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.

Concept of dual-resolution light field imaging using an organic photoelectric conversion film for high-resolution light field photography.

Light field imaging is an emerging technique that is employed to realize various applications such as multi-viewpoint imaging, focal-point changing, and depth estimation. In this paper, we propose a concept of a dual-resolution light field imaging system to synthesize super-resolved multi-viewpoint images. The key novelty of this study is the use of an organic photoelectric conversion film (OPCF), which is a device that converts spectra information of incoming light within a certain wavelength range into an electrical signal (pixel value), for light field imaging. In our imaging system, we place the OPCF having the green spectral sensitivity onto the micro-lens array of the conventional light field camera. The OPCF allows us to acquire the green spectra information only at the center viewpoint with the full resolution of the image sensor. In contrast, the optical system of the light field camera in our imaging system captures the other spectra information (red and blue) at multiple viewpoints (sub-aperture images) but with low resolution. Thus, our dual-resolution light field imaging system enables us to simultaneously capture information about the target scene at a high spatial resolution as well as the direction information of the incoming light. By exploiting these advantages of our imaging system, our proposed method enables the synthesis of full-resolution multi-viewpoint images. We perform experiments using synthetic images, and the results demonstrate that our method outperforms other previous methods.

Cacao bean husk: an applicable bedding material in dairy free-stall barns.

OBJECTIVE: The objectives of the study were to assess the effect of cacao bean husk as bedding material in free-stall barn on the behavior, productivity, and udder health of dairy cattle, and on the ammonia concentrations in the barn. METHODS: Four different stall surfaces (no bedding, cacao bean husk, sawdust, and chopped wheat straw) were each continuously tested for a period of 1 week to determine their effects on nine lactating Holstein cows housed in the free-stall barn with rubber matting. The lying time and the milk yield were measured between d 4 and d 7. Blood samples for plasma cortisol concentration and teat swabs for bacterial counts were obtained prior to morning milking on d 7. The time-averaged gas-phase ammonia concentrations in the barn were measured between d 2 and d 7. RESULTS: The cows spent approximately 2 h more per day lying in the stalls when bedding was available than without bedding. The milk yield increased in the experimental periods when cows had access to bedding materials as compared to the period without bedding. The lying time was positively correlated with the milk yield. Bacterial counts on the teat ends recorded for cows housed on cacao bean husk were significantly lower than those recorded for cows housed without bedding. Ammonia concentration under cacao bean husk bedding decreased by 6%, 15%, and 21% as compared to no bedding, sawdust, and chopped wheat straw, respectively. The cortisol concentration was lowest in the period when cacao bean husk bedding was used. We observed a positive correlation between the ammonia concentrations in the barn and the plasma cortisol concentrations. CONCLUSION: Cacao bean husk is a potential alternative of conventional bedding material, such as sawdust or chopped wheat straw, with beneficial effects on udder health and ammonia concentrations in the barns.

Involvement of insulin-like peptide in long-term synaptic plasticity and long-term memory of the pond snail Lymnaea stagnalis.

The pond snail Lymnaea stagnalis is capable of learning taste aversion and consolidating this learning into long-term memory (LTM) that is called conditioned taste aversion (CTA). Previous studies showed that some molluscan insulin-related peptides (MIPs) were upregulated in snails exhibiting CTA. We thus hypothesized that MIPs play an important role in neurons underlying the CTA-LTM consolidation process. To examine this hypothesis, we first observed the distribution of MIP II, a major peptide of MIPs, and MIP receptor and determined the amounts of their mRNAs in the CNS. MIP II was only observed in the light green cells in the cerebral ganglia, but the MIP receptor was distributed throughout the entire CNS, including the buccal ganglia. Next, when we applied exogenous mammalian insulin, secretions from MIP-containing cells or partially purified MIPs, to the isolated CNS, we observed a long-term change in synaptic efficacy (i.e., enhancement) of the synaptic connection between the cerebral giant cell (a key interneuron for CTA) and the B1 motor neuron (a buccal motor neuron). This synaptic enhancement was blocked by application of an insulin receptor antibody to the isolated CNS. Finally, injection of the insulin receptor antibody into the snail before CTA training, while not blocking the acquisition of taste aversion learning, blocked the memory consolidation process; thus, LTM was not observed. These data suggest that MIPs trigger changes in synaptic connectivity that may be correlated with the consolidation of taste aversion learning into CTA-LTM in the Lymnaea CNS.

