Preserving the Ankle by the Masquelet Technique for the Large Bone Defect in the Distal Tibia in Children: A Case Report.

CASE: A 14-year-old adolescent girl sustained an open fracture of the foot after jumping off a building. Initial radiographs revealed large bone defect in the distal metaphysis of the comminuted tibia. The comminuted distal tibia was reconstructed by external fixation and internal fixation with bridge plating, followed by the Masquelet technique. After 12 months, the fracture healed without infection, and the patient could walk independently. CONCLUSION: In the case of a comminuted fracture of the contaminated distal tibia with a large bone defect, plate fixation and the Masquelet technique produced good outcomes.

Chronic and Acute Oral Administration of Caffeine Enhance Performance of Olfactory Learning in Crickets.

Crickets were arbitrarily fed a caffeine-containing diet during their lifetime or were orally administered caffeine in solution once during the conditioning session. These chronically or acutely treated crickets were used to test whether there were positive effects of caffeine on their learning performance. The effects of chronic exposure to caffeine on crickets' growth were also examined by measuring their body weight and counting the number of adults that emerged from larvae. Chronic treatment with relatively high concentrations of caffeine (5 mg/g and 0.5 mg/g in the diet) impaired the growth of crickets. Mortality was higher with 5 mg/g caffeine, and the survivors could not attain the final molt (eclosion). Body weight gain and eclosion rate were also negatively influenced in the group fed 0.5 mg/g caffeine. The olfactory learning paradigm was used to examine the effects of chronic caffeine treatment. Of the caffeine concentrations tested (0.5 µg/g to 0.5 mg/g), significant improvement in long-term memory (LTM) formation was observed only with 50 µg/g caffeine. Acute effects on olfactory learning were examined after oral administration of 0.1 nM to 10 µM caffeine solutions during the conditioning session. Administration of caffeine ≥ 1 nM consistently resulted in a significant improvement in LTM formation. These results suggest that chronic exposure to caffeine enhances learning performance in crickets at a specific dose (50 µg/g in the diet), although it inhibits their growth at higher doses (≥ 0.5 mg/g). In contrast, acute oral administration of caffeine in naive crickets may enhance their learning performance even at a very low (nanomolar) concentration.

Effects of elevated carbon dioxide on contraction force and proteome composition of sea urchin tube feet.

This study examined how contraction force and protein profiles of the tube feet of the sea urchin (Pseudocentrotus depressus) were affected when acclimated to 400 (control), 2000 and 10,000µatm CO2 for 48days. Acclimation to higher CO2 conditions significantly reduced contraction force of the tube feet. Two-dimensional gel electrophoresis showed that eight spots changed in protein volume: six up-regulated and two down-regulated. Using matrix-assisted laser desorption/ionization-quadrupole ion trap-time of flight mass spectrometry, three up-regulated spots (tubulin beta chain, tropomyosin fragment, and actin N-terminal fragment) and two down-regulated spots (actin C-terminal fragment and myosin light chain) were identified. One possible interpretation of the results is that elevated CO2 weakened contraction of the tube feet muscle through an alteration of proteome composition, mainly associated with post-translational processing/proteolysis of muscle-related proteins.

Effects of Caffeine on Olfactory Learning in Crickets.

Caffeine is a plant-derived alkaloid that is generally known as a central nervous system (CNS) stimulant. In order to examine the effects of caffeine on higher CNS functions in insects, we used an appetitive olfactory learning paradigm for the cricket Gryllus bimaculatus. Crickets can form significant long-term memories (LTMs) after repetitive training sessions, during which they associate a conditioned stimulus (CS: odor) with an unconditioned stimulus (US: reward). Administration of hemolymphal injections of caffeine established LTM after only single-trial conditioning over a wide range of caffeine dosages (1.6 µµg/kg to 39 mg/kg). We investigated the physiological mechanisms underlying this enhancement of olfactory learning performance pharmacologically, focusing on three major physiological roles of caffeine: 1) inhibition of phosphodiesterase (PDE), 2) agonism of ryanodine receptors, and 3) antagonism of adenosine receptors. Application of drugs relevant to these actions resulted in significant effects on LTM formation. These results suggest that externally applied caffeine enhances LTM formation in insect olfactory learning via multiple cellular mechanisms.

