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Calcium release from intracellular stores has various actions in neurons, but its effects on network oscillation have not been well understood. The olfactory center (procerebrum, PC) of the terrestrial slug Limax valentianus shows a regular oscillation in the local field potential (LFP). Here we report that caffeine, which is an agonist for ryanodine receptors and triggers calcium release from intra-cellular stores, has strong modulatory effects on the PC. In isolated PC neurons, caffeine enhanced the cytoplasmic calcium concentration, and this was blocked by ryanodine. Caffeine elevated the frequency and amplitude of the LFP oscillation, which was also blocked by ryanodine. The time lag between the frequency and amplitude effects suggests distinct mechanisms for the modulation of these two parameters. These results suggest that calcium release from intracellular stores through ryanodine receptors activates network activity in the PC.

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.

In many animals, neurons in the olfactory system have been shown to respond not only to odorants but also to air movements. However, the manner in which the mechanical dynamics of odor stimulation affect olfactory processing remains poorly understood. Using a series of flow rates and odor concentrations from clean air to high-concentration vapors, we systematically analyzed the effects of air movement and odor concentration on olfactory processing. We extracellularly recorded local field potentials and spike units from the olfactory epithelium (OE) and tentacular nerve (TN), which connects the first and second relay centers of olfactory information, in the terrestrial slug Limax marginatus. We found that clean air puffs at a flow rate of 0.18 ml/s (gentle wind), but not high-concentration odor puffs at lower flow rates, induced electro-olfactogram (EOG) oscillations in the OE with a constant frequency (2.5 Hz), regardless of the odor. Surgically isolated OE preparations also showed these EOG oscillations, indicating that the oscillations arose from the OE independently of the downstream circuits. The EOG oscillations entrained the slower spontaneous TN oscillations (1-2 Hz) to the fixed rhythm (2.5 Hz). Spontaneous and odor-evoked units were phase-locked to the TN oscillation peaks. This TN oscillation entrainment by the EOG oscillations caused stronger phase-locking, specifically TN oscillation peaks and EOG oscillation troughs. Taken together, these results suggest that when odors are carried by a gentle wind, the air movement induces EOG oscillations and modulates rhythmic spike patterning of olfactory outputs to the second olfactory relay center in Limax.

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

Sensory systems show behavioral state-dependent gating of information flow that largely depends on the thalamus. Here we examined whether the state-dependent gating occurs in the central olfactory pathway that lacks a thalamic relay. In urethane-anesthetized rats, neocortical EEG showed a periodical alternation between two states: a slow-wave state (SWS) characterized by large and slow waves and a fast-wave state (FWS) characterized by faster waves. Single-unit recordings from olfactory cortex neurons showed robust spike responses to adequate odorants during FWS, whereas they showed only weak responses during SWS. The state-dependent change in odorant-evoked responses was observed in a majority of olfactory cortex neurons, but in only a small percentage of olfactory bulb neurons. These findings demonstrate a powerful state-dependent gating of odor information in the olfactory cortex that works in synchrony with the gating of other sensory systems. They suggest a state-dependent switchover of signal processing modes in the olfactory cortex.

The digit-like extensions (the digits) of the tentacular ganglion of the terrestrial slug Limax marginatus are the cell body rich region in the primary olfactory system, and they contain primary olfactory neurons and projection neurons that send their axons to the olfactory center via the tentacular nerves. Two cell clusters (the cell masses) at the bases of the digits form the other cell body rich regions. Although the spontaneous slow oscillations and odor responses in the tentacular nerve have been studied, the origin of the oscillatory activity is unknown. In the present study, we examined the contribution of the neurons in the digits and cell masses to generation of the tentacular nerve oscillations by surgical removal from the whole tentacle preparations. Both structures contributed to the tentacular oscillations, and surgical isolation of the digits from the whole tentacle preparations still showed spontaneous oscillations. To analyze the dynamics of odor-processing circuits in the digits and tentacular ganglia, we studied the effects of gamma-aminobutyric acid, glutamate, and acetylcholine on the circuit dynamics of the oscillatory network(s) in the peripheral olfactory system. Bath or local puff application of gamma-aminobutyric acid to the cell masses decreased the tentacular nerve oscillations, whereas the bath or local puff application of glutamate and acetylcholine to the digits increased the digits' oscillations. Our results suggest the existence of two intrinsic oscillatory circuits that respond differentially to endogenous neurotransmitters in the primary olfactory system of slugs., (Copyright 2004 Wiley Periodicals, Inc.)