Excitatory effects of GABA on procerebrum neurons in a slug.

Classical neurotransmitters, such as glutamate and γ-aminobutyric acid (GABA), often have different actions on invertebrate neurons from those reported for vertebrate neurons. In the terrestrial mollusk Limax, glutamate was found to function as an inhibitory transmitter in the procerebrum (PC), but it has not yet been clarified how GABA acts in the PC. We thus examined what effects GABA exerts on PC neurons in the present study. For this purpose, we first applied GABA to isolated PC preparations and recorded postsynaptic currents and potentials in PC neurons. The GABA application reduced the amplitude of inhibitory postsynaptic currents and depolarization-induced outward currents recorded in nonbursting neurons and increased the number of spontaneous spikes of nonbursting neurons. However, direct GABA-induced currents were not observed in either bursting or nonbursting neurons. These results suggest a potential direct effect of GABA on outward currents resulting in enhanced excitability of PC neurons. Next, we measured the change in [Ca(2+)](i) in cultured PC neurons by application of GABA. The GABA application increased spontaneous Ca(2+) events in cultured neurons. These Ca(2+) events were ascribable to the influx of extracellular Ca(2+). We then confirmed the presence of GABA and GABA receptors in the PC. The GABA-like immunoreactivity was observed in the neuropil layers of the PC, and the mRNAs for both GABA(A) and GABA(B) receptors were expressed in the PC. In particular, GABA(B) receptor mRNA, rather than GABA(A), was found to be more abundantly expressed in the PC. These results suggest that GABA functions as an excitatory modulator for PC neurons via mainly GABA(B) receptors.

Competition and disturbance affect elevational distribution of two congeneric conifers.

Climatic change will affect elevational vegetation distribution because vegetation distribution is related to thermal conditions. However, how elevational species distributions are determined by biotic and abiotic factors is not clear. The long-term plot census along an elevational gradient is indispensable to clarify mechanisms of elevational distribution of tree species. Two congeneric conifers, the less shade-tolerant Abies veitchii and shade-tolerant A. mariesii, dominate at low and high elevations, respectively, in the subalpine zone in Japan. This study investigated the population dynamics of the two species at three elevations (low, middle, high) for 13 years to examine why the two species dominated the different elevations from the viewpoints of competition and disturbance. This study showed that growth and survival rates were not highest at the most dominant elevations for each species. At the high elevation where A. mariesii dominated and small disturbances frequently occurred, the recruitment rate of A. mariesii was highest among the three elevations and that of A. veitchii was largely decreased by tree competition. However, A. veitchii was dominant earlier than A. mariesii at the low elevation after large disturbances by the high growth rate of individual trees. Therefore, A. mariesii was superior to A. veitchii at the high elevation because of its high recruitment rate and large reduction of recruitment of A. veitchii due to competition, while A. veitchii was superior to A. mariesii at the low elevation after large disturbances because of higher growth rate than A. mariesii. It is suggested that the elevational distributions of the two species were determined by elevational changes in population dynamics in relation to competition and disturbance. Long-term observational studies of forest dynamics among various elevations are indispensable to predict the effects of climatic change on vegetation distribution.

Two pairs of tentacles and a pair of procerebra: optimized functions and redundant structures in the sensory and central organs involved in olfactory learning of terrestrial pulmonates.

