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First published online May 1, 2006
Journal of Experimental Biology 209, 1827-1836 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02212

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Common and specific inhibitory motor neurons innervate the intersegmental muscles in the locust thorax

Peter Bräunig¹, Michael Schmäh² and Harald Wolf^2,*

¹ Institut für Biologie II, Rheinisch-Westfälische Technische Hochschule Aachen, Kopernikusstraße 16, D-52074 Aachen, Germany
² Abteilung Neurobiologie, Universität Ulm, Albert-Einstein-Allee 11, D-89081 Ulm, Germany

View larger version (75K): [in a new window]   Fig. 1. Anatomy of the locust (pro- and meso-) thorax. Muscles relevant in the present report are highlighted in reddish colours (adapted from fig. 8 in Campbell, 1961). Muscle numbers follow the Snodgrass terminology (Snodgrass, 1929). The nervous system is marked in blue. Note nerve supplies of muscles: M59 (sterno-pleural intersegmental muscle) supplied by a branch of N1B, M60 (2nd ventral longitudinal muscle) supplied by N1A1, M61 (sterno-spinal muscle) supplied by N1A2, M81 (dorsal longitudinal muscle) and M82 (oblique dorsal muscle) supplied by N1D1 (for details, also see Fig. 6). The larval dorsal longitudinal muscle DLM3 (hidden) is located just below the posterior ventral corner of M81, indicated by grey shading. Scale bar, 1 mm.

View larger version (59K): [in a new window]   Fig. 2. Anti-GABA immunocytochemistry of peripheral nerves and muscles. (A) Three GABA-immunoreactive axons (arrowheads) enter nerve 6 (N6) as it branches off the posterior pro-mesothoracic connective. (B) Branching point of mesothoracic nerve 1 (N1) as it divides into N1B (here still including N1C: N1B/C) and N1D (compare Figs 1 and 6). A sensory branch that supplies the sternum (see ventralmost label in Fig. 1) also buds off at this point. Two immunoreactive axons run from the prothoracic ganglion through N1 and into N1B to supply M59. Only the larger-diameter axon gives off a branch into N1D to supply M81 and M82. (C) GABA-immunoreactive axonal branches and terminal boutons on M59. Two immunoreactive axons supply the muscle (arrowheads). Scale bars, 35 µm (A,B); 100 µm (C).

View larger version (55K): [in a new window]   Fig. 3. Backfills reveal anatomical details of (inhibitory) innervation in the locust prothorax. (A) Backfilling the motor nerve to M59 stains two (presumptive inhibitory, see text) motor neurons in the prothoracic ganglion, the axons of which leave the ganglion through nerve 6 (N6) (confocal image of neurobiotin fill, developed with Cy2, ganglion outline indicated). Arrowheads indicate looping primary neurites. (B) Backfilling the motor nerve to M59 also stains axonal branches and terminal boutons on M82 (Ni2+-backfill, silver-intensified). (C) Backfilling the motor nerve to M59 finally reveals the axons of the two (inhibitory) motor neurons shown in A as they project into the periphery through N6. Only the smaller diameter axons send a branch into N1A to innervate M60 (confocal image, as in A). Scale bars, 200 µm (A); 150 µm (B); 25 µm (C).

View larger version (72K): [in a new window]   Fig. 4. Double labelling of inhibitory motor neurons through combination of backfilling nerve 6 (A; red fluorescence, Cy3) and anti-GABA immunocytochemistry (B; green fluorescence, Cy2). (Ai,Bi,Ci) A whole-mount preparation (anterior is to the top; ganglion midline indicated by dotted line, backfill labels are contralateral to the side of nerve 6 exit); (Aii,Bii,Cii) a cross section through the same area of the prothoracic ganglion in a different animal (ventral is to the top). The overlays in Ci and Cii show which neurons have acquired both labels (arrowheads indicate the same cells in Ai–Ci, and Aii–Cii, respectively). Note that the cell marked with an asterisk in Ci does not stain for GABA in Bi. It only appears orange because it is located on top of cells labelled in green. Scale bar, 50 µm.

View larger version (25K): [in a new window]   Fig. 5. Electrophysiological examination of motor nerve supplies and intracellular muscle fibre recordings support anatomical and immunocytochemical data. (A) Extracellular recordings from the nerves supplying M81 (bottom trace) and M59 (middle trace), and intracellular recording from an M59 muscle fibre (top trace) during spontaneous activity illustrate two major features. First, there are two classes of IPSPs in M59, with different amplitudes and shapes. Second, the large-amplitude IPSPs exhibit a 1:1 relationship to spikes in the nerve supplies of both, M59 and M81 (marked by arrowheads). The small-amplitude IPSPs are related only to spike activity in the M59 motor nerve. (Bi) Stimulating the motor nerve to M81 and M82 in a different experiment (N1D; stimulus artefacts marked by arrow) elicits spikes in N1B (bottom trace) and small-amplitude IPSPs in a fibre of M59 (top trace). (Bii) Large-amplitude IPSPs in this same muscle fibre are not stimulus-related but occur in 1:1 relationship to spontaneous spikes, with slightly different shape and amplitude when compared to those seen in Bi, bottom trace. Four traces each from the same recording are superimposed in Bi and Bii. (C) Stimulating the motor nerve to M59 (stimulus artefacts indicated by arrow) elicits (small-amplitude) IPSPs in fibres of M60. Large-amplitude IPSPs (arrowhead) occur spontaneously. (D) Simultaneous intracellular recordings from fibres of M59 (top trace) and M60 (bottom trace) illustrate common inhibitory input. 1:1 relationship of IPSPs in the two muscle fibres is clearly discernible (dotted lines mark the beginnings of IPSPs in M60), as is the unrelated occurrence of larger-amplitude IPSPs in M59 (arrowheads; uncorrelated IPSPs were also observed in M60 in other experiments). Scale bars, 20 mV, 275 ms (A); 10 mV, 32.5 ms (B); 10 mV, 25 ms (C); 20 mV, 250 ms (D).

View larger version (23K): [in a new window]   Fig. 6. Summary diagram of the inhibitory motor neuron supply to prothoracic muscles, as revealed by the combination of immunocytochemical, neuroanatomical and electrophysiogical techniques. The three inhibitory motor neurons are each marked by a particular colour: CI59/81/82, green; CI59/60, blue; SI60, red. Compare also to the more realistic representation of nerves and muscles in Fig. 1. T1, T2, pro- and mesothoracic ganglia; TN, transverse nerve (remaining nerve and muscle labels see text).