Procerebrum Research Articles

Spatiotemporal Neural Activity Changes in the Molluscan Olfactory Center Specifically Induced by Innately Aversive and <i>In Vitro</i> Aversively Conditioned Odors

In the procerebrum (PC), the olfactory center, of the land slug Limax, an oscillation of local field potential (LFP) with 0.5 - 1 Hz is observed by electrophysiological extracellular recording. The oscillation has a phase delay along the distal-proximal axis, resulting in the propagation of waves from the distal to proximal region. One important advantage of nervous systems of mollusks such as Limax is that their nervous systems in vitro retain several types of computational properties found in vivo (e.g. learning and memory). A previous study showed that the LFP frequency in the PC of Limax increased specifically in response to innately aversive and in vitro aversively conditioned odors. In the present study, we examined spatiotemporal neural activity changes induced in the PC by those odors using the fluorescent voltage imaging technique. The results showed that innately aversive (onion and hexanol) and in vitro aversively conditioned (carrot, which is innately attractive) odors specifically induced an increase in propagation speed of the neural activity in the PC, while innately attractive odors did not induce it. The results also suggested that the avoidance behavior by those odors might be induced by the increase of propagation speed and the following increases in the discharges of the partial nerve that transmits the motor output.

Phase-Dependent Modulation of Oscillatory Phase and Synchrony by Long-Lasting Depolarizing Inputs in Central Neurons.

Oscillatory neural activities have been implicated in various types of information processing in the CNS. The procerebral (PC) lobe of the land mollusk Limax valentianus shows an ongoing oscillatory local field potential (LFP). Olfactory input increases both the frequency and spatial synchrony of the LFP oscillation by a nitric oxide (NO)-mediated mechanism, but how NO modulates the activity in a specific manner has been unclear. In the present study, we used electrical stimulation and NO uncaging to systematically analyze the response of the LFP oscillation and found phase-dependent effects on phase shifting and synchrony. The neurons that presumably release NO in the PC lobe preferentially fired at phases in which NO has a synchronizing effect, suggesting that the timing of NO release is regulated to induce a stereotyped response to natural sensory stimuli. The phase-response curve (PRC) describes the timing dependence of responses of an oscillatory system to external input. PRCs are usually constructed by recording the temporal shifts of the neural activity in response to brief electrical pulses. However, NO evokes a long-lasting depolarization persisting for several cycles of oscillation. The phase-response relationship obtained by NO stimulation was approximately the integral of the PRC. A similar relationship was also shown for regular firing of mouse cerebellar Purkinje cells receiving step depolarization, suggesting the generality of the results to oscillatory neural systems with highly distinct properties. These results indicate novel dynamic effects of long-lasting inputs on network oscillation and synchrony, which are based on simple and ubiquitous mechanisms.

Nitric Oxide-Mediated Modulation of Central Network Dynamics during Olfactory Perception.

Nitric oxide (NO) modulates the dynamics of central olfactory networks and has been implicated in olfactory processing including learning. Land mollusks have a specialized olfactory lobe in the brain called the procerebral (PC) lobe. The PC lobe produces ongoing local field potential (LFP) oscillation, which is modulated by olfactory stimulation. We hypothesized that NO should be released in the PC lobe in response to olfactory stimulation, and to prove this, we applied an NO electrode to the PC lobe of the land slug Limax in an isolated tentacle-brain preparation. Olfactory stimulation applied to the olfactory epithelium transiently increased the NO concentration in the PC lobe, and this was blocked by the NO synthase inhibitor L-NAME at 3.7 mM. L-NAME at this concentration did not block the ongoing LFP oscillation, but did block the frequency increase during olfactory stimulation. Olfactory stimulation also enhanced spatial synchronicity of activity, and this response was also blocked by L-NAME. Single electrical stimulation of the superior tentacle nerve (STN) mimicked the effects of olfactory stimulation on LFP frequency and synchronicity, and both of these effects were blocked by L-NAME. L-NAME did not block synaptic transmission from the STN to the nonbursting (NB)-type PC lobe neurons, which presumably produce NO in an activity-dependent manner. Previous behavioral experiments have revealed impairment of olfactory discrimination after L-NAME injection. The recording conditions in the present work likely reproduce the in vivo brain state in those behavioral experiments. We speculate that the dynamical effects of NO released during olfactory perception underlie precise odor representation and memory formation in the brain, presumably through regulation of NB neuron activity.