Terrestrial pulmonates can learn olfactory-aversion tasks and retain them in their long-term memory. To elucidate the cellular mechanisms underlying learning and memory, researchers have focused on both the peripheral and central components of olfaction: two pairs of tentacles (the superior and inferior tentacles) and a pair of procerebra, respectively. Data from tentacle-amputation experiments showed that either pair of tentacles is sufficient for olfactory learning. Results of procerebrum lesion experiments showed that the procerebra are necessary for olfactory learning but that either one of the two procerebra, rather than both, is used for each olfactory learning event. Together, these data suggest that there is a redundancy in the structures of terrestrial pulmonates necessary for olfactory learning. In our commentary we exemplify and discuss functional optimization and structural redundancy in the sensory and central organs involved in olfactory learning and memory in terrestrial pulmonates.

Multiple subtypes of serotonin receptors in the feeding circuit of a pond snail.

In the central nervous system of the pond snail Lymnaea stagnalis, serotonergic transmission plays an important role in controlling feeding behavior. Recent electrophysiological studies have claimed that only metabotropic serotonin (5-HT(2)) receptors, and not ionotropic (5-HT(3)) receptors, are used in synapses between serotonergic neurons (the cerebral giant cells, CGCs) and the follower buccal motoneurons (the B1 cells). However, these data are inconsistent with previous results. In the present study, we therefore reexamined the serotonin receptors to identify the receptor subtypes functioning in the synapses between the CGCs and the B1 cells by recording the compound excitatory postsynaptic potential (EPSP) of the B1 cells evoked by a train of stimulation to the CGC in the presence of antagonists: cinanserin for 5-HT(2) and/or MDL72222 for 5-HT(3). The compound EPSP amplitude was partially suppressed by the application of these antagonists. The rise time of the compound EPSP was longer in the presence of MDL72222 than in that of cinanserin. These results suggest that these two subtypes of serotonin receptors are involved in the CGC-B1 synapses, and that these receptors contribute to compound EPSP. That is, the fast component of compound EPSP is mediated by 5-HT(3)-like receptors, and the slow component is generated via 5-HT(2)-like receptors.

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.

Identification and classification of innexin gene transcripts in the central nervous system of the terrestrial slug Limax valentianus.

Intercellular gap junction channels and single-membrane channels have been reported to regulate electrical synapse and the brain function. Innexin is known as a gap junction-related protein in invertebrates and is involved in the formation of intercellular gap junction channels and single-cell membrane channels. Multiple isoforms of innexin protein in each species enable the precise regulation of channel function. In molluscan species, sequence information of innexins is still limited and the sequences of multiple innexin isoforms have not been classified. This study examined the innexin transcripts expressed in the central nervous system of the terrestrial slug Limax valentianus and identified 16 transcripts of 12 innexin isoforms, including the splicing variants. We performed phylogenetic analysis and classified the isoforms with other molluscan innexin sequences. Next, the phosphorylation, N-glycosylation, and S-nitrosylation sites were predicted to characterize the innexin isoforms. Further, we identified 16 circular RNA sequences of nine innexin isoforms in the central nervous system of Limax. The identification and classification of molluscan innexin isoforms provided novel insights for understanding the regulatory mechanism of innexin in this phylum.

FMRFamide regulates oscillatory activity of the olfactory center in the slug.

In the olfactory center of terrestrial animals, changes in the oscillatory frequency of the local field potential (LFP) are thought to be involved in olfaction-based behavior and olfactory memory. The terrestrial slug Limax has a highly developed olfactory center, the procerebrum, in which the LFP spontaneously oscillates. Although changes in the oscillatory frequency are thought to correspond to the preference for specific odors, our knowledge about the mechanism of this frequency regulation is limited. To clarify the mechanism of the bidirectional frequency changes in the procerebrum, we focused on the neuropeptide Phe-Met-Arg-Phe-NH2 (FMRFamide), which is known to have neuromodulatory functions in invertebrate nervous systems. Application of FMRFamide decreased the oscillatory frequency via G-protein-mediated cascades. Immunohistochemistry showed that FMRFamide-like-immunoreactive neuronal cell bodies are located in the cell mass layer of the procerebrum, projecting their neurites to the neuropile layers. The procerebrum was shown to also receive innervation from other regions of the cerebral ganglion. Furthermore, according to their morphological and projection characteristics, FMRFamide-containing neurons belong to the subpopulations of both bursting and nonbursting neurons in the procerebrum. The mRNA splice variant encoding multiple copies of canonical FMRFamide was specifically expressed in the procerebrum. Taking into account previous results showing that serotonin increases the oscillatory frequency, our results indicate that FMRFamide and serotonin both regulate the LFP frequency but in exactly the opposite direction in the olfactory center of the terrestrial slug.

