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First published online February 29, 2008
Journal of Experimental Biology 211, 1000-1011 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.015222
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A newly identified extrinsic input triggers a distinct gastric mill rhythm via activation of modulatory projection neurons

Dawn M. Blitz1, Rachel S. White1, Shari R. Saideman1,*, Aaron Cook1, Andrew E. Christie2,3, Farzan Nadim4,5 and Michael P. Nusbaum1,{dagger}

1 Department of Neuroscience, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA
2 Department of Biology, University of Washington, Seattle, WA 98195-1800, USA
3 Mount Desert Island Biological Laboratory, Salisbury Cove, ME 04672, USA
4 Department of Mathematical Sciences, New Jersey Institute of Technology, Newark, NJ 07102, USA
5 Department of Biological Sciences, Rutgers University, Newark, NJ 07102, USA


Figure 1
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  		Fig. 1. Schematic of the isolated stomatogastric nervous system, including the axon projections of MCN1 and CPN2 to the STG. The two lines with arrowheads projecting posteriorly from the STG neuropil represent the projection pattern of most STG motor neurons. Ganglia: CoG, commissural ganglion; OG, oesophageal ganglion; STG, stomatogastric ganglion; TG, thoracic ganglion. Nerves: cocTG, circumoesophageal connective from the CoG to the TG; cocB, circumoesophageal connective from the CoG to the brain; dgn, dorsal gastric nerve; dpon, dorsal posterior oesophageal nerve; ion, inferior oesophageal nerve; lgn, lateral gastric nerve; lvn, lateral ventricular nerve; mvn, medial ventricular nerve; pdn, pyloric dilator nerve; poc, post-oesophageal commissure; son, superior oesophageal nerve. Neurons: CPN2, commissural projection neuron 2; MCN1, modulatory commissural neuron 1.

 

Figure 2
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  		Fig. 2. The gastric mill rhythm is triggered by poc nerve stimulation. (Left) Prior to poc stimulation, there was an ongoing pyloric rhythm [medial ventricular nerve (mvn) and pdn], but no gastric mill rhythm (dgn, lgn). The large, tonically active unit in the dgn corresponds to the activity of the anterior gastric receptor (AGR) neuron. AGR is a muscle tendon proprioceptor neuron that is spontaneously active in the isolated STNS (Combes et al., 1995Go; Smarandache and Stein, 2007Go). (Middle) 2 min after tonic poc stimulation (15 Hz, 30 s), the gastric mill rhythm was triggered, as is evident from the rhythmic bursting in the protractor LG neuron that alternated with the retractor phase activity of the DG, VD and IC neurons. Note the pyloric-timed bursting in the LG neuron. (Right) This expanded section of the middle panel shows more explicitly that each protractor LG burst is time-locked to the pyloric rhythm. Each period of inactivity in LG starts with a pyloric dilator (PD) neuron burst (grey bars). Pro., protraction, Ret., retraction.

 

Figure 3
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  		Fig. 3. The poc-triggered gastric mill rhythm is long-lasting. (Left) Before poc stimulation, there was an ongoing pyloric rhythm (pdn) but no gastric mill rhythm (lgn, dgn). (Middle) 2 min after tonic poc stimulation (15 Hz, 30 s), the gastric mill rhythm had been triggered and was ongoing. Note the pyloric-timed LG bursts. (Right) This rhythm persisted for more than 15 min after poc stimulation.

 

Figure 4
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  		Fig. 4. The poc-triggered gastric mill rhythm requires the activation of CoG projection neurons. (A) During normal saline superfusion of the CoGs, tonic poc stimulation (15 Hz, 30 s) triggered the gastric mill rhythm. (B) During superfusion of 5x Mg2+/5x Ca2+ saline selectively to the CoGs and OG (grey shading in STNS schematic), the same poc stimulation did not trigger the gastric mill rhythm. (C) After washout of the 5x Mg2+/5x Ca2+ saline, poc stimulation again triggered the gastric mill rhythm. Note that the black bar in each STNS schematic represents a Vaseline wall that enabled separate saline superfusion of the anterior (CoGs, OG) and posterior (STG) aspects of the STNS. All panels are from the same preparation.

 

Figure 5
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  		Fig. 5. Activation of the CoG projection neurons CPN2 and MCN1, as well as the gastric mill rhythm, is triggered by poc stimulation. (Left) Before stimulation, CPN2 and MCN1 were weakly active and there was an ongoing pyloric rhythm (pdn) but no gastric mill rhythm (lgn, dgn). (Middle) After poc stimulation (15 Hz, 30 s), CPN2 and MCN1 were excited and the gastric mill rhythm was triggered. (Right) Expanded section from the middle panel showing that the activity of LG, MCN1 and CPN2 is interrupted in pyloric-time. Note that each such interruption occurs during activity of the pyloric pacemaker PD neuron (grey shading). Most hyperpolarized membrane potential: CPN2, –45 mV.