Voltage-gated membrane currents in neurons involved in odor information processing in snail procerebrum

The procerebrum (PC) of the snail brain is a critical region for odor discrimination and odor learning. The morphological organization and physiological function of the PC has been intensively investigated in several gastropod species; however, the presence and distribution of ion channels in bursting and non-bursting cells has not yet been described. Therefore, the aim of our study was to identify the different ion channels present in PC neurons. Based on whole cell patch-clamp and immunohistochemical experiments, we show that Na(+)-, Ca(2+)-, and K(+)-dependent voltage-gated channels are differentially localized and expressed in the cells of the PC. Different Na-channel subtypes are present in large (10-15μm) and small (5-8μm) diameter neurons, which are thought to correspond to the bursting and non-bursting cells, respectively. Here, we show that the bursting neurons possess fast sodium current (I NaT) and NaV1.9-like channels and the non-bursting neurons possess slow sodium current (I NaT) and NaV1.8-like channels in addition to the L-type Ca(-), KV4.3 (A-type K-channel) and KV2.1 channels. We suggest that the bursting and/or non-bursting character of the PC neurons is at least partly determined by the battery of ion-channels present and their cellular and subcellular compartmentalization.

Coherency Evaluation of Spatiotemporal Neural Activities in the Molluscan Olfactory Center Applying Extracellular Recording with Wavelet Analysis

The procerebrum (PC) of the land slug Limax is the olfactory center involved in olfactory discrimination and learning. In the PC, an oscillation of local field potential (LFP) with 0.5 - 1 Hz is observed by electrophysiological extracellular recording. Additionally, spatiotemporal neural activities in the PC have been examined using optical recordings. However, extracellular recording is preferable to measure neural activities for a long time with a high speed, while it is not abundant in spatial information. In this study, we therefore attempted to elicit spatial information from extracellular recording. For this purpose, we evaluated spatial information included in the LFP compared with the spatiotemporal neural activities measured by the fluorescent voltage imaging. As a result, aversive odors induced the coherent spatiotemporal neural activities in the PC, and the increase in coherency was observed as a change in the LFP waveform. It was also evaluated as a decrease in entropy by analyzing the LFP oscillation patterns and wavelet analysis. Thus, although the LFP provides only one series of signals, the coherency of the spatiotemporal neural activities in the PC can be evaluated by applying extracellular recording with wavelet analysis.

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.

Nitric oxide synthase in the olfactory center of the snail, Helix pomatia. A light- and electronmicroscopic histochemical study