Glutamate transporters in the central nervous system of a pond snail.

Previous studies on glutamate (GLU) and its receptors in the pond snail Lymnaea stagnalis have suggested that GLU functions as a neurotransmitter in various behaviors, particularly for generation of feeding rhythm. The uptake mechanism of GLU is not yet known in Lymnaea. In the present study, we characterized the GLU transporters and examined their functions in the feeding circuits of the central nervous system (CNS) in Lymnaea. First, measurement of the accumulation of (3)H-labeled GLU revealed the presence of GLU transport systems in the Lymnaea CNS. The highest accumulation rate was observed in the buccal ganglia, supporting the involvement of GLU transport systems in feeding behavior. Second, we cloned two types of GLU transporters from the Lymnaea CNS, the excitatory amino acid transporter (LymEAAT) and the vesicular GLU transporter (LymVGLUT). When we compared their amino acid sequences with those of mammalian EAATs and VGLUTs, we found that the functional domains of both types are well conserved. Third, in situ hybridization revealed that the mRNAs of LymEAAT and LymVGLUT are localized in large populations of nerve cells, including the major feeding motoneurons in the buccal ganglia. Finally, we inhibited LymEAAT and found that changes in the firing patterns of the feeding motoneurons that have GLUergic input were similar to those obtained following stimulation with GLU. Our results confirmed the presence of GLU uptake systems in the Lymnaea CNS and showed that LymEAAT is required for proper rhythm generation, particularly for generation of the feeding rhythm.

Raphe modulation of the pre-Bötzinger complex respiratory bursts in in vitro medullary half-slice preparations of neonatal mice.

Spontaneous respiratory bursts which begin in the pre-Bötzinger complex were recorded from the hypoglossal (XIIth) nerve rootlets of in vitro slices prepared from newborn mice. First, we examined the respiratory bursts before and after a midline or para-midline transection which spared the caudal raphe nuclei: the raphe obscurus and raphe pallidus. After a midline transection, the respiratory bursts in both half-slices were desynchronized and had slightly decreased amplitudes and frequencies. After a para-midline transection, the bursts continued with similar frequencies in the half slice containing the raphe obscurus and raphe pallidus. Second, to analyze the effects of modulation by the raphe obscurus and raphe pallidus, a dorsal or ventral midline lesion was used to damage either the raphe obscurus or raphe pallidus. After a dorsal lesion, the synchronized respiratory bursts persisted with slightly decreased frequencies. In contrast, after a ventral lesion, the bursts were almost completely abolished, but recovered significantly after the addition of 5-HT. The present results demonstrated that the pre-Bötzinger complex on each side of the medulla can independently generate rhythmic respiratory activity. It is suggested that the 5-HT released from the ventral part of the raphe nuclei (predominantly the raphe pallidus) plays a critical role in sustaining rhythmic respiratory bursts.

Effects of tentacle amputation and regeneration on the morphology and activity of the olfactory center of the terrestrial slug Limax valentianus.

