Masaki Sakai

Morphological and Functional Recovery of the Planarian Photosensing System during Head Regeneration

Abstract When exposed to light, planarians display a distinctive light avoidance behavior known as negative phototaxis. Such behavior is temporarily suppressed when animals are decapitated, and it is restored once the animals regenerate their heads. Head regeneration and the simple but reproducible phototactic response of planarians provides an opportunity to study the association between neuronal differentiation and the establishment of behavior in a simple, experimentally tractable metazoan. We have devised a phototaxis assay system to analyze light response recovery during head regeneration and determined that light evasion is markedly re-established 5 days after amputation. Immunohistological and in situ hybridization studies indicate that the photoreceptors and optic nerve connections to the brain begin by the fourth day of cephalic regeneration. To experimentally manipulate the light response recovery, we performed gene knockdown analysis using RNA interference (RNAi) on two genes (1020HH and eye53) previously reported to be expressed at 5 days after amputation and in the dorso-medial region of the brain (where the optic nerves project). Although RNAi failed to produce morphological defects in either the brain or the visual neurons, the recovery of the phototactic response normally observed in 5-day regenerates was significantly suppressed. The data suggest that 1020HH and eye53 may be involved in the functional recovery and maintenance of the visual system, and that the phototaxis assay presented here can be used to reliably quantify the negative phototactic behavior of planarians.

Differential staining of insect neurons with nickel and cobalt

Abstract Pairs of neurons were differentially stained by intracellular injection of the divalent cations, nickel and cobalt. Pairs of neuronal tracts could be similarly labelled following immersion of their axon bundles in the markers described. Use of nickel and a 4 : 6 mixture of cobalt and nickel according to the protocols presented here revealed the morphology of cricket neuron processes as thin as 1 μm in diameter and yielded a favourable contrast between the cells marked, as well as with respect to the background of unstained tissue.

Copulation sequence and mating termination in the male cricket Gryllus bimaculatus DeGeer

Abstract Mating in the male cricket Gryllus bimaculatus proceeds as a result of a stimulus-esponse chain. The final act, spermatophore extrusion, is elicited by mechanical stimulation of small sensilla in the cavity enclosed by the epiphallus. In normal copulation, stimulation for this seems to be made by insertion of the females copulatory papilla into the cavity. To induce spermatophore extrusion efficiently, the bodily tonus had to be heightened, eliciting male copulatory actions by giving key stimuli to the body surface in a sexually excited state. Mating termination, which is defined as spermatophore protrusion, is not initiated by peripheral feedback from the genitalia but is apparently due to some central events in the terminal abdominal ganglion. Control experiments with many adverse stimuli confirm that mimetic stimulation can induce mating termination normally as in natural copulation. Some case in which the male cycle is renewed after spermatophore extrusion without genitalia coupling are also described.

Behaviorally significant immobile state of so-called thanatosis in the cricket Gryllus bimaculatus DeGeer: its characterization, sensory mechanism and function

We have found that the cricket Gryllus bimaculatus shows an immobile posture, so-called thanatosis. Thanatosis was reflexly elicited by gently holding the forelegs and pronotum of the cricket. During thanatosis, the respiration rate decreased markedly while the heart rate doubled compared with the resting state. Animals in the state of extreme rigidity were unresponsive to the external disturbances but easily aroused by mechanical stimulation such as prodding. The immobile posture usually persisted for 2–4 min, but occasionally for more than 20 min, and then suddenly ended. Catalepsy was induced during thanatosis when a leg was passively forced to the extended position. For elicitation of the flexion reflex, either campaniform sensilla and femoral chordotonal organs (FCOs) in the forelegs, and spine-like sensilla on the pronotum were necessary. Among these receptors, however, only the FCOs were involved in inducing the immobile state. Centrally, the brain was indispensable for thanatosis to be maintained. In semi-natural conditions the thanatotic state did occur spontaneously while the cricket struggled to get into a small crevice according to the nature of this species. This sudden immobilization could help the cricket get out of danger of predators like reptiles and amphibians in the natural habitat.