Event Abstract Back to Event Nitric oxide synthase in the olfactory center of the snail, Helix pomatia. A light- and electronmicroscopic histochemical study K. Nacsa1, K. Elekes1 and Z. Serfőző1* 1 Balaton Limnological Research Institute, Hungarian Academy of Sciences, Department of Experimental Zoology, Hungary Nitric oxide (NO) contributes to the generation of oscillatory waves which are related to the process of memory formation in the procerebrum (PC), the olfactory center of terrestrial snails. The anatomy of NO synthase (NOS) containing neurons is known very little in the PC. Therefore in this study we localized NOS using both NADPH-diaphorase (NADPH-d) histochemical reaction (NADPH-d) and NOS immunohistochemistry in the PC of Helix pomatia. Labeling alternate cryostat sections obtained from the PC revealed an identical NADPH-d and NOS localization; NOS was detected both in the globuli cell bodies and axons, and the different neuropil areas. A gradual from top to base appearance of NOS was seen in the terminal neuropil, whereas it was evenly and strongly expressed in the internal neuropil. A lateral neuropil region receiving NOS positive axon terminals both from the upper and lower tentacles was also intensively labeled. At ultrastructural level NADPH-d cytochemistry revealed that NOS was bound to the nuclear envelope and endoplasmic reticulum elements in the globuli cells, whereas it was attached to the membrane of agranular synaptic vesicles and medium electron dense granular vesicles in the neuropil. These observations refer to the synthesis of NO in globuli cells, and support the involvement of NO both in the process of transmitter release and intercellular signaling in the different neuropil regions. Acknowledgements Support: OTKA, Nos. 78224 (K.E.), PD75276 (Z.S.) Keywords: Sensory and motor activity, Neuroscience Conference: 13th Conference of the Hungarian Neuroscience Society (MITT), Budapest, Hungary, 20 Jan - 22 Jan, 2011. Presentation Type: Abstract Topic: Sensory and motor activity Citation: Nacsa K, Elekes K and Serfőző Z (2011). Nitric oxide synthase in the olfactory center of the snail, Helix pomatia. A light- and electronmicroscopic histochemical study. Front. Neurosci. Conference Abstract: 13th Conference of the Hungarian Neuroscience Society (MITT). doi: 10.3389/conf.fnins.2011.84.00003 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 03 Mar 2011; Published Online: 23 Mar 2011. * Correspondence: Dr. Z. Serfőző, Balaton Limnological Research Institute, Hungarian Academy of Sciences, Department of Experimental Zoology, Tihany, Hungary, serfozo@tres.blki.hu Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers K. Nacsa K. Elekes Z. Serfőző Google K. Nacsa K. Elekes Z. Serfőző Google Scholar K. Nacsa K. Elekes Z. Serfőző PubMed K. Nacsa K. Elekes Z. Serfőző Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Internal representation and memory formation of odor preference based on oscillatory activities in a terrestrial slug

The terrestrial slug Limax exhibits a highly developed ability to learn odors with a small nervous system. When a fluorescent dye, Lucifer Yellow (LY), is injected into the slug's body cavity after odor-taste associative conditioning, a group of neurons in the procerebral (PC) lobe, an olfactory center of the slug, is labeled by LY. We examined the relationships between conditioning strategies and LY labeling. The positions of LY-labeled neurons in the PC lobe after appetitive conditioning were more apical than those after aversive conditioning and did not depend on the conditioned odor, suggesting that the biological value of odors affected the position of LY-labeled neural clusters. A simple computational model consisting of two layers of oscillators with electrical synaptic interaction was constructed based on physiological features of the PC lobe, and showed that the oscillatory phase difference between the layers contributed to determination of the positions of LY-labeled neurons, suggesting that phase difference in oscillatory activity plays a role in the association of odors and the preference for them.

Spontaneous Recovery of the Injured Higher Olfactory Center in the Terrestrial Slug Limax

BackgroundOf all organs and tissues in adult mammals, the brain shows the most limited regeneration and recovery after injury. This is one reason why treating neurological damage such as ischemic injury after stroke presents such a challenge. Here we report a novel mode of regeneration which the slug's cognitive center, the procerebrum, shows after surgical lesioning in the adult. It is well known that the land slug Limax possesses the capacity to demonstrate conditioned food aversion. This learning ability critically depends on the procerebrum, which is the higher olfactory center in the brain of the terrestrial mollusk.Principal FindingsIn the present study, after a 1-month recovery period post-surgical lesioning of the procerebrum we investigated whether the brain of the slug shows recovery from damage. We found that learning ability, local field potential oscillation, and the number of cells in the procerebrum (PC) all recovered spontaneously within 1 month of bilateral lesioning of the PC. Moreover, neurogenesis was enhanced in the lesioned PC. However, memory acquired before the surgery could not be retrieved 1 month after surgery although the procerebrum had recovered from injury by this time, consistent with the notion that the procerebrum is the storage site of odor-aversion memory, or deeply involved in the memory recall process.SignificanceOur findings are the first to demonstrate that a brain region responsible for the associative memory of an adult organism can spontaneously reconstitute itself, and can recover its function following injury.

