Article Figures & Data
Figures
- Fig. 1.
Schematic of the stomatogastric nervous system and the response of the gastric mill and pyloric circuits to individual stimulation of two different modulatory projection neurons.A, The stomatogastric nervous system includes four ganglia plus their connecting and peripheral nerves. The ganglia are the stomatogastric ganglion (STG), oesophageal ganglion (OG), and the paired commissural ganglia (CoGs). The STG contains the gastric mill and pyloric circuits, whereas modulatory projection neurons that innervate the STG are located in the OG and CoGs. Modulatory commissural neuron 1 (MCN1) occurs as a single copy in each CoG, whereas the modulatory proctolin neuron (MPN) occurs as an equivalent pair of somata in the OG. The axonal projection patterns of only one MCN1 and MPN are shown. The inferior (ion) and superior oesophageal nerves (son) are severed (as indicated by the broken lines) to eliminate the modulatory influence from CoG projection neurons that innervate the STG. Anterior is to the left. B,Schematic of the cotransmitter complements of MCN1 and MPN, plus their actions on the STG circuits. Also illustrated are the two synaptic actions that occur onto the STG terminals of MCN1 that are pivotal for enabling this neuron to elicit the gastric mill rhythm (Coleman et al., 1995; Blitz et al., 1999). T-bars represent transmitter-mediated excitation, filled circlerepresents synaptic inhibition, and resistor symbolrepresents electrical coupling. C, Tonic stimulation of MCN1 and MPN elicits distinct STG motor patterns. MCN1 stimulation enhances and modifies the pyloric rhythm (lvn, mvn) and activates the gastric mill rhythm (dgn,LG). MPN stimulation enhances and modifies the pyloric rhythm but does not activate a gastric mill rhythm. Note that the pyloric rhythm during stimulation of these two projection neurons is distinct (e.g., mvn). Most hyperpolarized LGVm: saline superfusion (left), −66 mV; MCN1 stimulation (middle), −72 mV; MPN stimulation (right), −69 mV. Nerves: dpon, dorsal posterior oesophageal nerve; dgn, dorsal gastric nerve;ion, inferior oesophageal nerve; lgn, lateral gastric nerve; lvn, lateral ventricular nerve;mvn, medial ventricular nerve; pdn, pyloric dilator nerve; pyn, pyloric constrictor nerve;son, superior oesophageal nerve; stn, stomatogastric nerve. STG neurons: DG, dorsal gastric neuron; IC, inferior cardiac neuron; LG, lateral gastric neuron; LP, lateral pyloric neuron;PD, pyloric dilator neuron; PY, pyloric neuron; VD, ventricular dilator neuron.
- Fig. 2.
The modulatory neurons MCN1 and MPN elicit distinct pyloric rhythms. A, Extracellular recordings of the pyloric rhythm response to comparable stimulation of MCN1 and MPN.B, The pyloric cycle frequency and the activity level of several pyloric neurons are significantly enhanced by stimulation of either MCN1 or MPN, relative to ongoing pyloric rhythms during saline superfusion in the absence of stimulation. Additionally, there are significant differences in each of the analyzed parameters during MCN1 versus MPN stimulation. Each bar represents the mean ± SD for the indicated parameter. MCN1 stimulations: n = 44; MPN stimulations: n = 17. Comparison between MCN1 and saline: * p ≤ 0.05. MPN is significantly different from both saline alone and MCN1 stimulation: ▵ p≤ 0.05.
- Fig. 3.
Application of the tachykinin receptor antagonist Spantide I effectively weakens the gastric mill and pyloric circuit responses to stimulation of MCN1. During saline superfusion, MCN1 stimulation elicited the gastric mill rhythm (dgn,LG) and enhanced the pyloric rhythm (lvn,mvn). When MCN1 was stimulated during Spantide I (2 × 10−5m) superfusion, the gastric mill rhythm was not activated. Note that, during Spantide I superfusion, the LG neuron response to MCN1 was subthreshold and consisted of only unitary electrical EPSPs, whereas the DG neuron fired tonically instead of rhythmically. Also, the pyloric rhythm response was reduced relative to MCN1 stimulation in saline. The effects of Spantide I were reversible with saline wash. The middle amplitude unit in the dgn represents activity of the anterior gastric receptor (AGR) sensory neuron, whereas the smallest units are stimulus artifacts resulting from tonic stimulation of the ions to activate MCN1. Most hyperpolarized LGVm: saline, −71 mV; MCN1 during saline, −73 mV; MCN1 during Spantide, −68 mV; saline after Spantide, −73 mV.
- Fig. 4.