Characterization of locomotor-related spike activity in protocerebrum of freely walking cricket.

To characterize the neural elements involved in the higher-order control of spontaneous walking in insects, we recorded extracellular spike activity in the protocerebrum of freely walking crickets (Gryllus bimaculatus). Locomotor behavior was simultaneously recorded using a newly developed motion tracking system. We focused on spike units that altered their firing patterns during walking. According to their activity patterns with reference to walking bouts, these locomotor-related spike units were classified into the following four types: continuously activated unit during walking (type 1); continuously inhibited unit during walking (type 2); transiently activated unit at the onset of walking (type 3); and transiently activated unit at the termination of walking (type 4). The type 1 unit was the most dominant group (25 out of 33 units), whereas only a few units each were recorded for types 2-4. Some of the locomotor-related units tended to change firing pattern before the onset or termination of walking bouts. Spike activity in some type 1 units was found to be closely correlated with walking speed. When firing timing was compared between unit pairs, their temporal relationships (synchronization/desynchronization) altered, depending on the behavioral state (standing/walking). Mechanical stimuli applied to the body surface elicited excitatory responses in the majority of the units. Histological observations revealed that the recorded sites were concentrated near or within the mushroom body and central complex in the protocerebrum.

Behavioral response to antennal tactile stimulation in the field cricket Gryllus bimaculatus.

We examined behavioral responses of the field cricket Gryllus bimaculatus to tactile stimuli to the antennae. Three stimulants of similar shape and size but different textures were used: a tibia from the hunting spider Heteropoda venatoria (potential predator), a tibia from the orb-web spider Argiope bruennichi (less likely predator), and a glass rod. Each stimulus session comprised a first gentle contact and a second strong contact. The evoked behavioral responses were classified into four categories: aversion, aggression, antennal search, and no response. Regardless of the stimulants, the crickets exhibited antennal search and aversion most frequently in response to the first and second stimuli, respectively. The frequency of aversion was significantly higher to the tibia of H. venatoria than to other stimulants. The most striking observation was that aggressive responses were exclusive to the H. venatoria tibia. To specify the hair type that induced aggression, we manipulated two types of common hairs (bristle and fine) on the tibia of the predatory spider. When bristle hairs were removed from the H. venatoria tibia, aggression was significantly reduced. These results suggest that antennae can discriminate the tactile texture of external objects and elicit adaptive behavioral responses.

Refuge size preference in the field cricket Gryllus bimaculatus.

Size preference for artificial refuges was examined in the adult field cricket Gryllus bimaculatus under laboratory conditions. Blinded crickets were placed individually in a container consisting of a circular arena and six different-sized artificial refuges (triangular tent-like shelters). The crickets were allowed to walk freely inside the container for a constant period. Size preference was evaluated by determining cumulative stay period in each shelter. When the depth of the shelters varied from 60 to 160 mm at 20-mm intervals, and the width was fixed at 30 mm, both males and females tended to remain in relatively longer shelters (≥ 140 mm). Females, in particular, exhibited a distinct preference for the longest shelter (160 mm). The width of the shelters was then varied from 20 to 40 mm at 4-mm intervals, and the depth was fixed at 100 mm. Although males did not show selectivity to specific shelters, females tended to select a shelter with a particular width (32 mm). These results suggest that adults of G. bimaculatus have size preferences for refuges under blinded conditions. However, the preferences may involve sexual differences as well.

Sensitive profiling of biogenic amines in urine using CE with transient isotachophoretic preconcentration.

