c-Fos expression during temporal order judgment in mice.

The neuronal mechanisms for ordering sensory signals in time still need to be clarified despite a long history of research. To address this issue, we recently developed a behavioral task of temporal order judgment in mice. In the present study, we examined the expression of c-Fos, a marker of neural activation, in mice just after they carried out the temporal order judgment task. The expression of c-Fos was examined in C57BL/6N mice (male, n = 5) that were trained to judge the order of two air-puff stimuli delivered bilaterally to the right and left whiskers with stimulation intervals of 50-750 ms. The mice were rewarded with a food pellet when they responded by orienting their head toward the first stimulus (n = 2) or toward the second stimulus (n = 3) after a visual "go" signal. c-Fos-stained cell densities of these mice (test group) were compared with those of two control groups in coronal brain sections prepared at bregma -2, -1, 0, +1, and +2 mm by applying statistical parametric mapping to the c-Fos immuno-stained sections. The expression of c-Fos was significantly higher in the test group than in the other groups in the bilateral barrel fields of the primary somatosensory cortex, the left secondary somatosensory cortex, the dorsal part of the right secondary auditory cortex. Laminar analyses in the primary somatosensory cortex revealed that c-Fos expression in the test group was most evident in layers II and III, where callosal fibers project. The results suggest that temporal order judgment involves processing bilateral somatosensory signals through the supragranular layers of the primary sensory cortex and in the multimodal sensory areas, including marginal zone between the primary somatosensory cortex and the secondary sensory cortex.

Wireless voltammetry recording in unanesthetised behaving rats.

In vivo voltammetry is a valuable technique for rapid measurement of dopamine in the brain of freely behaving rats. Using a conventional voltammetry system, however, behavioural freedom is restricted by cables connecting the head assembly to the measurement system. To overcome these difficulties, we developed a wireless voltammetry system utilizing radio waves. This system consisted of a potentiostat and transmitter system that was mounted on the back of the rat, and a receiver and analysis system. A single-step pulse (100-250 mV) was applied at 4 Hz after an activation pulse to a carbon fibre recording electrode (diameter: 7 microm). Measurement of dopamine (detection limit: 2.7 x 10(-7)M) was demonstrated in vitro. In vivo experiment was performed at least 1 week after the recording electrode was implanted in the rat striatum. Administration of 2-phenylethylamine to rats increased dopamine signal current, which was consistent with the result in the microdialysis measurement. During a resident-intruder test, dopamine signal current in a resident rat increased upon introduction of an intruder rat. These results show that the present wireless system is useful for a long-term measurement of dopamine in behaving rats.

Referral of tactile stimuli to action points in virtual reality with reaction force.

When we touch something with a tool, we feel the touch at the tip of the tool rather than at the hand. Yamamoto and Kitazawa [Yamamoto, S., Kitazawa, S., 2001b. Sensation at the tips of invisible tools. Nat. Neurosci. 4, 979-980] previously showed that the judgment of the temporal order of two successive stimuli, delivered to the tips of sticks held in each hand, was dramatically altered by crossing the sticks without changing the positions of the hands. This provided evidence for the referral of tactile signals to the tip of a tool in hand. In this study, we examined importance of force feedback from the tool in the referral by manipulating the direction of force feedback in a virtual reality. The virtual tool consisted of a spherical action point that was moved with a stylus in hand. Subjects held two styli, one in each hand, put each action point on each of two buttons in the virtual reality, and were required to judge the order of successive taps, delivered to the two styli. We manipulated the direction of reaction force from each button so that it was congruent or incongruent to the visual configuration of the button. When the arms were uncrossed, judgment primarily depended on whether the action points were crossed or not in the visual space. But when the arms were crossed, judgment critically depended on the direction of force feedback. The results show that tactile signals can be referred to the action point in the virtual reality and that the force feedback becomes a critical factor when the arms are crossed.

Referral of tactile sensation to the tips of L-shaped sticks.

When we touch something with a tool, we feel the touch at the tip of the tool rather than at the hand that holds the tool. We reported previously that judging the temporal order of two successive stimuli delivered to the tips of straight sticks held in each hand was dramatically altered by crossing the sticks without changing hand position. The results suggested that tactile signals are referred to the tip of a tool held in the hand. Here we examined temporal order judgement using L-shaped sticks instead of straight ones to determine whether the shape of a tool affects the way tactile signals are referred. Subjects reported the order of stimuli correctly in most trials when the tip of each L-shaped stick occupied the hemispace ipsilateral to the anatomical laterality of the hand holding the L-shaped stick. The subjects, however, misreported the order of stimuli presented at moderately short intervals (<300 ms) when the tip of the stick occupied the hemispace contralateral to the anatomical laterality of the hand holding it. The judgment reversal occurred irrespective of the number of physical crossings between the sticks and the arms (0, 1, and 3), as long as the tips of L-shaped tools were placed in the contralateral hemispace. Our results suggest that our brain refers tactile signals from the hand directly to the location of the tip without much accounting for the route that connects hand and tip.

Temporal order judgment in mice.

A temporal order judgment task was developed for mice. After training male mice (C57BL6NCrj, n=15) to poke their noses into a hole, two stimuli (brief puffs of air) were delivered to the whiskers with a fixed interval of 750 ms in one of four orders: right (R)-left (L), L-R, L-L, and R-R. The mice were rewarded when they oriented their heads toward the first (n=5) or second (n=10) stimulus after a visual go signal. The mice were trained for up to 50 days. All mice met the criterion for task achievement (daily correct response rate >70% on 3 consecutive days) in response to unilateral stimuli (L-L and R-R), and 9 of the 15 mice met the criterion for task achievement in response to bilateral stimuli (L-R and R-L). The median periods for task achievement were 15 and 34 days for unilateral and bilateral stimuli, respectively. The correct response rate dropped to approximately the chance level after all whiskers had been removed. The nine successful mice were trained further and tested with smaller interstimulus intervals. The probability of right-first judgment plotted against the stimulation interval was fitted with a sigmoid function (r2=0.92) with asymptotes of 0.29 and 0.73 and a temporal resolution of 160 ms. The sigmoid curve was biased horizontally by 133 ms, reflecting the fact that stimuli delivered simultaneously were judged as left-first rather than right-first. The results show that mice can be trained to judge the temporal order of tactile stimuli delivered to whiskers and that such judgment might be lateralized to the right hemisphere.