The optical recording method using voltage-sensitive dye is one of the powerful techniques for two-dimensional detection of mass neuronal activity in the brain and for the analysis of spatiotemporal properties [1][2][3][4]. Respiratory neuron activity in the lower brain stem consists of widely extending neuron networks, including respiratory neurons in the ventrolateral medulla (VLM) [5][6][7][8][9][10]. Therefore the optical recordings could also be a very effective method for a spatiotemporal analysis of neuronal activity in the medullary respiratory center. Using this method, inspiratory burst activity, induced by electrical stimulation, was analyzed in medullary slice preparations [11]. The goal of our optical recording study was to understand the macroscopic behavior of respiratory neuron networks in the medulla, for example, by revealing the sites of initiation of each burst cycle and the manner of the burst propagation. Therefore as the next step in the optical analysis of the respiratory activity, a detailed spatiotemporal analysis of "spontaneous" respiratory activity is important. In the present study we show that this method is also available for analysis of spontaneous respiratory activity in the medulla. The results have been partly presented in abstract form [12]. Key words: respiratory center, optical imaging, ventrolateral medulla, newborn rat, in vitro preparation. Abstract:We report on the optical imaging of spontaneous respiratory neuron bursts in the ventrolateral medulla (VLM) of medullary slices or brain stem-spinal cord preparations. A medullary slice with a thickness of 1.0-1.4 mm or brain stem-spinal cord from 0-to 4-d-old rats was stained with fluorescent voltage-sensitive dye, RH795. Optical signals were recorded as a fluorescence change by using an optical recording apparatus with a 128ϫ128 photodiode array and a maximum time resolution of 0.6 ms. Motoneuronal activity was simultaneously recorded at the hypoglossal nerve roots or fourth cervical ventral roots. Fluorescence changes corresponding to the spontaneous inspiratory burst activity were detected in the hypoglossal nucleus and VLM in slice preparations, and in a limited area extending rostrocaudally in the VLM of the brain stem-spinal cord preparation. These measurements did not require signal averaging by multiple trials. Results suggest that inspiratory neurons are localized in more compact form at the level of the nucleus ambiguus than at the more rostral VLM, and that peak activity during the inspiratory phase propagates from the caudal to the rostral VLM. In 60% of brain stem-spinal cord preparations, weak and scattered fluorescence changes preceding the inspiratory burst activity were detected more predominantly in the rostral part of the VLM. The present findings show the feasibility of optical recordings for the in vitro analysis of spontaneous respiratory neuron activity in the medulla.

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Fig.1.Recording chamber.

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