A transient ITP-CZE system is proposed for the determination of biogenic amines in urine. The complete separation and identification of dopamine, tyramine (TA), tryptamine (T), serotonin, epinephrine, norepinephrine, and normetanephrine have been achieved in a twofold diluted urine sample (in which the analytes were below the LODs by normal CZE). The tITP preconcentration conditions were created by introducing a 30 mM solution of NaOH, containing 0.1% hydroxypropylcellulose (pH 6.5 adjusted with MES), and 0.1 M HCl before and after the sample zone to work as leading and terminating electrolytes, respectively. This allowed the LODs of direct UV absorption detection to be decreased down to the 10(-8) M level that is comparable with the sensitivity thresholds of LIF detection or inline SPE-CE. The RSDs of migration time and peak area for real-biofluid analysis were in the range of 0.1-4.5% and 0.8-16% (n=3), respectively. Quantification of dopamine, TA, T, and serotonin was performed using internal calibration. To the best of our knowledge, this is the first report on probing urinal biogenic amines and their metabolites by tITP-CZE.

Antennal motor activity induced by pilocarpine in the American cockroach.

The antennal motor system is activated by the muscarinic agonist pilocarpine in the American cockroach Periplaneta americana, and its output patterns were examined both in restrained intact animals and in isolated CNS preparations. The three-dimensional antennal movements induced by the hemocoelic drug injection were analyzed in in vivo preparations. Pilocarpine effectively induced prolonged rhythmic movements of both antennae. The antennae tended to describe a spatially patterned trajectory, forming loops or the symbol of infinity (infinity). Such spatial regularity is comparable to that during spontaneous tethered-walking. Rhythmic bursting activities of the antennal motor nerves in in vitro preparations were also elicited by bath application of pilocarpine. Cross-correlation analyses of the bursting spike activities revealed significant couplings among certain motor units, implying the spatial regularity of the antennal trajectory. The pilocarpine-induced rhythmic activity of antennal motor nerves was effectively suppressed by the muscarinic antagonist atropine. These results indicate that the activation of the antennal motor system is mediated by muscarinic receptors.

Antennal and locomotor responses to attractive and aversive odors in the searching cockroach.

The behavioral responses to attractive and aversive odors were examined in blinded adult male cockroaches under tethered-walking conditions. A sex pheromone-like stimulant derived from adult virgin females and artificially synthesized limonene were used as attractive and aversive odor sources, respectively. When a searching animal was stimulated with the attractive female-derived odor, the horizontal deflections of both the antennae were increased, and in most cases the vertical antennal positions were shifted downward. The stimulation also significantly decreased the walking speed of the animal. These behavioral changes imply a careful search in the immediate surroundings. The aftereffect of the sex pheromone was more pronounced on locomotion than on antennal movement. On the other hand, stimulation with the aversive odor (limonene) tended to suppress active antennal movement, and also increased the walking speed. Immediately after the withdrawal of the aversive odor, the active movement of the antennae was resumed, and the walking speed rapidly decreased to a level approximately the same as that of the control period. These results indicate that the responses to the qualitatively opposite types of odor are reciprocal to each other with regard to both antennal movement and locomotion.

Active tactile sensing for localization of objects by the cockroach antenna.

Antennal movement during tactile orientation behavior was examined three-dimensionally in American cockroaches during tethered walking. When a wooden rod was presented to the tip of one antenna in an upright orientation at one of the three different horizontal positions (30 degrees , 60 degrees , or 90 degrees from the center of the head), the animal touched it repeatedly with the antenna, and tried to approach it (positive thigmotaxis). Positional shifts were also observed for the contralateral unstimulated antenna. The ipsilateral antenna tended to touch the object during inward movement (adduction) at all three test angles. The cumulative turn angle made during a continuous test period of 24 s clearly depended on the object's position; however, the contact frequencies were almost the same regardless of the position. The relationships between contact frequency and some locomotion parameters were also investigated on a shorter time scale of 3 s. The contact frequency positively correlated with the turn angle, with the accuracy of orientation at all three test angles, and with the translation velocity at test angles of 30 degrees and 60 degrees . It is concluded that the performance during tactile orientation can be represented effectively by the frequency with which the antennae touch the attractive objects.