Quantitative comparison of pyloric rhythm parameters during tonic stimulation of MCN1 before and during Spantide I application (2–5 × 10−5m). The pyloric cycle frequency was significantly increased when MCN1 was stimulated, whether or not Spantide I was present. However, MCN1 stimulation elicited a significantly faster cycle frequency during saline superfusion than when Spantide I was applied. The same results pertained to the activity level of at least three pyloric neurons, including the lateral pyloric (LP), inferior cardiac (IC), and ventricular dilator (VD) neurons. Significant difference from saline control indicated by * (p ≤ 0.001;n = 29). Significant difference from saline control and MCN1 stimulation in saline indicated by ▵ (p ≤ 0.001; n = 29).
- Fig. 5.
The MCN1-elicited pyloric rhythm is still altered when CabTRP Ia actions are blocked with Spantide I during suppression of the gastric mill rhythm. A, When the gastric mill rhythm was suppressed by maintained hyperpolarization of the LG neuron (note the lack of gastric mill-timed activity in the IC and VD neurons), the pyloric rhythm (lvn, mvn) was still enhanced by MCN1 stimulation during superfusion of saline either alone or with Spantide I (2 × 10−5m). However, the MCN1-elicited pyloric rhythm is slower and weaker during the latter condition. B, Quantitative comparison of several pyloric rhythm parameters under these conditions shows that the pyloric cycle frequency and activity level of the LP, IC, and VD neurons are all enhanced by MCN1 stimulation. However, all of these parameters are significantly reduced during Spantide I application. Significant difference from saline control indicated by * (p ≤ 0.05; n = 22). Significant difference from saline control and MCN1 activation with LG hyperpolarized in normal saline is indicated by ▵ (p ≤ 0.05; n = 22).
- Fig. 6.
The pyloric circuit response to MPN stimulation is not altered by application of Spantide I. A, The MPN (20 Hz)-elicited pyloric rhythm was not changed when Spantide I (2 × 10 −5m) was added to the bath. B, Comparison of parameters of the MPN-elicited pyloric rhythm before and during Spantide I application shows no significant differences in the pyloric rhythm cycle frequency or the number of spikes per burst fired by the LP, IC, or VD neurons. Significant difference from saline control indicated by * (p ≤ 0.05; n = 8). Prestimulation Vm: MPN, −55 mV.
- Fig. 7.
The tachykinin receptor blocker Spantide I selectively blocks gastric mill rhythm activation by MCN1.A, Tonic stimulation of MCN1 elicited the gastric mill rhythm during saline superfusion but not during Spantide I application (10−5m). The gastric mill rhythm is represented by rhythmic alternating impulse bursts in the LG and DG neurons. The two nonrhythmic units recorded on the dgn are the AGR sensory neuron and the gastric mill (GM) motor neurons. B, Stimulation of the dorsal posterior oesophageal nerve (dpon) during saline superfusion activated a different version of the gastric mill rhythm, which then persisted for ∼10 min after stimulation. MCN1 did not contribute to this gastric mill rhythm because the ionswere transected. This gastric mill rhythm was again activated bydpon stimulation in the presence of Spantide I (10−5m). Note that, in contrast to the MCN1-elicited rhythm, the GM neurons (dgn) participated in the dpon-elicited gastric mill rhythm. A and B were from the same experiment, and stimulations occurred during the same applications of Saline (middle) and Spantide I (right). In each case, dpon stimulation was performed after the effects of MCN1 stimulation subsided. Most hyperpolarized LG Vm: Prestimulation, −64 mV; Saline, −67 mV; Spantide, −68 mV.
- Fig. 8.
The endopeptidase inhibitor phosphoramidon selectively enhances the actions of focally applied CabTRP Ia on the STG. A, Focal application of CabTRP Ia (10−4m in puffer pipette, 6 PSI) onto the desheathed STG neuropil excited the LG neuron and enhanced the pyloric rhythm (lvn). The response of both the pyloric neurons and the LG neuron to CabTRP Ia was increased in strength and duration during superfusion of phosphoramidon (10−5m), in a reversible manner. B, Focal application of proctolin (10−4m in puffer pipette, 6 PSI) onto the STG neuropil excited the pyloric rhythm (lvn) but did not excite the LG neuron. Note that there was no sustained depolarizing response in the LG neuron. The onset of the pyloric-timed subthreshold oscillations in the LG neuron after proctolin application was an indirect result of proctolin excitation of the pyloric circuit. There was no change in the response of either the pyloric neurons or the LG neuron to proctolin application during phosphoramidon (10−5m) superfusion. Most hyperpolarized LGVm: −62 mV. C, Superfusion of phosphoramidon significantly prolonged the excitatory actions of focally applied (2 sec, 6 PSI) CabTRP Ia, but not proctolin, on the pyloric rhythm and the LG neuron. The asterisks indicate that, relative to pre and postapplication saline controls, phosphoramidon significantly increased the duration of CabTRP Ia action on the pyloric cycle frequency (p < 0.05;n = 7), the number of LP spikes/pyloric-timed burst (p < 0.01; n = 7), and the depolarization of the LG neuron (p < 0.01; n = 8). In contrast, phosphoramidon neither enhanced nor prolonged the pyloric rhythm response to proctolin application (p > 0.05;n = 7). Duration of response of all three parameters was measured from the end of the peptide application to the return of that parameter to prepeptide application levels.