Mechanism of execution of sequential motor acts during copulation behavior in the male cricketGryllus bimaculatus DeGeer

SummaryThe roles of the mechanosensory afferents from the wings, cerci, tergites and genitalia in copulation behavior were examined by ablation and stimulation in the male cricketGryllus bimaculatus DeGeer.The sexually excited male cricket exhibited an intense posture (IP) upon contact stimulation of the elytra and the 4th to 9th abdominal tergites. This posture allowed the backward slipping (BWS) or hooking to take place subsequently.Backward slipping (BWS), which is the movement to get under the female, was elicited during IP by contact stimulation to the middle and distal regions of the dorsal surface of the cercus.Hooking, the coordinated movements for hanging the epiphallus onto the females subgenital plate could be induced during IP by contact either on the dorsum, periproct or proximal 2 mm regions of the cercus. The latter two regions played a role in performing hooking accurately.Among four types of mechano-sensilla on the cercus the trichoid type was crucial for the initiation of BWS and hooking. Calculations revealed that about 60 trichoid hairs (4% of all the trichoid hairs in one cercus) were sufficient for the male to carry out hooking normally.The input from the bristle hairs on the epiphallus initiated the spermatophore extrusion (SPE) by swelling the endophallus.These results demonstrated that copulation behavior in the male cricket consisted of several motor acts and each act is triggered by specific input from the contact-sensitive sensilla on the elytra, tergites, cerci and genitalia. The sequential execution of each motor act is achieved because one motor act results in a positional change in contact with the female which in turn gives rise to another act. This type of motor control is a model of the so-called chain reaction in instinct behavior.

Post-copulatory sexual refractoriness is maintained under the control of the terminal abdominal ganglion in the male cricket Gryllus bimaculatus DeGeer

Abstract The location of the center that controls the change underlying mating termination and subsequent sexual refractoriness was investigated in the male cricket Gryllus bimaculatus DeGeer. In intact males, hooking movements of the genitalia did not recommence after the spermatophore was extruded during copulation, while other copulatory movements such as body thrusting and cereal vibration recommenced under the female. In males with the connectives cut anywhere between the brain and the terminal abdominal ganglion (TAG) immediately after spermatophore extrusion, hooking movements were no longer elicited by artificial stimulation of the abdominal tergites and cerci, while other copulatory movements were still evoked. The isolated adbomen containing 5 ganglia, or even only the TAG showed a time-fixed (about 50 min) sexually unresponsive state when separated immediately after spermatophore protrusion (new spermatophore formation). TAG-separated males all recommenced courtship and copulation attempts many hours or days after the operation. Spermatophores in pre-copulatory operated males were spontaneously extruded some time after operation. They showed copulatory movements to artificial stimulation of the tergites but no further hooking movements. Intact males paired with uncopulatable females, whose genital chambers were closed with wax, entered the sexually refractory stage 1–3 times within 5 h through abnormal self-spermatophore extrusion, while TAG-separated males did not, and remained permanently sexually receptive to females. Pre-copulatory males exhibiting spermatophore extrusion during cryogenic inactivation of the connectives were all in the sexual refractory stage when they recovered. These results suggest that the switching and timer functions underlying the male reproductive cycle are contained in the TAG.

Auto-spermatophore extrusion in male crickets

SUMMARY The reproductive cycle of the male cricket consists of the mating stage and the sexually refractory stage. The latter is further divided into the first refractory stage (RS1) from spermatophore extrusion in copulation to spermatophore preparation after copulation, and the second refractory stage (RS2) from spermatophore preparation to recommencement of a calling song. RS2 is time-fixed and unaffected by the female or by stress, hence RS2 is assumed to be controlled by the reproductive timer. Previously, we suggested that the timer is located in the terminal abdominal ganglion (TAG), because functional inactivation of the TAG by local cooling lengthened RS2 in proportion to cooling time. To obtain further evidence of timer localization and to examine the operation of the timer in dissected animals, we investigated the characteristics of auto-spermatophore extrusion, a phenomenon in which males eject the mature spermatophore themselves without any prior courtship. The occurrence of auto-spermatophore extrusion was 100% in dissected males with the TAG separated, compared to 1.7% in intact males. The time interval (SPaSE) between spermatophore preparation and auto-spermatophore extrusion was comparable to RS2 measured by the calling song. Spike recording from a genital motor neurone in the separated TAG indicated that burst discharge associated with auto-spermatophore extrusion occurred with a SPaSE comparable to RS2. Other efferent neurones, some of which were identified as dorsal unpaired median (DUM) neurones, showed a time-dependent spike frequency increase during SPaSE. These results strengthen our previous conclusion that the reproductive timer is located within the TAG, and demonstrate that the timer functions normally even when the TAG is separated from the central nervous system.

