Studies of neuronal network emergence during sensory processing and motor control are greatly facilitated by technologies that allow us to simultaneously record the membrane potential dynamics of a large population of neurons in single cell resolution. To achieve whole-brain recording with the ability to detect both small synaptic potentials and action potentials, we developed a voltage-sensitive dye (VSD) imaging technique based on a double-sided microscope that can image two sides of a nervous system simultaneously. We applied this system to the segmental ganglia of the medicinal leech. Double-sided VSD imaging enabled simultaneous recording of membrane potential events from almost all of the identifiable neurons. Using data obtained from double-sided VSD imaging, we analyzed neuronal dynamics in both sensory processing and generation of behavior and constructed functional maps for identification of neurons contributing to these processes.

Operant discrimination learning has been extensively utilized in the study on the perceptual ability of animals and their higher-order brain functions. We tested in this study whether American lobster Homarus americanus, which was previously found to possess ability of operant learning with claw gripping, could be trained to discriminate light stimuli of different intensities. For the current purpose, we newly developed a PC-controlled operant chamber that allowed the animal under a body-fixed condition to perform operant reward learning with claw gripping. Lobsters were first reinforced when they gripped the sensor bar upon presentation of a light cue. Then they were trained to grip the bar only when the light stimulus of a specific intensity was presented to obtain food reward while the stimuli of three different intensities including the reinforced one were presented in a random order. Finally, they were re-trained to grip the bar only when the light stimulus of another intensity that was not rewarded in the preceding training to obtain food while other intensities including the one that was rewarded previously were not rewarded any more. In these training procedures, the operant behavior occurred more frequently in response to the rewarded cue than to the non-rewarded one. The action latency for the reinforced stimuli showed a significant decrease in the course of training.These data demonstrate that lobsters can be trained with the light cues of different intensity as discriminative stimuli under a restrained condition that would allow application of electrophysiological techniques to the behaving subjects.-3-

Operant conditioning is a common tool for studying cognitive aspects of brain functions. As the first step toward understanding those functions in simple invertebrate microbrains, we tested whether operant conditioning could be applied to train American lobster Homarus americanus that has been extensively adopted as an animal model for neurophysiological analyses of nervous system functions and behavioral control. The animal was trained by food rewarding for gripping of a sensor bar as the operant behavior. Lobsters were first reinforced when they acted on the bar with a stronger grip than a pre-set value. After this reinforcement, the animal learnt to grip the bar for food pellets. The yoked control experiment in which the animal received action-independent reinforcement excluded the possibility of pseudoconditioning that the food simply drove the animal to frequent gripping of the sensor bar. The association of the bar grip with food was extinguished by rewarding nothing to the operant behavior, and was restored by repeating the reinforcement process as before. In addition, lobsters successfully carried out differential reinforcement regarding the gripping force: their gripping force changed depending on the increased force threshold for food reward. These data demonstrate that lobsters can be trained by operant conditioning paradigms involving acquisition and extinction procedures with the precise claw gripping even under the force control.

In this protocol, we introduce an effective method for voltage-sensitive dye (VSD) loading and imaging of leech ganglia as used in Tomina and Wagenaar (2017). Dissection and dye loading procedures are the most critical steps toward successful whole-ganglion VSD imaging. The former entails the removal of the sheath that covers neurons in the segmental ganglion of the leech, which is required for successful dye loading. The latter entails gently flowing a new generation VSD, VF2.1(OMe).H, onto both sides of the ganglion simultaneously using a pair of peristaltic pumps. We expect the described techniques to translate broadly to wide-field VSD imaging in other thin and relatively transparent nervous systems.

Animals generally exhibit circadian rhythms of locomotor activity. They initiate locomotor behavior not only reflexively in response to external stimuli but also spontaneously in the absence of any specific stimulus. The neuronal mechanisms underlying circadian locomotor activity can, therefore, be based on the rhythmic changes in either reflexive efficacy or endogenous activity. In crayfish Procambarus clarkii, it can be determined by analyzing electromyographic (EMG) patterns of walking legs whether the walking behavior is initiated reflexively or spontaneously. In this study, we examined quantitatively the leg muscle activity that underlies the locomotor behavior showing circadian rhythms in crayfish. We newly developed a chronic EMG recording system that allowed the animal to freely behave under a tethered condition for more than 10 days. In the LD condition in which the animals exhibited LD entrainment, the rhythmic burst activity of leg muscles for stepping behavior was preceded by non-rhythmic tonic activation that lasted for 1323 ± 488 msec when the animal initiated walking. In DD and LL free-running conditions, the pre-burst activation lasted for 1779 ± 31 and 1517 ± 39 msec respectively. In the mechanical stimulus-evoked walking, the pre-burst activation ended within 79 ± 6 msec. These data suggest that periodic changes in the crayfish locomotor activity under the condition of LD entrainment or free-running are based on activity changes in the spontaneous initiation mechanism of walking behavior rather than those in the sensori-motor pathway connecting mechanoreceptors with leg movements.