Unilateral memory storage in the procerebrum of the terrestrial slug Limax

In most sensory modalities, neuronal inputs are bilaterally processed in a higher center. In some animal species, however, functional lateralization is sometimes observed in the sensory processing at the higher level. For the terrestrial slug Limax, olfaction is the most important sensory modality and this slug can acquire odor-aversion memories. Previously, it has been demonstrated in bilateral PC ablation experiments that the procerebrum (PC) is necessary for odor-aversion memory, and that the PC is the memory storage site. On the other hand, it has been hypothesized that only the unilateral PC is used for odor-aversion learning. Here we demonstrated that the number of the slugs with intact memory performance was reduced by approximately 50% when the PC was surgically ablated only unilaterally before or after conditioning. There was no difference in the memory performance of the right vs. the left PC-ablated slugs. However, memory deficit from unilateral PC ablation was not observed when the ipsilateral tentacles were also amputated at the same time. We also showed that there was no lateral memory transfer from one PC to the other, after up to 7 days post-conditioning. Our results demonstrated clearly that either the left or right PC is randomly used for olfactory learning, and that the side of use is determined at the level of the olfactory ascending pathway to the PC.

A novel nitric oxide synthase expressed specifically in the olfactory center

Nitric oxide (NO) plays important roles in the olfactory center of various animals. In the terrestrial slug, NO is indispensable for field potential oscillation in the higher olfactory center, the procerebrum (PC), and also for odor learning. Here we identify a novel NO synthase (NOS) gene, limNOS2, in the terrestrial slug. The mRNA (∼10kb) of limNOS2 encodes a protein consisting of 1616 amino acids, including a PDZ domain. The protein has 70.0% sequence identity with the previously identified limNOS1 gene. In contrast to limNOS1, however, limNOS2 is expressed specifically in the PC. Moreover, most of the cells in the PC contain limNOS2 mRNA, indicating that the nonbursting neurons, the major constituent of the PC, have this mRNA. The expression pattern of limNOS2 conforms well to the pattern of NOS enzymatic histochemical staining. Our present findings indicate that limNOS2 is responsible for most of the NO generation in the PC.

Changes in frequency of spontaneous oscillations in procerebrum correlate to behavioural choice in terrestrial snails.

The aim of our study was to understand functional significance of spontaneous oscillations of local field potential in the olfactory brain lobe of terrestrial snail, the procerebrum (PC). We compared changes in frequency of oscillations in semi-intact preparations from snails trained to percept the same conditioned odor as positive (associated with food reinforcement) or negative (associated with noxious reinforcement). In vivo recordings in freely behaving naïve snails showed a significant decrease of spontaneous PC oscillations frequency during a stage of tentacle withdrawal to odor presentation. In in vitro preparations from naïve snails, a similar decrease in frequency of the PC oscillations to odor presentation was observed. Changes in frequency of the oscillations to cineole presentations in the “aversive” group of snails (demonstrating withdrawal) were much more pronounced than in naïve snails. No significant difference in responses to 5% and 20% cineole was noted. Changes in the spontaneous oscillations frequency in the snails trained to respond with positive reaction (approach) to cineole depended on the concentration of the applied odor, and these responses were qualitatively similar to responses of other groups during the first 10 s of responses to odor, but significantly different (increase in PC oscillations frequency) from the responses of the aversively trained and naïve snails in the interval 11–30 s, which corresponds to the end of the tentacle withdrawal and timing of decision making (approach or escape) in the free behaving snails. Obtained results suggest that frequency of the PC lobe spontaneous oscillations correlate to the choice of behavior in snails: withdrawal (decrease in frequency) or approach (increase in frequency) to the source of odor.