- Fig. 9.
Phosphoramidon does not enable neurally released CabTRP Ia to overcome the blocking actions of Spantide I.A, The presence of the peptidase inhibitor phosphoramidon (10−5m) enhanced the LG neuron response to MCN1 stimulation. Note the considerable increase in both the number of action potentials and the burst duration in the LG neuron when MCN1 was stimulated in the presence of phosphoramidon. As in Figure 3, the larger tonically active unit is the AGR neuron, whereas the smaller tonic unit represents stimulus artifacts from ion stimulation. Most hyperpolarized LGVm: −68 mV. B, Phosphoramidon did not rescue the LG neuron response to MCN1 stimulation in the presence of Spantide I. Activation of MCN1 (20 Hz) readily elicited the gastric mill rhythm during saline superfusion (Sal.) or in the presence of phosphoramidon (P; 10−5m). However, during phosphoramidon superfusion, both LG neuron activity (number of LG spikes per burst) and the gastric mill cycle period were significantly increased relative to MCN1 stimulation during saline superfusion (both parameters: * representsp < 0.02; n = 5). In contrast, there was neither LG neuron activity nor a gastric mill rhythm elicited when MCN1 was stimulated in the presence of Spantide I (S; 2–5 × 10−5m), with or without coapplication of phosphoramidon (10−5m) (n = 5). Neither the LG neuron nor the gastric mill rhythm were active without MCN1 stimulation during saline superfusion.C, Pre-MCN1 stimulation controls during saline superfusion; Sal. 1, saline superfusion before Spantide I application; S, Spantide I application;P, phosphoramidon application; P/S, phosphoramidon and Spantide I coapplication; Sal. 2, saline superfusion after washout from P/S.
- Fig. 10.
The MCN1- and MPN-elicited pyloric rhythms remain distinct during Spantide I (2 × 10−5m) application.A, An example of the MCN1-elicited pyloric rhythm during Spantide I application. LG neuron activity was suppressed with hyperpolarizing current injection. B, An example of the MPN-elicited pyloric rhythm from the same preparation shown inA. LG neuron activity was suppressed as inA. Note the differences between this rhythm and the MCN1-elicited rhythm in A. For example, the VD neuron was only active during the MCN1-elicited pyloric rhythm, whereas the IC neuron was more active during the MPN-elicited rhythm.Vm: MPN, −55 mV. C,Quantitative comparison of several parameters from these two pyloric rhythms reveals there is no difference in the pyloric cycle frequency, but that differences persist in the activity levels of the LP, IC, and VD neurons. The IC and LP neurons produced more spikes per burst during MPN stimulation, whereas the VD neuron was active only during MCN1 stimulation. Significant difference from saline control indicated by * (p ≤ 0.05; n = 20). Significant difference from saline control and MCN1 stimulation indicated by ▵ (p ≤ 0.05;n = 17).
- Fig. 11.
Intraburst firing frequency in some pyloric neurons is different during the MPN- and MCN1-elicited pyloric rhythms with Spantide I (2–5 × 10−5m) present and the LG neuron hyperpolarized. The PD, LP, and IC neurons exhibited increased firing intensity when MCN1 or MPN was stimulated, relative to their prestimulation values (indicated by *p ≤ 0.05; n = 7). PD and IC neuron firing frequency was significantly higher during MPN than MCN1 stimulation. Significant difference during MPN stimulation, relative to saline controls and MCN1 activation indicated by ▵ (p ≤ 0.05;n = 7).
- Fig. 12.
Distinct pyloric motor patterns were elicited by MPN and MCN1 stimulation when CabTRP Ia actions were blocked by Spantide I (2–5 × 10−5m). The phase relationships (the fraction of a normalized pyloric cycle during which each neuron is active) are shown as a function of the normalized pyloric cycle period. Pyloric cycle period is arbitrarily designated as beginning with burst onset in the PD neuron, and ending with the onset of the next PD burst. Top panel, Pyloric motor pattern during tonic MPN stimulation (20 Hz). Bottom panel, Activity pattern of the same neurons during tonic MCN1 stimulation (20 Hz). The phase of burst onset and offset for the LPG neuron, and offset of the PD neuron, was not significantly different between the conditions (p > 0.05; n = 6). There were also no differences in the phase of burst offset of the IC, LP, and PY neurons when either projection neuron was stimulated, but the phase of burst onset for each of these neurons occurred earlier during MPN stimulation (▵: p ≤ 0.05;n = 6). Burst onset and offsets phases for the VD neuron were not compared because VD was not active during MPN activation (n = 6 of 6).
