Tatsuhiko Sekiguchi

Effects of electrical stimulation of the tentacular digits of a slug upon the frequency of electrical oscillations in the procerebral lobe

To find the primary mechanism for the frequency changes of electrical oscillations in the procerebral (PC) lobe of a slug, we electrically stimulated the tip, middle and basal regions of the digits of the superior and inferior tentacles and recorded the local field potentials from the PC lobe. Stimuli to the middle and basal regions of the digits of the inferior tentacle significantly decreased the frequency of electrical oscillations in the PC lobe, whereas those to the tip regions of the digits of the inferior tentacle and all regions of the digits of the superior tentacle increased it. These findings suggest that the change in the frequency of electrical oscillations in the PC lobe depends on the excited region in the digits, providing the first presentation of the physiological difference in the olfactory function between the superior and inferior tentacles.

Cooling-induced retrograde amnesia reflexes Pavlovian conditioning associations in Limax flavus

The relationships between cooling-induced retrograde amnesia and associations in Pavlovian conditioning in the terrestrial mollusk Limax flavus were studied. In the first experiment, the slugs were conditioned to avoid carrot odor and the experimental conditions required for amnesia induction were studied. Memory reactivation before cooling was found to be necessary for amnesia induction and the induced amnesia was selective for the reactivated memory. In the subsequent experiments, slugs were conditioned to avoid both carrot and cucumber odors using one of three Pavlovian conditioning paradigms, namely two-independent first-order conditioning, phase-2-sequential second-order conditioning and phase-2-simultaneous second-order conditioning, after which amnesia was induced by cooling immediately after presentation of one of the conditioning odors. The amnesia pattern induced differed depending upon the conditioning procedure used, which indicated that amnesia induction was related closely to stimulus associations in slugs. The possible role of cooling-induced retrograde amnesia as a tool for studying memory associations is also discussed.

Properties of Wave Propagation in the Oscillatory Neural Network in Limax marginatus

Abstract The olfactory center (procerebral lobe; PC lobe) of the terrestrial slug, Limax marginatus, shows oscillatory behavior with a frequency of about 1 Hz and an activity wave that propagates from the apical to distal regions of the PC surface. To study the dynamic properties of this oscillatory network, serotonin, glutamate and acetylcholine were applied to the PC lobe. Serotonin and acetylcholine increased the frequency of PC oscillation and decreased the velocity of wave propagation. The effect of serotonin on the frequency was long-lasting and there was a delay before it caused a decrease in the wave propagation velocity. In contrast, the effect of acetylcholine on the frequency was phasic, and no delay was observed. Glutamate first decreased, then increased, the frequency. However, specific changes in the wave propagation velocity were not observed. From these experimental results, it is suggested that the oscillatory neural network of PC lobe has a potential to represent odor information as a series of spatially and temporally distributed ensembles of coherent firing neurons.

Contributions of Superior and Inferior Tentacles to Learned Food-Avoidance Behavior in Limax marginatus

Abstract We investigated learned food-avoidance behavior of conditioned slugs in detail, and examined the effect of removing the inferior or superior tentacles (ITs and STs) on the avoidance behavior. The conditioning procedure significantly lowered the preference level of slugs to the conditioned odor, and the decrease of the preference was maintained for at least 48 hr. Video analysis experiments showed that change in the crawling direction during an aversion response occurred consistently accompanied by an average of 1.2 head swings. The first head swing was usually observed within 1 cm from the conditioned odor. Removal of the ITs after conditioning reversed the trend towards a lowered preference level. On the other hand, removal of the STs did not change the level. These results suggest that the IT inputs are needed to induce conditioned behavior. The contribution of STs to the conditioning behavior is indicated by the significant increase in the average number of head swings (3.6 swings) in a trial after their removal. In some trials, the slugs lacking STs avoided the odor source after crossing it. These observations indicate that removal of STs decreases the orientation ability of conditioned slugs.

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 slugs 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.

