2A). At both sides of A3, the primary neurite gave off a large anterior and a smaller posterior dendrite. The ipsilateral and contralateral dendrites branched out bilateral symmetrically in the very dorsal neuropile of A3. In each neuromere

of the three thoracic ganglia, one prominent anterior lifescience branch and one or two posterior branches arose from the main axon and projected dorsally toward the midline of the respective ganglion. A3-AO occurred as a bilateral pair of sibling neurons, and in all three neurobiotin-labeled specimen, the Inhibitors,research,lifescience,medical mirror-image sibling neuron was clearly stained as well (Fig. 3). Such dye coupling may indicate electrical coupling between the left and right A3-AO interneuron via gap junctions (cf. Ewadinger et al. 1994; Fan et al. 2005; Anava et al. 2009). Figure 2 Structure and activity of the

abdominal ascending opener-interneuron A3-AO. (A) Morphology of A3-AO with cell body and dendrites in A3 and axonal projections in thoracic ganglia (ventral view). (B–D) Inhibitors,research,lifescience,medical Singing motor pattern (top trace) and activity … Figure 3 Dye coupling of the two A3-AO sibling neurons with neurobiotin. Extended focus views (maximum intensity projections of confocal image stacks) showing the fluorescence-labeled (neurobiotin-avidinCy3) arborizations of the two bilateral symmetrical A3-AO … During fictive singing, the membrane potential of A3-AO, as recorded in its main dendrite, oscillated with the syllable pattern (Fig. 2B). Inhibitors,research,lifescience,medical In the opener phase of each syllable, it depolarized by 20–25 mV, and in the closer phase, it hyperpolarized 5–10 mV beneath resting potential. The depolarization preceding the first syllable of a chirp was up to 3.5 mV higher than the following. Every depolarization gave rise to a volley Inhibitors,research,lifescience,medical of 4–6 action potentials with a spike frequency of up to 380 Hz during the first syllable and 360 Hz during the following syllables of a chirp. The spike activity Inhibitors,research,lifescience,medical in A3-AO preceded the next opener burst in the wing nerve by 10.1 ± 0.8 msec (mean ± SD; N = 10) and the following closer burst by 29.2 ± 2.2 msec (mean ± SD; N = 10). Therefore, we refer to A3-AO as an opener interneuron. Interestingly, we never observed any

synaptic inputs or spike activity in the A3-AO dendrite before and after singing episodes or during chirp intervals. When a constant depolarizing current of 5 nA was injected into the dendrite of A3-AO, the interneuron responded with consistently repeated depolarization–hyperpolarization oscillations of its membrane potential generating a burst Org 27569 of 2–6 action potentials during each depolarization. This rhythmic interneuron activity reliably elicited alternating opener–closer motoneuron activity reflecting the normal syllable pattern in the ipsi- and contralateral wing nerves. Activation of a single A3-AO interneuron is therefore sufficient to continuously drive the motor pattern of the syllable rhythm. Current pulses of 500 msec elicited long chirps with 14–15 syllables (Fig.