Spatio-temporal patterns of antennal movements in the searching cockroach.

To characterize the spatio-temporal patterns of antennal behavior in insects, the voluntary movement of both right and left antennae was examined in the cockroach Periplaneta americana. The position of the tip of the antenna (flagellum) is controlled by two mobile joints at its base (the scape and the pedicel) and by the neck. Horizontal and vertical components of movement at the antennal basal joints exhibited rhythmic activities during locomotory (walking) and non-locomotory (pausing) states in the searching animal. In both states, the horizontal component was slower than vertical one. Joint-manipulation experiments suggested that the faster vertical component is due mainly to movements of the scape-pedicel joint, while the slower horizontal component may originate from the head-scape joint. Large horizontal deflections of the antenna corresponded consistently with the yaw component of head movement. The trajectories of the antennae showed little patterned regularity in most animals. In a few cases, however, loop-like patterns appeared. The area scanned by an antenna was narrower in the walking state than in the pausing state, mainly because of a decrease in the horizontal angular range. Cross-correlation analyses revealed that the coupling between right and left horizontal antennal motor systems and that for the vertical systems were both significantly stronger in the walking state than during pausing. These results indicate that the spatio-temporal pattern of antennal movements changes dynamically depending on the animal's behavioral state.

Intracellular responses of antennal chordotonal sensilla of the American cockroach.

The responses of mechanoreceptor neurons in the antennal chordotonal organ have been examined in cockroaches by intracellular recording methods. The chordotonal organ was mechanically stimulated by sinusoidal movement of the flagellum. Stimulus frequencies were varied between 0.5 and 150 Hz. Receptor neurons responded with spike discharges to mechanical stimulation, and were classed into two groups from plots of their average spike frequencies against stimulus frequency. Neurons in one group responded to stimulation over a wide frequency range (from 0.5 to 150 Hz), whereas those in a second group were tuned to higher frequency stimuli. The peak stimulus frequency at which receptor neurons showed maximum responses differed from cell to cell. Some had a peak response at a stimulus frequency given in the present study (from 0.5 to 150 Hz), whereas others were assumed to have peak responses beyond the highest stimulus frequency examined. The timing for the initiation of spikes or of a burst of spikes plotted against each stimulus cycle revealed that spike generation was phase-locked in most cells. Some cells showed phase-independent discharges to stimulation at lower frequency, but increasing stimulus frequencies spike initiation began to assemble at a given phase of the stimulus cycle. The response patterns observed are discussed in relation to the primary process of mechanoreception of the chordotonal organ.

Mechanosensory control of antennal movement by the scapal hair plate in the American cockroach.

The voluntary movement of antennae of blinded cockroaches was examined in the tethered-walking condition. An object of metal plate was presented to a tip of a single antenna in order to induce tactile orientation behavior. Horizontal movements of the antenna before and during the object presentation were analyzed both before and after ablation of a mechanosensory organ, the scapal hair plate (S-HP), at the base of antenna. The resting antennal position shifted outwardly by about 20 degrees after the S-HP ablation. Spontaneous antennal movements in ablated animals became stiff and wider ranged. The tactile object was set at two different horizontal positions, 45 degrees and 90 degrees clockwise to the head, for the right side antenna. The number of contacts in a constant test period was significantly decreased in the tests at 45 degrees after ablation. Trajectories of antennal movements before and after contacts were categorized into four patterns. In the case that an antenna made contact with the object during its abduction (outward) movement, it then passed the object outwardly or withdrew inwardly. These were termed "outward-pass (O-P)" and "outward-withdrawal (O-W)" patterns, respectively. Similarly, contacts during the adduction (inward) movement were divided into "inward-pass (I-P)" or "inward-withdrawal (I-W)" pattern. Significant effects of the S-HP ablation appeared in the tests at 90 degrees : the I-P pattern mostly disappeared and was replaced by the I-W pattern. The results strongly suggest that the S-HP has crucial roles for controlling both spontaneous and stimulated movements of the cockroach antenna.