A newly identified extrinsic input triggers a distinct gastric mill rhythm via activation of modulatory projection neurons

Abstract

Neuronal network flexibility enables animals to respond appropriately to changes in their internal and external states. We are using the isolated crab stomatogastric nervous system to determine how extrinsic inputs contribute to network flexibility. The stomatogastric system includes the well-characterized gastric mill (chewing) and pyloric (filtering of chewed food) motor circuits in the stomatogastric ganglion. Projection neurons with somata in the commissural ganglia (CoGs) regulate these rhythms. Previous work characterized a unique gastric mill rhythm that occurred spontaneously in some preparations, but whose origin remained undetermined. This rhythm includes a distinct protractor phase activity pattern, during which a key gastric mill circuit neuron (LG neuron) and the projection neurons MCN1 and CPN2 fire in a pyloric rhythm-timed activity pattern instead of the tonic firing pattern exhibited by these neurons during previously studied gastric mill rhythms. Here we identify a new extrinsic input, the post-oesophageal commissure (POC) neurons, relatively brief stimulation (30 s) of which triggers a long-lasting (tens of minutes) activation of this novel gastric mill rhythm at least in part via its lasting activation of MCN1 and CPN2. Immunocytochemical and electrophysiological data suggest that the POC neurons excite MCN1 and CPN2 by release of the neuropeptide Cancer borealis tachykinin-related peptide Ia (CabTRP Ia). These data further suggest that the CoG arborization of the POC neurons comprises the previously identified anterior commissural organ (ACO), a CabTRP Ia-containing neurohemal organ. This endocrine organ thus appears to also have paracrine actions, including activation of a novel and lasting gastric mill rhythm.

Figure 1

Schematic of the isolated stomatogastric nervous system, including the axon projections of MCN1 and CPN2 to the STG. Abbreviations: Ganglia- CoG, commissural ganglion; OG, oesophageal ganglion; STG, stomatogastric ganglion; TG, thoracic ganglion. Nerves- coc, circumoesophageal connective; 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

The gastric mill rhythm is triggered by poc nerve stimulation. (Left) Prior to poc stimulation, there was an ongoing pyloric rhythm (mvn, pdn), but no gastric mill rhythm (dgn, lgn). The 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., 1995; Smarandache and Stein, 2007). (Middle) Two minutes after tonic poc stimulation (15 Hz, 30 sec), the gastric mill rhythm was triggered, as is evident from the rhythmic bursting in the protractor LG and GM neurons 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).

Figure 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) Two minutes after tonic poc stimulation (15 Hz, 30 sec), the gastric mill rhythm had been triggered and was ongoing. Note the pyloric-timed LG bursts. (Right) This rhythm persisted for more than 15 minutes after poc stimulation.

Figure 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 sec) triggered the gastric mill rhythm. (B) During superfusion of 5X Mg²⁺/5X Ca²⁺ saline to the CoGs, the same poc stimulation did not trigger the gastric mill rhythm.

Figure 5

POC stimulation triggers activation of the CoG projection neurons MCN1 and CPN2 as well as the gastric mill rhythm. (Left) Before stimulation, the projection neurons 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 sec), CPN2 and MCN1 were excited and the gastric mill rhythm was elicited. (Right) Expanded time scale 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 bars). Most hyperpolarized membrane potential: CPN2, −45 mV.

Figure 6

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

Figure 7

The POC neurons project through the medial aspect of the coc to influence MCN1 and CPN2 in the CoG. (A) STNS schematic indicating the location and extent of the coc transections that occurred in Panels B and C (grey boxes). (B) Transecting the medial aspect of the coc eliminated the ability of poc stimulation to trigger the gastric mill rhythm. (Left) POC stimulation before medial coc transection triggered the gastric mill rhythm. (Right) POC stimulation after medial coc transection did not trigger the gastric mill rhythm. (C) Transecting the lateral aspect of the coc did not alter the ability of poc stimulation to trigger the gastric mill rhythm. POC stimulation before (Left) and after (Right) lateral coc transection triggered the gastric mill rhythm.

Figure 8

A CabTRP Ia-immunoreactive (IR) axon bundle projects through the poc and medial aspect of the anterior coc to form terminal arborizations in the CoG. (A) CabTRP Ia-IR occurred in a tightly associated axon bundle in the medial aspect of the anterior coc (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. (B) The CabTRP Ia-lR axon bundle in the medial aspect of the anterior coc (filled arrowhead) projected past the poc towards the TG, and also projected through the poc (open arrowhead). (C) CabTRP Ia-IR bundle was transected in a preparation in which the medial coc was transected (arrowhead). (D) CabTRP Ia-IR bundle was not transected in a preparation in which the lateral coc was transected (arrowhead). Spatial axes in (C) are for (A-C). All scale bars=150 μm.

Figure 9

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

Figure 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 M) to the CoGs, CPN2 was weakly active before poc stimulation and LG was silent (left panel: top, middle, bottom). Thirty seconds after poc stimulation (15 Hz, 15 sec), the gastric mill rhythm was triggered (as indicated by the rhythmic LG bursting) and CPN2 activity was strengthened (middle panel: top, middle, bottom). Ninety seconds 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). In contrast, ninety seconds after poc stimulation during phosphoramidon superfusion, CPN2 activity remained strong and the gastric mill rhythm persisted. (B) The duration of LG bursting after poc stimulation in phosphoramidon (10⁻⁵ M; black bar) and saline wash (white bar) was normalized relative to its bursting duration in saline before phosphoramidon application. There was a significant increase in the normalized duration of LG bursting after poc stimulation (black bar: p<0.05, n=5; statistical analysis was performed on the raw data) in the presence of phosphoramidon. In contrast, phosphoramidon (10⁻⁵ M; black bar) did not alter the normalized duration of POC-triggered LG bursting after stimulation of the proprioceptor sensory GPR neurons relative to pre-application controls. White bar represents the normalized duration of LG bursting when GPR was stimulated after washout of phosphoramidon. Most hyperpolarized membrane potentials: CPN2_stn, −73mV; LG, −63 mV.