The tentacles of pulmonates regenerate spontaneously following amputation. The regenerated tentacle is equipped with all the elements necessary for normal olfactory functioning, and the slugs can behave as well as they did before the tentacle amputation. However, it is not known what changes occur to the olfactory center procerebrum in the brain at the morphological and physiological levels. Here, we investigated the innervation of tentacular nerves into the procerebrum by examining the size of the terminal mass (input layer from tentacular nerves) of the procerebrum and also by staining afferent nerves immunohistochemically at 15, 58 and 75 days following unilateral amputation of the superior and inferior tentacles. The size of the terminal mass was significantly decreased, and the Phe-Met-Arg-Phe-NH(2)ergic (FMRFamidergic) afferent nerves disappeared by 15 days following the tentacle amputation. However, the size of the terminal mass had recovered substantially by 58 days, as the tentacle regenerated. The FMRFamidergic innervation into the cerebral ganglion was also restored by this time. An extended recovery (75 days), however, did not result in any further increase in the size of the terminal mass. We also recorded the local field potential (LFP) oscillation in the procerebrum. We found that the oscillatory frequency of the LFP had decreased at 15 days following the tentacle amputation but had recovered at 58 and 75 days. These results suggest that the amputation and regrowth of the tentacle are accompanied by the respective degeneration and re-innervation of olfactory nerves, and these changes in the innervation status affect the basal state of LFP oscillation.

Glutamatergic neurotransmission in the procerebrum (olfactory center) of a terrestrial mollusk.

The terrestrial slug Limax has the ability to learn odor associations. This ability depends on the function of the procerebrum, the secondary olfactory center in the brain. Among the various neurotransmitters that are thought to be involved in the function of the procerebrum, glutamate is one of the most important molecules. However, the existence and function of glutamate in this system have been proposed solely on the basis of a few lines of indirect evidence from pharmacological experiments. In the present study, we demonstrated the existence and release of glutamate as a neurotransmitter in the procerebrum of Limax, by using three different techniques: 1) immunohistochemistry of glutamate, 2) in situ hybridization to mRNA of the vesicular glutamate transporter, and 3) real-time imaging of glutamate release within the procerebrum using the glutamate optical sensor EOS2. The release of glutamate within the cell mass layer of the procerebrum was synchronized with oscillation of the local field potential and had the same physiological properties as this oscillation; both were blocked by a serotonin antagonist and were propagated in an apical to basal direction in the procerebrum. Our observations suggest strongly that the oscillation of the local field potential is driven by the glutamate released by bursting neurons in the procerebrum.

Effects of propofol on IPSCs in CA1 and dentate gyrus cells of rat hippocampus: Propofol effects on hippocampal cells' IPSCs.

Propofol (2, 6-diisopropylphenol) is one of the most popular intravenous anesthetic agents. In this study, we compared the effects of propofol on inhibitory postsynaptic currents (IPSCs) induced by single and paired electrical stimulations in CA1 pyramidal cells (CA1-PCs) and dentate gyrus granule cells (DG-GCs) in rat hippocampal slices using the whole cell patch-clamp technique. In the absence of propofol, the amplitude of evoked IPSC by single stimulation and decay time constants were stable in both CA1-PCs and DG-GCs for 30 min. Propofol (1 µM and 10 µM) increased both IPSC amplitude in CA1-PCs, but not in DG-GCs. Further, using a paired pulse stimulation protocol, the ratio of IPSC amplitudes (the second response: A2/the first response: A1) was increased by propofol in CA1, but not in DG-GCs. These results suggest that propofol selectively affects IPSCs in CA1-PCs, which is similar to previously reported actions of benzodiazepines.

Requirement of new protein synthesis of a transcription factor for memory consolidation: paradoxical changes in mRNA and protein levels of C/EBP.

