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First published online March 17, 2006
Journal of Experimental Biology 209, 1285-1300 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02133

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Functional consequences of activity-dependent synaptic enhancement at a crustacean neuromuscular junction

Wolfgang Stein^*, Carmen R. Smarandache, Melanie Nickmann and Ulrike B. S. Hedrich

Abteilung Neurobiologie, Universität Ulm, D-89069 Ulm, Germany

View larger version (34K): [in a new window]   Fig. 1. Schematic diagram of the stomatogastric nervous system, illustrating the different experimental setups. Anterior is toward the top. The stomatogastric nervous system contains four ganglia, including the paired commissural ganglia (CoG), the oesophageal ganglion (OG) and the stomatogastric ganglion (STG), plus their connecting and peripheral nerves. The STG contains the motor neuron LG, which is part of the central pattern generator of the gastric mill rhythm. LG projects its axon through the dorsal ventricular nerve (dvn) to the lateral ventricular nerve (lvn) and the lateral gastric nerve (lgn). It is the only neuron that innervates the gm6 muscle. Experimental setups: (1) In some experiments, the inferior (ions) and superior (sons) oesophageal nerves were transected to eliminate descending modulatory input to the STG (example shown on top right). In these experiments, the ion was stimulated extracellulary to elicit a gastric mill rhythm (Bartos and Nusbaum, 1997; Stein et al., 2005). (2) With both CoGs intact, gastric mill rhythms were elicited by stimulation of one of the dorsal posterior oesophageal nerves (dpon; Beenhakker et al., 2004; Stein et al., 2005; example shown on top left). (3) When the electrical response of the gm6 muscle (shown for the right muscle in the diagram) or gm6 muscle force (shown for the left muscle) was recorded, the lvn was transected between stimulation compartment and STG (broken lines on lvn). The stump of the lvn was then stimulated extracellularly with various stimulus protocols to elicit action potentials in the LG axon.

View larger version (23K): [in a new window]   Fig. 2. Facilitation and summation affect the electrical response of the gm6 muscle to stimulation of LG. (A) Left: intracellular recording of gm6 muscle during stimulation of the LG motor neuron. Average of five sweeps with two-pulse stimulation. Interstimulus interval: 200 ms. Arrows show the times of stimulation. Right: average of five sweeps with two-pulse stimulation. The arrow points at the first EJP in each sweep. Interstimulus intervals: 100 ms, 200 ms, 400 ms, 800 ms, 1600 ms, 3200 ms. (B) Exponential decay function fit of the facilitation index. Average of nine animals. Arrow indicates maximum possible facilitation (as would occur at an interstimulus interval of 0; dotted lines, see Materials and methods section). ISI, interstimulus interval. F, facilitation index. (C) EJPs facilitate and summate with repetitive stimuli. 10 consecutive stimuli were used, followed by a test pulse. Left, 5 Hz stimuli; middle, 10 Hz stimuli; right, 20 Hz stimuli. (D) 5 Hz stimulation of LG. 10 stimuli plus a single test pulse with a delay of 500 ms were used. Left: original recording of 11 EJPs with different amplitudes. Middle: overlay of all EJPs. Right: Overlay of all EJPs after normalizing them the to peak depolarization of each EJP. (E) Left: original recording of a 20 Hz stimulus train. Circles indicate calculated baseline potential (white) and EJP amplitude (black). Gray circles mark the peak depolarization of the EJP. Right: the contribution of EJP amplitude (black circles) and baseline potential (white circles) to the peak amplitude (gray circles) of each EJP are given. (F–H) EJP amplitudes increased during 5 Hz (F), 10 Hz (G) and 20 Hz (H) train stimulation. Plot of 20 animals (white circles) plus average. Please note that averages were shifted slightly to the left for clarity. **Significantly different from first EJP, P<0.01. (I) Plot of averaged EJP amplitudes (N=20 for 5 Hz, 10 Hz and 20 Hz, N=10 for 30 Hz) over the time in the stimulus train. With higher stimulus frequencies, higher EJP amplitudes were reached in a shorter time. **Significantly different from stimulation with lower stimulus frequency, P<0.01. *Significantly different from stimulation with lower stimulus frequency, P<0.05.