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., (Copyright 2003 Wiley Periodicals, Inc. J Neurobiol 58: 369-378, 2004)

To explore neural mechanisms how olfactory information is processed in the brain and finally converted into behavior, it would be useful to have isolated whole brains that include both olfactory organs and motor output. In the present study, we identified an in vitro index of odor-evoked behavior in the terrestrial mollusk Limax and also studied the modulation of this in vitro index of the behavior. We determined that shortening of the mantle muscles is one of the withdrawal responses selectively induced by aversive odors and that the shortening is mediated by a pair of parietal nerves. We also identified a motoneuron (named the posterior visceral neuron, p-VN) that projects to the parietal nerve and innervates the mantle muscles. When we applied various odors to the nose in these isolated molluscan brains, only aversive odors induced discharges in the p-VN. These results indicate that p-VN discharges can serve as an in vitro index of odor-induced aversive behavior. We also identified a novel serotonergic neuron (named the posterior cerebral serotonergic cell, p-CSC). Discharges in the p-CSC released serotonin to the tentacle ganglion (TG); serotonin in the TG then inhibited odor-induced discharges in the p-VN, the in vitro index of aversive behavior. These results suggest that the serotonergic system is involved in the regulation of approach and avoidance behavior in Limax.

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

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

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.

We identified two classes of network oscillations with different frequency ranges in the tentacle ganglion (TG), the primary olfactory center of the terrestrial mollusk Limax marginatus, and investigated the responses of these oscillations to odor inputs. A recent study indicated that there are serotonergic terminals in the TG. We found that when serotonin was applied to the TG, the spontaneous network oscillation of about 1.5 Hz in the TG changed its oscillatory frequency to 0.5 Hz. These two oscillations are distinct, because 1) in most cases, the application of serotonin to the TG initially inhibited the 1.5-Hz oscillation and subsequently generated the slow 0.5-Hz oscillation; and 2) occasionally, the application of serotonin did not inhibit the spontaneous 1.5-Hz oscillation, resulting in the coexistence of two network oscillations. Thus the TG has two different oscillatory dynamics. We named the spontaneous 1.5-Hz oscillation the fast oscillation (FO), and the serotonin-induced 0.5-Hz oscillation the slow oscillation (SO). By calculating the spatial coherence of the TG oscillations, we found that the FO is a noncoherent oscillatory mode and the SO is a coherent oscillatory mode. Finally, odor presentation to the olfactory receptors selectively modulated the SO by decreasing the oscillatory amplitude, but the FO was not modulated by the odor input. These results indicate that 1) the TG has two oscillatory states (FO and SO) and these states are changed by the extracellular level of serotonin, and 2) these two oscillatory states have different responses to odors.

Synchronous oscillations in olfactory systems have been thought to play critical roles in encoding olfactory information. However, their role in determining behavior is unknown. As a first step toward understanding the decoding process of coherent oscillation, we looked for a neuron in the terrestrial slug Limax marginatus that receives output signals from the procerebrum (PC), which is the olfactory center of Limax. We identified a neuron in the metacerebrum that extends its neurites into both the PC and the metacerebrum, and named it the metacerebro-procerebral neuron (MPN). The MPN exhibited a membrane potential oscillation that was synchronous with the local field potential oscillation in the PC. When we cut the PC off, the membrane potential oscillation of the MPN disappeared. Numerous varicosities were found on the neurites in the metacerebrum, while no varicosities were found on the neurites inside the PC. From these morphological and physiological results, we conclude that the MPN is an output neuron from the PC. The MPN also receives monosynaptic inputs from the superior and inferior tentacle nerves. The MPN thus may receive olfactory information from two pathways, one directly from the sensory organ and the other by way of the PC, possibly functioning to integrate them.