Brain control of mating behavior in the male cricket Gryllus bimaculatus DeGeer: brain neurons responsible for inhibition of copulation actions.

We localized brain neurons responsible for the inhibition of copulation actions in the male cricket, Gryllus bimaculatus. Males with one connective-cut between the brain and the terminal abdominal ganglion (TAG) were able to perform normal reproductive behaviors such as courtship, copulation and spermatophore protrusion. However, copulation response tests using a female dummy showed that they failed to exhibit a transient inhibitory state after application of noxious stimulation in the mating stage and to exhibit a tonic inhibitory state in the post-copulatory sexual refractory stage. A partial cut of a single connective (hemilateral connective-cut) combined with the copulation response test and an axonal backfilling demonstrated that approximately 40 brain neurons (per one connective), whose somata were located in the posterior region of the protocerebrum and mainly contralaterally to their descending axons running through the dorso-medial part of the connective, were candidates for the inhibition of copulation actions, at least in the mating stage. In addition, we report here for the first time, that copulation actions can be elicited shortly after copulation even in intact males. This unusual behavior was frequently observed in fledglings after the final molt. The results are discussed from the perspective of the operation of the brain inhibition system on the central pattern generator (CPG) for copulation.

Copulation in the cricket is performed by chain reaction.

Abstract The male and female genitalia are finely designed to match each other for copulation in the cricket Gryllus bimaculatus. Copulatory acts of the male, stereotyped and time-fixed, are elicited by stimulation of mechanoreceptors on particular regions of the abdomen, cerci and genitalia. Sequential execution of each motor act proceeds as a chain reaction in which one act stimulates some receptors which in turn elicits another act and so on, while the female remains immobile on the males back. Each key stimulus for a motor act appears as a result of the males own act, except for copulatory papilla protrusion by the female. The final sequence of spermatophore extrusion and transfer are irreversible fixed motor actions which are triggered when the female copulatory papilla stimulates the epiphallic hairs. They proceed without continual central drive from the brain, and apparently without sensory feedback. In addition, they are well coordinated with movement and posture in the entire body. Some neural mechanisms of controlling mating behavior and switching the reproductive cycle are discussed.

Brain control of mating behavior in the male cricket Gryllus bimaculatus DeGeer: the center for inhibition of copulation actions.

We re-examined the functional role of the brain and suboesophageal ganglion (SOG) in inhibiting mating behavior in the male cricket Gryllus bimaculatus DeGeer. Experiments were conducted by using mimetic stimulation to elicit copulation actions. To induce a change in the male internal state from a sexually responsive state to a sexually unresponsive state in the mating stage, noxious stimulation, head injury and leg pinching were used. Males that sustained a head injury became sexually unresponsive but became responsive as soon as the brain was removed, while males that underwent the same treatment remained unresponsive after the SOG was destroyed by sectioning. The inhibitory effects of the brain were also demonstrated by the fact that further removal of the SOG in the decerebrated males did not change their copulatory responsiveness, while removal of the brain in SOG-sectioned males markedly increased copulatory responsiveness. Furthermore, decerebrated males did not become sexually unresponsive by leg pinching, while SOG-sectioned males that had recovered sexual responsivenss, did. These results suggest that the brain, and not the SOG, plays a key role in the inhibition of copulation in the male cricket during the mating stage.