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.

Metyrosine-associated endocrinological changes in pheochromocytoma and paraganglioma.

Objective: Metyrosine (alpha-methyl-para-tyrosine) effectively reduces catecholamine levels in patients with pheochromocytoma/paraganglioma. However, improvements in physiological and metabolic parameters and changes in endocrine function associated with metyrosine administration should be validated in comparison to surgery. This study was performed to confirm the effects of metyrosine on the physiological, metabolic, and endocrinological functions of patients with pheochromocytoma/paraganglioma in the perioperative period. Design: This retrospective cohort study was performed at a single university hospital. Methods: We included ten patients with pheochromocytoma/paraganglioma who received oral metyrosine after α-blocker therapy and consecutive surgeries. Urinary catecholamine metabolite levels and other clinical parameters were evaluated before and after metyrosine administration, and 1 week after surgery. Results: The mean age was 53.1 ± 16.1 years. Of the ten participants (four men and six women), nine had pheochromocytoma and one had paraganglioma. The median maximum metyrosine dose was 750 mg/day. Urinary catecholamine metabolite levels significantly decreased in a dose-dependent manner after metyrosine administration. Both systolic and diastolic blood pressure significantly decreased after metyrosine and surgical treatment. Metyrosine administration significantly improved insulin sensitivity, although surgery improved the the basal insulin secretion. Additionally, serum prolactin and thyroid-stimulatory hormone levels were significantly increased by metyrosine treatment, whereas plasma renin activity was decreased. Conclusions: Metyrosine significantly reduced catecholamines in patients with pheochromocytoma/paraganglioma and ensured the safety of the surgery. Adjustment of metyrosine administration may make surgical pretreatment more effective in achieving stabilized blood pressure and improving glucose metabolism. Endocrine parameters may manifest as the systemic effects of metyrosine administration.

Eruptive xanthomas as a marker for metabolic disorders: A specific form of xanthoma that reflects hypertriglyceridemia.

Eruptive xanthomas are skin manifestations associated with hypertriglyceridemia. Accordingly, the improvement of hypertriglyceridemia can ameliorate this condition. We report a case of a patient with type 2 diabetes mellitus who was diagnosed with this skin lesion. Clinicians should be aware that eruptive xanthomas could indicate metabolic disorders associated with atherosclerosis.

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.

Masked Diabetes Insipidus Hidden by Severe Hyponatremia: A Case of Pituitary Metastasis of Lung Adenocarcinoma.

BACKGROUND Hyponatremia is an electrolyte disorder frequently encountered by clinicians. Secondary adrenal insufficiency due to pituitary metastatic tumors should be considered as an alternative diagnosis when clinicians encounter patients with lung cancer who demonstrate hyponatremia. However, masked central diabetes insipidus should also be considered to prevent critical dehydration when glucocorticoid replacement therapy will be initiated. CASE REPORT A 70-year-old man with advanced lung adenocarcinoma demonstrated high-grade hyponatremia of 122 mmol/L. Magnetic resonance imaging disclosed a metastatic pituitary tumor and endocrinological examinations confirmed panhypopituitarism, including secondary adrenal insufficiency. Hydrocortisone replacement revealed masked diabetes insipidus with elevation of serum sodium levels that reached 151 mmol/L. Desmopressin administration was required to prevent water depletion and to immediately ameliorate the hypernatremia. CONCLUSIONS This is the first case report of masked diabetes insipidus that demonstrated high-grade hyponatremia. Secondary adrenal insufficiency can mask the hypernatremia that is a typical manifestation of diabetes insipidus. Physicians should consider adrenal insufficiency and diabetes insipidus due to pituitary metastasis of advanced malignancies, even when they encounter patients with hyponatremia.

Collagenous Alzheimer amyloid plaque component impacts on the compaction of amyloid-ß plaques.

Massive deposition of amyloid ß peptides (Aß) as senile plaques (SP) characterizes the brain pathology of Alzheimer's disease (AD). SPs exhibit a variety of morphologies, although little is known about the SP components that determine their morphology. Collagenous Alzheimer amyloid plaque component (CLAC) is one of the major non-Aß proteinaceous components of SP amyloid in AD brains. Here we show that overexpression of CLAC precursor (CLAC-P) in the brains of APP transgenic mice results in a significant remodeling of amyloid pathology, i.e., reduction in diffuse-type amyloid plaques and an increase in compact plaques laden with thioflavin S-positive amyloid cores. In vivo microdialysis revealed a significant decrease in Aß in the brain interstitial fluid of CLAC-P/APP double transgenic mice compared with APP transgenic mice. These findings implicate CLAC in the compaction of Aß in amyloid plaques and the brain dynamics of Aß.

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.

Unsupervised low-intensity home exercises as an effective intervention for improving physical activity and physical capacity in the community-dwelling elderly.

[Purpose] The purpose of this study was to examine the effectiveness of unsupervised low-intensity home exercises in improving physical activity and physical capacity for daily activities among the community-dwelling elderly. [Participants and Methods] We included 24 female older participants and divided them into two groups: 14 in the resistance training group and 10 in the fast walking group. The resistance training group performed shoulder joint flexion and abduction exercises using a resistance tube twice daily (in the morning and afternoon). Participants in the fast walking group walked fast for 3,000 steps once daily. Both groups continued the exercise intervention for 6 months. We measured the forced vital capacity, respiratory muscle strength, physical activity, walking distance in the 6-min walk test, grip strength, and knee extension strength before and after the intervention. [Results] The forced vital capacity significantly increased in the resistance training group, whereas the moderate-intensity physical activity time significantly increased in both groups and the vigorous-intensity physical activity time increased in the fast walking group only. We observed no intergroup differences in respiratory muscle strength, 6-min walking distance, grip strength, or knee extension strength. [Conclusion] The results of this study suggest that low-intensity home exercises can improve pulmonary function and physical activity and should be recommended for promoting health in the community-dwelling elderly.