View larger version (29K): [in a new window]   Fig. 3. Augmentation caused an increase in EJP amplitudes even when stimuli were suspended for several seconds. (A) Original recording of the electrical response of the gm6 muscle to 20 Hz stimulation of LG with 10 trains of stimuli. Inter-train interval 4 s. The responses to the first, fifth and tenth stimulus trains are shown. Dotted lines indicate maximum depolarization and amplitude of test EJP. (B) Test EJPs increased in amplitude with repeated train stimulation. EJP amplitudes were normalized to the test EJP after the first stimulus train and plotted over the number of the stimulus train. 20 Hz stimulations were used. For further details and N numbers, see Table 1. Significances are shown for the test EJP after the tenth stimulus train. Significantly different from amplitude of test EJP after the first stimulus train, P<0.05. **Significantly different from amplitude of test EJP after the tenth stimulus train with longer inter-train durations, P<0.01. *Significantly different from amplitude of test EJP after the tenth stimulus train with longer inter-train durations, P<0.05. Significantly different from amplitude of test EJP after the tenth stimulus train with 32 s inter-train duration, P<0.05. (C) The first, fifth and tenth EJP increased in amplitude when stimulus trains (20 Hz) were repeated. EJP amplitudes were normalized to the tenth EJP of the first stimulus train. Average of N=11 animals. (D) Development of the amplitude of the tenth EJP during repeated train stimulation (20 Hz) with inter-train intervals of 1–32 s. EJP amplitudes were normalized to the tenth EJP of the first stimulus train. Significances as in B. For details and N numbers, see Table 2. (E) Exponential decay function fit of the augmentation index A (as revealed by the normalized amplitude of the tenth EJP of the tenth stimulus train during 20 Hz stimulation) over inter-train interval. Average of nine animals (32 s inter-train interval) and 11 animals (all other intervals), respectively. Significantly different from A of longer inter-train interval (P<0.05). (F) Decrement of EJP amplitudes after the end of a series of 10 train stimulations (20 Hz). EJPs were elicited at delays of 2, 4, 6, 8, 10, 12, 14, 16 and 18 s after the end (arrow) of the last stimulus train. Average of three sweeps. (G) Exponential decay function fit of decrement of EJP amplitudes after the tenth stimulus train (20 Hz). Average of N=14 animals. Arrow, first EJP of the first stimulus train (control EJP). For details see Table 3. Dotted line indicates amplitude of control EJP. **Significantly different from control, P<0.01. *Significantly different from control, P<0.05. Significantly different from EJP amplitudes with longer delays, P<0.05. (H) Development of EJP amplitudes (average of N=11 animals) during repetitive train stimulation (20 Hz). All EJPs of all stimulus trains are shown. Amplitudes were normalized to the last EJP of the first stimulus train. After the third stimulus train, no further enhancement of EJP amplitudes was obtained. (I) Within-train facilitation of EJP amplitudes is affected by augmentation. The amplitudes of all EJPs in each stimulus train are shown. Amplitudes were normalized separately to the last EJP of each particular stimulus train (20 Hz). Average of N=11 animals. F, facilitation index.

View larger version (42K): [in a new window]   Fig. 4. Augmentation contributes to the muscle response during gastric mill rhythms recorded in vitro and in vivo. (A) Top: extracellular recording of the lateral gastric nerve (lgn) showing the activity of LG during a gastric mill rhythm that was elicited by dpon stimulation in the isolated nervous system (in vitro). Bottom: extracellular recording of lgn during a gastric mill rhythm that was elicited by ion stimulation (20 Hz) in the isolated nervous system. (B) Extracellular recordings of the lateral ventricular nerve (lvn) in intact animals (in vivo). The recordings show the activity of the lateral pyloric (LP), pyloric dilator (PD) and LG motor neurons. Three different gastric mill rhythms are shown (weak, intermediate, strong). (C) Intracellular recordings of gm6 muscle showing its response to LG stimulation with standardized in vitro rhythms. Left: ion elicited gastric mill rhythm. Right: dpon elicited gastric mill rhythm. In each panel the first and the tenth stimulus trains are shown. Dotted lines indicate the difference in amplitude of the first EJP and the peak depolarization between first and tenth stimulus train. Please note that amplitude scaling is different for the different types of stimulation. (D) Intracellular recordings of gm6 muscle during stimulation with standardized in vivo gastric mill rhythms. Left: weak rhythm, middle: intermediate rhythm, right: strong rhythm. In each panel the first and the tenth stimulus train are shown. Dotted lines indicate the difference in amplitude of the first EJP and the peak depolarization between first and tenth stimulus train.

View larger version (31K): [in a new window]   Fig. 5. Augmentation increases EJP amplitudes during various gastric mill-like stimulations. (A) Comparison of the amplitudes of the first EJPs of the first (white bars) and the tenth stimulus train (black/gray bars) during ion, dpon, weak in vivo, intermediate in vivo and strong in vivo rhythms. Average of N=6 animals. (B) Comparison of the amplitudes of the last EJPs of the first (white bars) and the tenth stimulus train (black/gray bars) during ion, dpon, weak, intermediate and strong rhythms. (C) Plot of augmentation index A (as revealed from the normalized first EJP of the tenth stimulus train) over train number. A is shown for ion and dpon rhythms. (D) Plot of augmentation index A of the first EJP over train number for weak, intermediate and strong in vivo rhythms. (E) Plot of augmentation index A of the last EJP over train number. A is shown for ion and dpon rhythms. (F) Plot of augmentation index A of the last EJP over train number for weak, intermediate and strong in vivo rhythms.

View larger version (25K): [in a new window]   Fig. 6. Within-train facilitation is affected by augmentation. (A–E) The development of the facilitation index F within single stimulus trains is shown for the first (black circles) and tenth (white circles) stimulus train for different stimulation protocols. EJP amplitudes were normalized to the last EJP of the first or tenth stimulus train, respectively. Average of N=6 animals.

View larger version (16K): [in a new window]   Fig. 7. Muscle force is enhanced by augmentation. (A) Original recording of gm6 muscle force during intermediate in vivo rhythm stimulation. Top trace: stimulus. Bottom trace: force. The first, tenth and eleventh stimulus trains are shown. Tenth and eleventh stimulus trains were separated by a pause of 100 s. Dotted lines indicate peak force. (B) Comparison of maximum force during first and tenth stimulus train. (C) Comparison of maximum slope of force production during first and tenth stimulus train.