The characteristics of GABAergic neurons involved in respiratory control have not been fully understood because identification of GABAergic neurons has so far been difficult in living tissues. In the present in vitro study, we succeeded in analysing the electrophysiological as well as morphological characteristics of GABAergic neurons in the pre-Bötzinger complex. We used 67-kDa isoform of glutamic acid decarboxylase-green fluorescence protein (GAD67-GFP) (Delta neo) knock-in (GAD67(GFP/+)) mice, which enabled us to identify GABAergic neurons in living tissues. We prepared medullary transverse slices that contained the pre-Bötzinger complex from these neonatal mice. The fluorescence intensity of the pre-Bötzinger complex region was relatively high among areas of the ventral medulla. Activities of GFP-positive neurons in the pre-Bötzinger complex were recorded in a perforated whole-cell patch-clamp mode. Six of 32 GFP-positive neurons were respiratory and the remaining 26 neurons were non-respiratory; the respiratory neurons were exclusively inspiratory, receiving excitatory post-synaptic potentials during the inspiratory phase. In addition, six inspiratory and one expiratory neuron of 30 GFP-negative neurons were recorded in the pre-Bötzinger complex. GFP-positive inspiratory neurons showed high membrane resistance and mild adaptation of spike frequency in response to depolarizing current pulses. GFP-positive inspiratory neurons had bipolar, triangular or crescent-shaped somata and GFP-negative inspiratory neurons had multipolar-shaped somata. The somata of GFP-positive inspiratory neurons were smaller than those of GFP-negative inspiratory neurons. We suggest that GABAergic inhibition not by expiratory neurons but by inspiratory neurons that have particular electrophysiological and morphological properties is involved in the respiratory neuronal network of the pre-Bötzinger complex.

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Spontaneous body movement starts at early fetal stage, at embryonic day (E) 12-15 in mice. In the present study, the movement of the head region was studied in E13-14 mice by in utero ultrasound imaging, together with the in vitro recording of underlying neural activities in the hypoglossal nerve and the ventral root of the upper cervical cord of an isolated brainstem-spinal cord preparation. The role of gamma-aminobutyric acid (GABA) in the generation of fetal movement was assessed using mice lacking GABA-synthesizing glutamate decarboxylase 67 (GAD67). At E14, mouth opening and tongue withdrawal were observed independently at frequency of 14/h each. This movement was rarely observed in the GAD67-deficient mouse. The intraventricular administration of picrotoxin or 3-mercaptopropionic acid abolished mouth opening in the wild-type mice. In a brainstem-spinal cord preparation, three types of neural discharge were recorded: mouth/tongue-moving burst, respiratory burst and irregular activity on the basis of their waveform, regularity in occurrence and concomitant muscle activity. In the GAD67-deficient mice, the occurrence of mouth/tongue-moving burst and irregular activity was inhibited to about 15 and 40% of those in the wild-type mice, respectively. Respiratory burst was slightly inhibited but the difference was not significant. Picrotoxin greatly reduced the frequency of mouth/tongue-moving burst. These results indicate that GABA is involved in rhythm generation in movement of the head region and support the hypothesis that cleft palate in the GAD67-deficient mouse is due to the impairment of mouth or tongue movement that assists palate formation.