Specific spatio-temporal activities in the cerebral ganglion of Incilaria fruhstorferi in response to superior and inferior tentacle nerve stimulation

In terrestrial gastropod mollusks (slugs and snails), olfaction is the dominant sensory modality guiding various kinds of behavior. Anatomical studies indicate that olfactory information is processed in the brain (the cerebral ganglion) in two lobes in particular: the procerebrum (PC) and the metacerebrum (MtC). This implies that olfactory functions emerge from simultaneous and cooperative processing in the PC and the MtC. However, no previous physiological study has investigated the activity in these two lobes simultaneously. In the present study, the activity evoked by electrical stimulation of the olfactory nerves, the superior and inferior tentacle nerves, was recorded optically from the whole cerebral ganglion of the terrestrial slug, Incilaria fruhstorferi. The results indicated that the evoked activity in the PC and the MtC showed two specific spatio-temporal patterns. First, when either set of nerves was stimulated, the activity of the medial neuropilar region of the MtC (the mMtC) always preceded the activity in the PC. Second, stimulation of the superior tentacle nerves activated the medial and lateral halves of the mMtC almost evenly, whereas stimulation of the inferior tentacle nerves activated the lateral half of the mMtC more strongly than the medial half. These results suggest that the activated region of the mMtC plays an important role in olfactory processing, especially with respect to the functional differences between the superior and inferior tentacles.

Recovery of learning ability after the ablation of the procerebrum in the terrestrial slug,Limax Valentianus

The procerebrum (PC) is indispensable for odor-aversion learning in Limax. On the other hand, the central nervous system (CNS) of some Pulmonata shows robustness against injury, recovering from nerve injury both at the histological and functional levels. To investigate whether the PC of Limax also shows robustness against nerve injury, we tested whether or not the slugs can acquire and retrieve odor-aversion memory after a long recovery period from PC ablation. When the recovery period is short (7 days), the PC-ablated slugs failed to avoid the conditioned odor. But when the recovery period is long (1 month), the PC-ablated slugs successfully avoided the conditioned odor. These results indicate that the CNS including the PC can recover from injury at least at the functional level.

Selective calcium imaging of olfactory interneurons in a land mollusk

The procerebrum (PC) of the land slug Limax is the olfactory center involved in olfactory discrimination and learning. The PC contains two types of neurons, bursting (B) and nonbursting (NB) neurons. B neurons are local interneurons and show spontaneous bursting while NB neurons directly receive input from olfactory nerve fibers, and are possibly involved in odor coding. In order to observe activity of NB neurons, selective calcium imaging was made by retrograde staining of NB neurons with the calcium sensitive dye, rhod-2 AM, from the internal mass (IM) of the PC. The stained cells, presumably NB neurons, appeared to be mostly silent, but showed transient calcium elevation in response to electrical stimulus to the tentacle nerve. Stimulation of the tentacle with an odorant also evoked calcium events in some of the stained cells. These included one or multiple calcium events, and these events were synchronized with each other. These results revealed characteristics of the activity of NB neurons.

The procerebrum is necessary for odor-aversion learning in the terrestrial slug Limax valentianus

The terrestrial slug Limax has a highly developed ability to associate the odor of some foods (e.g., carrot juice) with aversive stimuli such as the bitter taste of quinidine solution. The procerebrum (PC) is a part of the slug's brain thought to be involved in odor-aversion learning, but direct evidence is still lacking. Here we present evidence showing that the PC is essential for odor-aversion learning. Unlike sham-operated slugs, PC ablation 7 d prior to conditioning showed that most slugs did not avoid carrot juice in the memory retention test conducted 24 h after the conditioning. Slugs with the PC ablated 3 h, 1 d, 3 d, or 7 d after conditioning and examined by the memory retention test at 3 d after the PC ablation were also less likely to avoid carrot juice than sham-operated slugs. The PC ablation did not damage the ability of the slugs to sense attractive odor (everyday food) or innately aversive odor (onion or garlic). These results demonstrate that the PC is a necessary component in the retention and/or retrieval of odor-aversion memory.