Aversive Conditioning to a Compound Odor Stimulus and Its Components in a Terrestrial Mollusc

Abstract To understand the perception of an odor mixture by the slug Limax marginatus, a mixture of two odors (carrot and cucumber) was used to condition the slugs, and internal representation of the odor mixture and its components was determined by cooling-induced retrograde amnesia. Slugs conditioned with the odor mixture showed aversive behavior, not only towards the mixture, but also towards the individual odor components. When the conditioned slugs were cooled after presentation of one of the odor components, odor preferences for both components recovered, suggesting that the slugs perceived the odor mixture as an entity. However, when the slugs were exposed to the components of the odor mixture after conditioning with the mixture, cooling treatment resulted in amnesia, which was specific towards the odor presented before the cooling treatment. This suggests that slugs exposed to odor components after conditioning were able to recognize the odor components individually. Thus, slugs learn a binary mixture as an entity as long as they have no experience about the individual components of the mixture. These results are discussed in relation to other conditioning strategies, such as second-order conditioning or blocking, where a mixture of cues is used.

Controlling the Orientation of Purple Membrane Fragments on an Air/Water Interface by a New Method of Direct Electric Field Application during Purple Membrane Spreading

The orientation of purple membrane (PM) fragments on an air/water interface was controlled by a direct electric field applied across the interface during spreading of a PM solution. We call this the direct electric field application Langmuir-Blodgett (LB) technique, and found it effective in increasing the population of preferentially oriented PM fragments at the interface. Using this technique with an electric field exceeding 4 kV/cm and an initial coverage C i of 0.2 (i.e., the ratio of the surface area of the air/water interface covered by PM fragments to the total surface area of the interface before the PM was spread over the interface), we produced a PM LB film in which all the constituent PM fragments were preferentially oriented. We successfully demonstrated that this technique is a powerful tool for producing LB films in which proteins are perfectly oriented (i.e., all the constituent PM fragments are preferentially oriented).

Determination of the amount of native structural bacteriorhodopsin in purple membrane Langmuir-Blodgett films by a spectroscopic surface denaturation quantifying technique

Purple membrane (PM) shows denaturation when spread over an air/water interface. We established a technique, which we call the spectroscopic surface denaturation quantifying (SSDQ) technique, that uses infrared linear dichroism to determine the amount of native structural bacteriorhodopsin (BR) in PM Langmuir-Blodgett (LB) films. Using the SSDQ technique we found that the conformational change after surface denaturation of BR was the same as that caused by ethanol treatment. By extrapolating the data of the amount of non-denatured BR molecules in PM LB films vs. the area of a single BR molecule on an air/water interface, we also found that the surface area of a single non-denatured BR molecule was 11.5 nm2, which is consistent with that determined by high-resolution electron cryo-microscopy and electron diffraction (EMD). These results demonstrate that the SSDQ technique is effective in quantifying the amount of native structural BR in PM LB films. The SSDQ technique is also applicable to other types of protein consisting of alpha-helical conformation.

Limited rotational diffusion caused by frictional interaction among purple membrane fragments in a highly concentrated purple membrane solution spread over a water surface

Purple membrane (PM) fragments spread over an air-water interface show both preferential orientation and reverse orientation (i.e., the cytoplasmic side of bacteriorhodopsin being oriented toward the water and toward the air, respectively). We used a spectroscopic surface denaturation quantifying technique and a current-measurement technique to evaluate the ratio of the amount of preferentially oriented bacteriorhodopsin (BR) to the amount of all BR in a PM film. This ratio decreased as the initial coverage (the ratio of the total area of all the PM fragments spread on the air-water interface to the area of the air-water interface before the PM was spread over the interface) increased, when the initial coverage was high (more than 0.3). We concluded that this decrease was due to the limited rotational diffusion caused by frictional interaction among PM fragments in a highly concentrated PM solution spread over a water surface.

Simulation of the oscillatory potential propagation in the slug’s brain with an array of nonlinear oscillators

We simulate an oscillatory propagating waves in the olfactory center (procerebrum:PC) of a terrestrial slug Limax marginatus with an array of nonlinear oscillators. We search the conditions where the entrained waves are constantly propagating from one terminal of the array to the other one. As regards the interaction of the oscillators, the inputs which are proportional to the differences between amplitudes of oscillators next to each other are needed for constant propagation. In addition to the condition, the oscillators are necessary to be arranged regularly according to their proper frequencies. We can simulate the PC with an array of nonlinear oscillators in the way which is consistent with the morphological and physiological measurements such as gap junctions or frequency gradient.