Optical study of interactions among propagation waves of neural excitation in the rat somatosensory cortex evoked by forelimb and hindlimb stimuli.

Multisite optical recording has revealed that the neural excitation wave induced by a sensory stimulation begins at a focus and propagates in the cortex. This wave is considered to be important for computation in the sensory cortex, particularly the integration of sensory information; however, the nature of this wave remains largely unknown. In the present study, we examined the interaction between two waves in the rat sensory cortex induced by hindlimb and forelimb stimuli with different interstimulus intervals. We classified the resultant patterns as follows: 1) the collision of two waves, 2) the hindlimb response being evoked while the forelimb-induced wave is passing the hindlimb focus, and 3) the hindlimb response being evoked after the forelimb-induced wave has passed the hindlimb focus. In pattern 1, the two waves fused into a single wave, but the propagation pattern differed from that predicted by the superimposition of two singly induced propagation courses. In pattern 2, the state of the interaction between the two waves varied depending on the phase of optical signals constituting the forelimb-induced wave around the hindlimb focus. Although no hindlimb-induced wave was observed in the rising phase, the propagating velocity of the forelimb-induced wave increased. At the peak, neither the hindlimb-induced response nor a modulatory effect on the forelimb-induced wave was detected. In pattern 3, the hindlimb-induced wave showed a reduced amplitude and spatial extent. These results indicate that the state of the interaction between waves was strongly influenced by the relative timing of sensory inputs. NEW & NOTEWORTHY Sensory stimulation-induced cortical excitation propagates as a wave and spreads over a wide area of the sensory cortex. To elucidate the characteristics of this relatively unknown phenomenon, we examined the interaction between two individually induced waves in the somatosensory cortex. Either the waves collided or the preceding wave affected the emergence of the following one. Our results indicate that the state of the interaction was strongly influenced by the relative timing of sensory inputs.

Optical Analysis of Acute Changes after Peripheral Nerve Injury in Spatio-Temporal Pattern of Neural Response to Forelimb Stimulation in Rat Somatosensory Cortex.

Peripheral nerve injury induces functional reorganization of the central nervous system. The mechanisms underlying this reorganization have been widely studied. Our previous study involving multiple-site optical recording reported that a neural excitatory wave induced by somatic stimulation begins in a small area and propagates in the cortex. In the present study, to examine the possible role of this propagation wave in cortical reorganization, we analyzed the early changes in the spatio-temporal pattern of the sensory-evoked wave immediately, and 30 min, after nerve injury. The response to hypothenar stimulation, innervated by the ulnar nerve and adjoining the median nerve area, persisted after injury to either the ulnar or median nerve. Initially, we assessed changes in the response pattern at the focus. The latency increased after ulnar nerve injury, whereas no change was observed after median nerve injury. Similarly, no change was noted in the duration of the response signal with either nerve injury. Second, changes in the propagation wave pattern were analyzed. Ulnar nerve injury decreased the propagation velocity in the medial direction but the median nerve injury induced no changes. These results indicated that the propagation wave pattern is readily altered, even immediately after nerve injury, and suggest that this immediate change in the spatio-temporal pattern is one of the factors contributing to the cortical reorganization.

Concave-shaped transparent electrode to simultaneously monitor electrical activity from multiple sites within the optical sampling area of the intact rat cerebral cortex.

We have developed a concave-shaped transparent electrode unit that enables the placement of several electrodes within the optical sampling area on the spherical surface of the rat brain. This concave-shaped transparent electrode unit consists of an insulator base (a plano-concave lens) and a gallium-doped zinc oxide film that is a transparent conductor coating the base. Most of the unit is wrapped in an insulator film made of silicon dioxide, and the few areas left unwrapped act as electrodes. In the study reported here this newly developed transparent electrode unit worked well within the optical detection area without affecting optical recording. We applied this unit to our multiple-site optical recording system for membrane potential in order to eliminate pulsation artifacts and succeeded in optically recording spontaneous neural activity, including small changes in membrane potential, in the cerebral cortex in a single-sweep recording.

Optical Recording of the Pacemaking Activity of a Congenital Double-heart in an Early Chick Embryo: (embryonic double-heart/pacemaking activity/optical monitoring/potential-sensitive dye).