In Vitro Odor-Aversion Conditioning in a Terrestrial Mollusk

We developed an in vitro odor-aversion conditioning system in the terrestrial mollusk, Limax, and found a behavioral correlate of network oscillation in the olfactory CNS. We first examined the odor-induced behavior of Limax, after odor-aversion conditioning in vivo. Shortening of mantle muscles was specifically observed in response to aversively conditioned odors. We previously identified that parietal nerves, which project to the mantle muscle in Limax, regulate shortening of the mantle muscle. We therefore isolated whole brains containing noses (sensory organs) and parietal nerves (motor output), and applied an odor-aversion conditioning paradigm to these in vitro preparations. Before the in vitro conditioning, application of attractive odors to the noses did not elicit any discharge in the parietal nerves. However, after odor-aversion conditioning, discharges in the parietal nerves were observed in response to the natively attractive but aversively conditioned odors. We also found that network oscillation frequency in the procerebrum (PC), the olfactory CNS of Limax, increased specifically in response to the aversively conditioned odors that elicited avoidance behavior. In naive (nonconditioned) preparations, increases in the PC oscillation frequency were observed specifically in response to innately aversive odors. These results indicate that the isolated brains have an ability of odor learning. They also suggest that changes in PC network oscillation are associated with aversively conditioned and innately aversive odors, both of which elicit avoidance behavior. This in vitro conditioning system would be an effective approach for exploring the neural mechanism to determine the aversion to odors.

Optical recording of oscillatory neural activities in the molluscan brain

The procerebrum (PC) of the terrestrial slug shows a coherent oscillatory activity. Information is encoded in the PC by neurons with synchronized oscillatory activity, and the oscillatory activity will propagate to other brain regions as the PC transmits information. Using the optical recording of membrane potentials and the correlation analysis, we showed that the metacerebrum/mesocerebrum (MC) region also shows an oscillatory activity coherent with that of the PC. The MC oscillation was either inphase or antiphase with the PC oscillation, and its amplitude was larger when it was antiphase than it was inphase. These results indicate that the MC is capable of producing an oscillatory activity, possibly driven by synaptic input from the PC.

Odor-evoked responses in the olfactory center neurons in the terrestrial slug.

The procerebrum (PC) of the terrestrial mollusk Limax is a highly developed second-order olfactory center consisting of two electrophysiologically distinct populations of neurons: nonbursting (NB) and bursting (B). NB neurons are by far the more numerous of the two cell types. They receive direct synaptic inputs from afferent fibers from the tentacle ganglion, the primary olfactory center, and also receive periodic inhibitory postsynaptic potentials (IPSPs) from B neurons. Odor-evoked activity in the NB neurons was examined using perforated patch recordings. Stimulation of the superior tentacle with odorants resulted in inhibitory responses in 45% of NB neurons, while 11% of NB neurons showed an excitatory response. The specific response was reproducible in each neuron to the same odorant, suggesting the possibility that activity of NB neurons may encode odor identity. Analysis of the cycle-averaged membrane potential of NB neurons revealed a correlation between the firing rate and the membrane potential at the plateau phase between IPSPs. Also, the firing rate of NB neurons was affected by the frequency of the IPSPs. These results indicate the existence of two distinct mechanisms for the regulation of NB neuron activity.

Contribution of Excitatory Chloride Conductance in the Determination of the Direction of Traveling Waves in an Olfactory Center

Traveling waves have been found in the CNS of vertebrates and invertebrates. In the olfactory center [procerebrum (PC)] of the terrestrial slug Limax, periodic waves travel from the apex to the base with a frequency of approximately 0.7 Hz. The oscillation and propagation of waves have been thought to be mediated by the mutual connections of bursting neurons in the PC. The direction of the wave is Cl(-) dependent, because lowering the Cl(-) concentration in the medium reverses the direction. The bursting neurons have a Cl(-) channel-coupled glutamate receptor (GluClR), and, using a calcium imaging technique, the receptor was found to be excitatory. Activation of the GluClR with its selective agonist ibotenate resulted in an increased frequency of the oscillatory neural activity recorded as a periodic local field potential. Depletion of cytoplasmic Cl(-) with Cl(-)-free saline abolished all of the ibotenate-induced effects. Perforated-patch-clamp recording in single PC neurons revealed a spatial difference in the Cl(-)-dependent periodic depolarizations in the bursting neurons, with a higher amplitude in the apical region. These results suggest the involvement of excitatory GluClRs in the unidirectional propagation of waves in the PC.