Some specific transcription factors are essential for memory consolidation across species. However, it is still unclear whether only the activation of constitutively expressed forms of these conserved transcription factors is involved in memory consolidation or their de novo synthesis also occurs after learning. This question has remained unanswered partly because of the lack of an efficient method for the determination of copy numbers of particular mRNAs in single neurons, which allows the detection of new transcription at the cellular level. Here we applied a newly developed protocol of single-cell quantitative real-time polymerase chain reaction (qRT-PCR) to single neurons playing an important role in associative learning. Specifically, we examined the changes in the mRNA and protein expression levels of a highly conserved transcription factor, CCAAT/enhancer binding protein (C/EBP), in the paired B2 motoneurons of the pond snail Lymnaea stagnalis. These buccal neurons are involved in the motor control of feeding behavior, with a potentially important role in conditioned taste aversion (CTA). Single-cell qRT-PCR revealed a significant decrease in LymC/EBP mRNA copy numbers in the B2 motoneurons during memory consolidation after CTA training. By contrast, isoelectric focusing and immunoblotting of extracts of the buccal ganglia showed that translation and phosphorylation levels of LymC/EBP significantly increased during memory consolidation. The C/EBP-like immunoreactivity in the B2 motoneurons, which are the major immunopositive component in the buccal ganglia, also significantly increased during memory consolidation, suggesting that the main source of increase in the level of protein in the buccal ganglia are the B2 motoneurons. Thus, early memory consolidation after CTA learning in L.stagnalis involves both the rapid synthesis and phosphorylation of LymC/EBP as well as the rapid breakdown of LymC/EBP mRNA in the neural network controlling feeding, suggesting that all of these processes play a role in the function of C/EBP in memory consolidation.

Development of a one-run real-time PCR detection system for pathogens associated with bovine respiratory disease complex.

Bovine respiratory disease complex (BRDC) is frequently found in cattle worldwide. The etiology of BRDC is complicated by infections with multiple pathogens, making identification of the causal pathogen difficult. Here, we developed a detection system by applying TaqMan real-time PCR (Dembo respiratory-PCR) to screen a broad range of microbes associated with BRDC in a single run. We selected 16 bovine respiratory pathogens (bovine viral diarrhea virus, bovine coronavirus, bovine parainfluenza virus 3, bovine respiratory syncytial virus, influenza D virus, bovine rhinitis A virus, bovine rhinitis B virus, bovine herpesvirus 1, bovine adenovirus 3, bovine adenovirus 7, Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, Trueperella pyogenes, Mycoplasma bovis and Ureaplasma diversum) as detection targets and designed novel specific primer-probe sets for nine of them. The assay performance was assessed using standard curves from synthesized DNA. In addition, the sensitivity of the assay was evaluated by spiking solutions extracted from nasal swabs that were negative by Dembo respiratory-PCR for nucleic acids of pathogens or synthesized DNA. All primer-probe sets showed high sensitivity. In this study, a total of 40 nasal swab samples from cattle on six farms were tested by Dembo respiratory-PCR. Dembo respiratory-PCR can be applied as a screening system with wide detection targets.

Distribution of histaminergic neurons and their modulatory effects on oscillatory activity in the olfactory center of the terrestrial slug Limax.

Terrestrial mollusks can form an odor aversion memory following the simultaneous presentation of a food odor and an aversive stimulus. The local field potential oscillation recorded on the surface of the procerebrum (PC; the higher olfactory center) exhibits a frequency change in response to the detection of a learned odor; such a change is thus considered to reflect the internal state of the brain during memory recall. Thus far, dopamine and serotonin have been demonstrated to change the oscillatory frequency. Other monoamines, however, have not yet been studied. In the present study, we investigated the possible involvement of histamine (HA). Immunohistochemical staining of HA and in situ hybridization against histidine decarboxylase revealed the location of the cell bodies of HAergic neurons in all ganglia of the brain. The majority of them were located at the medial aspect of the pedal ganglia, and the cerebral ganglia also contained numerous HAergic neurons in their posterior regions. The neuropil layers of the PC received HAergic innervation from the neurons in the cerebral ganglion, as well as from a few neurons located in the dorsomedial part of the cell mass layer of the PC. The HAergic fibers, however, innervated spatially limited regions of the PC, and seemed to affect a small fraction of the PC neurons. HA exerted accelerating effects on the LFP oscillation in a dose-dependent manner, and this effect was suppressed by an H2 blocker, cimetidine. Our results support the involvement of HA in the functioning of the PC.

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.