A congenital double-hearted chick embryo was found among 16,171 embryos, which was at the 11 somite stage of development. The pacemaking activity of its double heart was monitored simultaneously from 9 different regions by optical methods. The right and left half hearts were tubular, and in both, spontaneous rhythmical action potentials and beating were detected, and differences were detected in their rhythms. Action potentials were also monitored in a malformed embryonic heart formed by partial fusion of the primordia. The results are discussed in relation to genesis of intrinsic pacemaking activity in cardiac primordia and to a spatial gradient of rhythmicity in the early stages of cardiogenesis.

Improvement of the optical imaging technique for intact rat brain using a plano-concave lens.

Use of a plano-concave lens improved the quality of optical signals from the rat cerebral cortex by improving the focus. When detecting neural activity from a curved surface of an in vivo brain by optical techniques, it is not possible to adjust the focus equally over the entire detecting area in the two-dimensional plane, since the active window of the optical detector is usually flat, while the intact brain surface is spherical. It has been known that the size of the optical signal is reduced as the distance of the real image to the active window of the detector increases; therefore, the level of the signal-to-noise ratio obtained from the unfocused area often becomes insufficient for quantitative physiological analyses. By placing a plano-concave lens on the cerebral cortex, we succeeded in obtaining a two-dimensional image that has no unfocused area over an entire image recorded by the detector.

An improved multiple-site optical membrane potential-recording system to obtain high-quality single sweep signals in intact rat cerebral cortex.

We improved our optical recording system to record epifluorescence optical signals from the intact cerebral cortex. Using custom-made fiber optic illumination equipment and tandem lens optics excluding a dichroic mirror, we successfully obtained high-quality single sweep optical signals in vivo in rat sensorimotor cortex. After reduction of pulsation artifacts with software originally designed for prolonged continuous recording under spontaneous breathing, the signal-to-noise ratio of the optical signal was sufficient to analyse evoked responses quantitatively. We were able to make isochrones for the onset of responses based on single sweep optical recordings. We further examined the effect of baclofen using this isochrone map, and succeeded in quantitatively demonstrating the inhibitory effect on the spatial pattern of neural activity in the rat sensorimotor cortex by calculating the area of response region on the isochrone map. Thus, our improved system provides sustained single sweep records suitable for quantitative analysis using epifluorescence optics.

Identification of critical structural determinants responsible for octopamine binding to the alpha-adrenergic-like Bombyx mori octopamine receptor.

Octopamine (OA) is a biogenic amine with a widespread distribution in the insect nervous system. OA modulates and/or regulates various behavioral patterns of insects as a neurotransmitter, neuromodulator, and neurohormone. OA receptors (OARs) belong to one of the families of G protein-coupled receptors (GPCRs). The binding of OA to OARs is coupled to the activation of the specific G proteins, which induces the release of intracellular second messengers such as cAMP and/or calcium. We previously reported the isolation of an OAR (BmOAR1) from Bombyx mori. In the study presented here, five mutated BmOAR1s were constructed with a point mutation in the putative binding crevice and expressed in HEK-293 cells. The S202A mutant receptor was found to retain the cAMP response to OA as does the wild-type receptor, but such function was impaired in the other four mutants (D103A, S198A, Y412F, and S198A/S202A). Furthermore, competition binding assays using [3H]OA and calcium mobilization assays gave results that were approximately consistent with those of the cAMP assays. Taken together, the results indicate that D103 and S198 are involved in the binding and activation of BmOAR1 with OA through electrostatic or hydrogen bond interactions, but S202 does not appear to participate in this process. Y412 seems to be involved in one of the active forms of BmOAR1. These findings should prove helpful in designing new pest control chemicals.

A long-time, high spatiotemporal resolution optical recording system for membrane potential activity via real-time writing to the hard disk.

Using real-time hard disk recording, we have developed an optical system for the long-duration detection of changes in membrane potential from 1,020 sites with a high temporal resolution. The signal-to-noise ratio was sufficient for analyzing the spreading pattern of excitatory waves in frog atria in a single sweep.