrum. Glutamate and acetylcholine are the major neurotransmitters used in the procerebrum. Phe-Met-Arg-Phe-NH2 (FMRFamide) has been shown to be involved in the regulation of the network activity of the procerebrum. Although there are thought to be various RFamide family peptides other than FMRFamide that are potentially recognized by anti-FMRFamide antibody in the central nervous system of mollusks, identifying the entire repertoire of RFamide peptides in Limax has yet to be achieved. METHODS: In the present study, we made a comprehensive search for RFamide peptide-encoding genes from the transcriptome data of Limax, and identified 12 genes. The expression maps of these RFamide genes were constructed by in situ hybridization in the cerebral ganglia including the procerebrum, and in the superior/inferior tentacles. RESULTS: Ten of 12 genes were expressed in the procerebrum, and nine of 12 genes were expressed in the tentacular ganglia. Gly-Ser-Leu-Phe-Arg-Phe-NH2 (GSLFRFamide), which is encoded by two different genes, LFRFamide1 (Leu-Phe-Arg-Phe-NH2-1) and LFRFamide2 (Leu-Phe-Arg-Phe-NH2-2), decreased the oscillatory frequency of the local field potential oscillation in the procerebrum when exogenously applied in vitro. We also found by immunohistochemistry that the neurons expressing pedal peptide send efferent projections from the procerebrum back to the tentacular ganglion. CONCLUSION: Our findings suggest the involvement of a far wider variety of RFamide family peptides in the olfactory information processing in Limax than previously thought.

Hepatocellular adenoma in a woman who was undergoing testosterone treatment for gender identity disorder.

A 32-year-old Japanese woman was admitted to our hospital for the diagnosis and treatment of multiple liver tumors. She had been receiving 125 mg testosterone enanthate every 2 weeks following female-to-male gender identity disorder (GID) diagnosis at 20 years of age. Ultrasonography, computed tomography, and magnetic resonance imaging showed 11 hepatic nodular tumors with a maximum diameter of 28 mm. Liver tumors with hepatocellular adenoma (HCA) were diagnosed with needle biopsy. Segmentectomy of the left lateral lobe including two lesions, subsegmentectomy of S6 including two lesions, enucleation of each tumor in S5 and S7, and open surgical radiofrequency ablation for each tumor in S4 and S7 were performed. Immunohistochemical specimens showed that the tumor cells were diffusely and strongly positive for glutamine synthetase and that the nuclei were ectopically positive for ß-catenin. Thus, the tumors were diagnosed as ß-catenin-activated HCA (b-HCA). Transcatheter arterial chemoembolization plus subsequent radiofrequency ablation was performed for the 3 residual lesions in S4 and S8. Although testosterone enanthate was being continued for GID, no recurrence was observed until at least 22 months after the intensive treatments. HCA development in such patients receiving testosterone should be closely monitored using image inspection.

Gastric Adenocarcinoma with Dual Differentiation toward Neuroendocrine and α-Fetoprotein-Producing Features: Report of 2 Cases.

Some gastric carcinomas show composite features of neuroendocrine carcinoma (NEC) and α-fetoprotein (AFP)-producing carcinoma, which are very rare; only a few cases have been reported to date. We reviewed an additional 2 such cases of gastric carcinoma, which were both advanced aggressive tumors showing regional lymph node metastasis and distant metastasis. Both cases were accompanied by ordinary adenocarcinoma forms, implying that they had preceded the NEC and AFP-producing carcinoma components. A distinctive feature was the finding suggestive of dual differentiation of tumor cells to neuroendocrine and AFP-producing phenotypes, which was identified even in the metastatic tumor in the regional lymph node. Because both tumors predominantly showed poorly differentiated forms, the final pathologic diagnosis must rely on the immunohistochemistry. Pathologists should always keep in mind the existence of such tumors for the correct diagnosis of some gastric carcinomas with specific phenotypes, especially in pathologic diagnosis on biopsy.

Context-Dependent Passive Avoidance Learning in the Terrestrial Slug Limax.

The terrestrial slug Limax has been used as a model animal for studying the neural mechanisms underlying associative olfactory learning. The slug also innately exhibits negative phototactic behavior using its eyes. In the present study, we developed an experimental paradigm for quantification of slug's negative phototaxis behavior, and investigated whether the nature of the negative phototaxis can be modified by learning experience. The experimental set-up consists of light and dark compartments, between which the slug can move freely. During conditioning, the slug was placed in the light compartment, and an aversive stimulus (quinidine sulfate solution) was applied when it reached the dark compartment. After a single conditioning session, the time to reach the dark compartment significantly increased when it was tested following 24 hr or one week. Protein synthesis inhibition immediately following the conditioning impaired the memory retention at one week but not at 24 hr. The retrieval of the memory was context-dependent, as the time to reach the dark compartment did not significantly increase if the slug was placed on a floor with a different texture in the memory retention test. If the aversive stimulus was applied when the slug was in the light compartment, the time to reach the dark compartment did not increase after 24 hr. This is the first report demonstrating the capability of the slug to form context-dependent passive avoidance memory that can be established in a single conditioning session.

Expression and light-dependent translocation of ß-arrestin in the visual system of the terrestrial slug Limax valentianus.