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.

Role of a new bioassay for thyroid-stimulating antibodies (aequorin TSAb) in Graves' ophthalmopathy.

Graves' ophthalmopathy (GO) is characterized by an autoimmune reaction against thyrotropin (TSH) receptors and is diagnosed by TSH receptor antibody (TRAb). A novel assay for thyroid-stimulating antibody (TSAb) was recently introduced using a frozen Chinese hamster ovary cell line expressing TSH receptors, cyclic adenosine monophosphate (cAMP)-gated calcium channel, and aequorin (aequorin TSAb). The aim of this study was to evaluate the role of aequorin TSAb in GO. We studied 136 Japanese patients with GO (22 euthyroid and 8 hypothyroid GO patients) at our hospital. TRAbs were estimated by first generation TRAb (TRAb 1st), second generation TRAb (hTRAb 2nd), conventional porcine TSAb, and the new aequorin TSAb assays. Aequorin TSAb, porcine TSAb, TRAb 1st, and hTRAb 2nd were positive in 125/136 (92%), 110/136 (81%), 81/130 (62%), and 93/114 (82%) patients, respectively. In patients with hyperthyroid GO, they were positive in 98/106 (98%), 96/106 (91%), 78/101 (77%), and 84/93 (90%) patients, respectively. In patients with euthyroid GO, they were positive in 19/22 (86%), 9/22 (41%), 1/21 (5%), and 6/17 (35%) patients, respectively. Aequorin TSAb levels were significantly related to TRAb 1st (r = 0.4172, p < 0.0001), hTRAb 2nd (r = 0.2592, p < 0.0001), and porcine TSAb (r = 0.4665, p < 0.0001). Clinical activity score (CAS) was significantly greater in patients with high titers of aequorin TSAb than in those with low titers. Aequorin TSAb levels were significantly related to the signal intensity ratio of the enlarged eye muscle and proptosis evaluated by MRI before steroid pulse therapy. Aequorin TSAb assay was more sensitive than the conventional assays, especially in euthyroid GO.

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.

Differential effects of diet- and genetically-induced brain insulin resistance on amyloid pathology in a mouse model of Alzheimer's disease.

BACKGROUND: Based on epidemiological and experimental studies, type 2 diabetes mellitus (T2DM), especially insulin resistance that comprises the core mechanism of T2DM, has been recognized as a significant risk factor for Alzheimer's disease (AD). Studies in humans and diabetic AD model mice have indicated a correlation between insulin resistance and increased amyloid deposition in the brain. Paradoxically, mice with targeted disruption of genes involved in the insulin signaling pathway showed protective effects against the AD-related pathology. These conflicting observations raise an issue as to the relationship between dysregulation of insulin signaling and AD pathophysiology. METHODS: To study the causal relations and molecular mechanisms underlying insulin resistance-induced exacerbation of amyloid pathology, we investigated the chronological changes in the development of insulin resistance and amyloid pathology in two independent insulin-resistant AD mouse models, i.e., long-term high-fat diet (HFD) feeding and genetic disruption of Irs2, in combination with dietary interventions. In addition to biochemical and histopathological analyses, we examined the in vivo dynamics of brain amyloid-ß (Aß) and insulin by microdialysis technique. RESULTS: HFD-fed diabetic AD model mice displayed a reduced brain response to peripheral insulin stimulation and a decreased brain to plasma ratio of insulin during the hyperinsulinemic clamp. Diet-induced defective insulin action in the brain was accompanied by a decreased clearance of the extracellular Aß in vivo and an exacerbation of brain amyloid pathology. These noxious effects of the HFD both on insulin sensitivity and on Aß deposition in brains were reversibly attenuated by dietary interventions. Importantly, HFD feeding accelerated Aß deposition also in the brains of IRS-2-deficient AD mice. CONCLUSIONS: Our results suggested a causal and reversible association of brain Aß metabolism and amyloid pathology by diet-dependent, but not genetically-induced, insulin-resistance. These observations raise the possibility that the causal factors of insulin resistance, e.g., metabolic stress or inflammation induced by HFD feeding, but not impaired insulin signaling per se, might be directly involved in the acceleration of amyloid pathology in the brain.

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.

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.

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.

Olfactory Memory Storage and/or Retrieval Requires the Presence of the Exact Tentacle Used During Memory Acquisition in the Terrestrial Slug Limax.

Terrestrial pulmonates can form odor-aversion memories once a food odor is presented in combination with an aversive stimulus. Most of the olfactory information ascends via a tentacular ganglion located in the tip of the two pairs of tentacles, and is then transmitted to the higher olfactory center, the procerebrum. The procerebrum is the locus of memory storage and has been shown to be necessary for odor-aversion learning. However, it is unknown whether the procerebrum is the sole locus in which the memory engram resides. By exploiting the regenerative ability of tentacles, here we investigated whether tentacles function merely in transmitting olfactory information to the procerebrum, or constitute a part of the memory engram. We showed that after removal of the tentacles used during memory acquisition, slugs were unable to retrieve the memory, even if these tentacles were regenerated sufficiently to subserve memory function. Our results support the view that tentacles are more than conduits of odor information; they also participate in the formation of the memory engram.