 

Figure 6
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  		Fig. 6. The pyloric rhythm in the STG is responsible for the pyloric-timed activity of the CoG projection neuron MCN1 and the gastric mill protractor neuron LG during the POC-triggered gastric mill rhythm. (Left) During the POC-triggered gastric mill rhythm, MCN1 and LG exhibited pyloric-timed activity. (Middle) When the pyloric rhythm was suppressed, by hyperpolarization of the pyloric pacemaker neurons, the POC-triggered gastric mill rhythm persisted but the activity of MCN1 and LG changed from pyloric-timed to tonic. (Right) After releasing the pyloric pacemaker neurons from hyperpolarization, the pyloric rhythm resumed and MCN1 and LG returned to exhibiting pyloric-timed activity.

 

Figure 7
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  		Fig. 7. The POC neurons project through the medial aspect of the cocTG to influence MCN1 and CPN2 in the CoG. (A) STNS schematic indicating the location and extent of the cocTG transections (grey boxes), the results of which are shown in B and C. (B) Transecting the medial aspect of the cocTG eliminated the ability of poc stimulation to trigger the gastric mill rhythm. (Left) Before medial cocTG transection, poc stimulation triggered the gastric mill rhythm. (Right) After medial cocTG transection, poc stimulation did not trigger the gastric mill rhythm. (C) Transecting the lateral aspect of the cocTG did not alter the ability of poc stimulation to trigger the gastric mill rhythm. The gastric mill rhythm was triggered both (left) before, and (right) after lateral cocTG transection by poc stimulation. B and C are from different preparations.

 

Figure 8
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  		Fig. 8. A CabTRP Ia-immunoreactive axon bundle projects through the poc and medial aspect of the anterior cocTG to form terminal arborizations in the CoG. (A) CabTRP Ia immunoreactivity (CabTRP Ia-IR) occurred in a tightly associated axon bundle in the medial aspect of the cocTG (arrowhead) that terminated as a dense arborization in the antero-medial CoG. There was also more diffuse CabTRP Ia-IR throughout the CoG neuropil and in a subset of CoG neuronal somata. Asterisk indicates area examined to determine the number of CabTRP Ia-IR fibers present in the cocTG (see text). (B) The CabTRP Ia-lR axon bundle in the medial aspect of the cocTG (filled arrowhead) projected past the poc towards the TG, and also projected through the poc (open arrowhead). Asterisk indicates area examined to determine the number of CabTRP Ia-IR fibers present in the poc (see text). (C) CabTRP Ia-IR bundle was transected in a preparation in which the medial cocTG was transected (arrowhead). (D) CabTRP Ia-IR bundle was not transected in a preparation in which the lateral cocTG was transected (arrowhead). Spatial axes in C are for A–C. All scale bars, 150 µm.

 

Figure 9
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  		Fig. 9. Exogenous CabTRP Ia mimics the POC activation of MCN1 and CPN2. A brief (500 ms) puff of CabTRP Ia (10–4 mol l–1) into the antero-medial aspect of the CoG neuropil excited MCN1 and CPN2 (monitored as excitatory postsynaptic potentials in GM; see text), and subsequently activated LG, GM and DG. Note that CabTRP Ia triggered pyloric-timed activity in MCN1, CPN2 and LG. Insets, showing an expanded time scale, indicate that the GM membrane potential was not pyloric-timed before CabTRP Ia application but exhibited barrages of excitatory postsynaptic potentials that were interrupted in pyloric-time after this application. Most hyperpolarized membrane potential: GM, –67 mV.

 

Figure 10
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  		Fig. 10. Blocking extracellular peptidase-mediated degradation of CabTRP Ia prolongs the actions of the POC neurons. (A) Before, during and after superfusion of the endopeptidase inhibitor phosphoramidon (10–5 mol l–1) to the CoGs, CPN2 was weakly active before poc stimulation and LG was silent (left panel: top, middle, bottom). CPN2 activity was monitored with an intra-axonal recording near the entrance to the STG (Beenhakker and Nusbaum, 2004Go). 30 s after poc stimulation (15 Hz, 15 s), the gastric mill rhythm was triggered (as indicated by the rhythmic LG bursting) and CPN2 activity was strengthened (middle panel: top, middle, bottom). 90 s after poc stimulation, the gastric mill rhythm had terminated and CPN2 activity had subsided during saline superfusion, both before and after phosphoramidon application (right panel: top, bottom). By contrast, 90 s after poc stimulation during phosphoramidon superfusion, CPN2 activity remained strong and the gastric mill rhythm persisted. (B) (Left) There was a significant increase in the duration of LG bursting after poc stimulation in the presence of phoshoramidon (Phos., 10–5 mol l–1; P<0.05, N=5), compared with its bursting duration in saline before phosphoramidon application (Ctl.). (Right) By contrast, phosphoramidon (10–5 mol l–1) did not alter the duration of LG bursting after stimulation of the proprioceptor sensory GPR neuron. Most hyperpolarized membrane potentials: CPN2stn, –73 mV; LG, –63 mV.

 

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© The Company of Biologists Ltd 2008