Vertebrates, cephalopods and arthropods are equipped with eyes that have the highest spatiotemporal resolution among the animal phyla. In parallel, only animals in these three phyla have visual arrestin specialized for the termination of visual signaling triggered by opsin, in addition to ubiquitously expressed ß-arrestin that serves in terminating general G protein-coupled receptor signaling. Indeed, visual arrestin in Drosophila and rodents translocates to the opsin-rich subcellular region in response to light to reduce the overall sensitivity of photoreceptors in an illuminated environment (i.e. light adaptation). We thus hypothesized that, during evolution, visual arrestin has taken over the role of ß-arrestin in those animals with eyes of high spatiotemporal resolution. If this is true, it is expected that ß-arrestin plays a role similar to visual arrestin in those animals with low-resolution eyes. In the present study, we focused on the terrestrial mollusk Limax valentianus, a species related to cephalopods but that has only ß-arrestin, and generated antibodies against ß-arrestin. We found that ß-arrestin is highly expressed in photosensory neurons, and translocates into the microvilli of the rhabdomere within 30 min in response to short wavelength light (400 nm), to which the Limax eye exhibits a robust response. These observations suggest that ß-arrestin functions in the visual system of those animals that do not have visual arrestin. We also exploited anti-ß-arrestin antibody to visualize the optic nerve projecting to the brain, and demonstrated its usefulness for tracing a visual ascending pathway.

C-Arm Cone Beam Computed Tomography Guidance for Radiofrequency Ablation in Hepatocellular Carcinoma.

OBJECTIVE: To assess the usefulness of C-arm cone beam computed tomography (CBCT) combined with ultrasound for the treatment of hepatocellular carcinoma (HCC) by radiofrequency ablation (RFA). METHODS: Patients underwent RFA following transcatheter arterial chemoembolization (TACE) or RFA alone under ultrasound or CBCT guidance combined with ultrasound-based techniques. They were divided into 2 groups based on the use (C group) and nonuse (NC group) of CBCT guidance. The technical success of RFA and local tumor progression after the first RFA session were evaluated by dynamic contrast-enhanced imaging methods. Between-group differences were assessed retrospectively. RESULTS: We enrolled 198 patients with 260 HCC nodules. The complete ablation rates were 63.0 and 89.4% in the NC and C groups, respectively. In log-rank testing, local tumor progression occurred significantly more often in the NC group when RFA was used without TACE, in males when des-gamma-carboxy prothrombin was ≥29 mAU/mL, and when the diameter of a nodule was ≥18 mm. On Cox proportional-hazards regression analysis, the NC group, RFA alone without TACE, and male gender were significant independent variables. CONCLUSION: TACE followed by RFA under CBCT and ultrasound guidance improves the reliability of ablation of target HCC nodules, reduces the need for additional treatment sessions, and prevents local tumor progression.

Length of the memory retention period depends on the extent of protein synthesis in the terrestrial slug Limax.

The terrestrial slug Limax can form an odor-aversion memory by the single simultaneous presentation of a food odor and an aversive stimulus. We have previously demonstrated that the long-term retention of this memory was impaired by a high-dose injection of a protein synthesis inhibitor 30min prior to the conditioning. However, the onset of amnesia was delayed if the dose of the inhibitor was reduced or a less potent protein synthesis inhibitor was used. We thus speculated that the persistence of memory depends on the amount of newly synthesized protein following learning. In the present study, we further elaborated on this idea by injecting a high dose of anisomycin at different timings before or after conditioning, and tested the memory retention at 1, 2, 3, 7, or 14days after the conditioning. We found that the injection of anisomycin 6h before, or 1h after the conditioning had no effect on memory retention for 7days, and an injection at 30min before and just following the conditioning impaired the memory retention at 3days. Interestingly, the injection at 3h before and 30min after the conditioning did not impair the retention at 3days but did impair retention at 7days. Taking into account the time course of protein synthesis inhibition in the brain, our results further support the idea that the memory retention period is dependent on the amount of protein synthesized following memory acquisition.

Octopaminergic system in the central nervous system of the terrestrial slug Limax.

The terrestrial slug Limax can learn to avoid the odor of some food (e.g., carrot juice) by the simultaneous presentation of an aversive stimulus (e.g., bitterness of quinidine). This type of associative memory critically depends on the higher olfactory center, the procerebrum in the central nervous system. The modulation of the local field potential (LFP) oscillation recorded on the procerebrum has been thought to reflect the information processing of the odor that elicits the behavioral change, such as avoidance of the aversively learned odor or approaching an attractive food's odor. Here we focused on octopamine, an important neuromodulator involved in learning and memory in invertebrates, and considered to be the invertebrate equivalent of noradrenaline. We identified a few octopaminergic neurons in the subesophageal and buccal ganglia, and a larger number near the procerebrum in the cerebral ganglia, using immunohistochmical staining and in situ hybridization of tyramine ß-hydroxylase, an octopamine-synthesizing enzyme. Application of octopamine reduced the frequency of LFP oscillation in a dose-dependent manner, and this effect was inhibited by preincubation with phentolamine. High-performance liquid chromatography analysis revealed the presence of octopamine, noradrenaline, and adrenaline in the central nervous system. Unexpectedly, noradrenaline and adrenaline both accelerated the LFP oscillation, in contrast to octopamine. Our results suggest that octopamine and noradrenaline have distinct functions in olfactory information processing, in spite of their structural similarity. J. Comp. Neurol. 524:3849-3864, 2016. © 2016 Wiley Periodicals, Inc.