Neural oscillations have been found to occur in the olfactory centers of some vertebrates and invertebrates, including the procerebrum of the terrestrial slug Limax marginatus. Using optical recording with the potential-sensitive dye di-4-ANEPPS, we analyzed the spatiotemporal pattern of procerebral neural activities in response to odorants applied to an in vitro brain-superior tentacle preparation. The odor of rat chow, on which the slugs were normally fed, increased the frequency of the oscillation. Garlic odor, which induced aversive behavior in the slug, caused a transient increase in oscillation frequency during stimulation, followed by a second increase in oscillation frequency when the stimulus was terminated. Wave propagation from the distal to the proximal region of the procerebrum was accelerated in parallel with modulation of the frequency. The cycle-by-cycle average of the optical signals showed that a large area of the cerebral ganglia, including the procerebrum, was depolarized during the initial increase in frequency. During the second increase, however, the net depolarization was most prominent in the terminal mass of the procerebrum. These results suggest that the level of depolarization generated by interactions among the neurites projecting to the terminal mass, such as the neurites of the nonbursting neurons, may control neural oscillations in the procerebrum.

We studied the oscillatory properties and the olfactory responses of the procerebral (PC) lobe of the cerebral ganglion in the terrestrial mollusc Limax marginatus. The PC lobe, a central olfactory organ in Limax, is a highly interconnected network of local interneurons that receives olfactory inputs from the inferior and superior tentacular noses. We used an optical recording technique with a voltage-sensitive dye to record the activity of the PC lobe from either the posterior or the dorsal surface. The recordings revealed that almost all PC interneurons showed spontaneous oscillatory activities that had been entrained with each other. Upon presentation to the nose of odors to which the slugs had been aversively conditioned, the basal level of the oscillation changed biphasically. In the early phase of the response, depolarization in the basal level of the oscillation occurred in one or more belt-shaped regions parallel to the dorsoventral axis. In the late phase of the response, hyperpolarization of basal potential level of the PC lobe oscillations occurred in a wider area. Such spatial and temporal modulation was not observed when the unpaired control odors were presented to the preparation, whereas the same preparations responded to the aversively conditioned stimuli. Thus, it was considered that the spatial and temporal response in the basal level of oscillation was specific to the aversively conditioned odors. Furthermore, the spatial pattern of the depolarization modulation in the early phase was repeatable in multiple trials performed using the same odor, although different odors produced different spatial patterns of the modulation. From these results, we conclude that in the PC lobe learned odors are represented as spatial and temporal activity patterns of oscillators that constitute a coherent network.

We compared behaviorally and physiologically the olfactory responses of slugs (Limax marginatus) that had been subjected to aversive, appetitive, or unpaired training with food odors (carrot or cucumber). In the aversive training, the slugs were exposed to the food odor as a conditioned stimulus (CS), and then quinidine sulfate solution as an unconditioned stimulus (UCS) was immediately applied to the lip of the slugs. This training caused a decrease in preference level for the CS. The unpaired training, in which the CS and the UCS were presented to the slugs with a 5-min interval, induced no change in the preference level for the CS. In the appetitive training, the slugs were allowed to eat the CS odor source without UCS application. When we used nonstarved slugs, it was found that the preference level for the CS increased upon the appetitive training. These results indicate that each training changed the preference for the odors in a characteristic manner. In the physiological experiments, we used brain-inferior tentacular nose preparations isolated from slugs and investigated the olfactory responses of the oscillations in the local field potential (LFP) of the procerebral (PC) lobe. We found that odor presentation induced various types of changes in the LFP oscillation frequency, although the rate of occurrence of the frequency modulation differed between odors used in the aversive and the unpaired training (aversive-conditioned and unpaired odors). The aversive-conditioned odors induced a decrease in the oscillatory frequency. Unpaired odors did not change it. Moreover, odors used in the appetitive training (appetitive-conditioned odors) induced an increase in the frequency. Thus, it was considered that those modulations of PC lobe oscillatory activity were independent of odor and reflected learned preference for odors.