Mechanosensory activation of a motor circuit by coactivation of two projection neurons

Abstract

Individual neuronal circuits can generate multiple activity patterns because of the influence of different projection neurons. However, in most systems it has been difficult to identify and assess the relative contribution of all upstream neurons responsible for the activation of any single activity pattern by a behaviorally relevant stimulus. To elucidate this issue, we used the stomatogastric nervous system (STNS) of the crab. The STNS includes the gastric mill (chewing) motor circuit in the stomatogastric ganglion (STG) and no more than 20 projection neurons that innervate the STG. We previously identified at least some (four) of the projection neurons that are activated directly by the ventral cardiac neuron (VCN) system, a population of mechanosensory neurons that activates the gastric mill circuit. Here we show that two of these projection neurons, the previously identified modulatory commissural neuron 1 (MCN1) and commissural projection neuron 2 (CPN2), are necessary and likely sufficient for the initiation/maintenance of the VCN-elicited gastric mill rhythm. Selective inactivation of either MCN1 or CPN2 still enabled a VCN-elicited gastric mill rhythm. However, because MCN1 and CPN2 have different actions on gastric mill neurons, these manipulations resulted in rhythms distinct from each other and from that occurring in the intact system. After removal of both MCN1 and CPN2, VCN stimulation failed to activate the gastric mill rhythm. Selective conjoint stimulation of MCN1 and CPN2, approximating their VCN-elicited activity patterns and firing frequencies, elicited a VCN-like gastric mill rhythm. Thus the VCN mechanosensory system elicits the gastric mill rhythm via its activation of a subset of the relevant projection neurons.

Figure 1.

The isolated stomatogastric nervous system and the gastric mill circuit. A, The isolated stomatogastric nervous system consists of the stomatogastric ganglion (STG), oesophageal ganglion (OG), and paired commissural ganglia (CoGs) plus their connecting and peripheral nerves. All identified CoG projection neurons occur as single copies in each CoG. Each of the bilaterally symmetrical VCN mechanosensory systems projects through the vcn, dpon, and son to innervate the ipsilateral CoG. Arrows with dotted lines point to ganglia. Arrows with full lines point to nerves and identified projection neurons. B, Schematic of the identified MCN1 and CPN2 synaptic actions on the gastric mill neurons. MCN1 data were obtained from Coleman and Nusbaum (1994), Coleman et al. (1995), and Beenhakker (2004). CPN2 data were obtained from Norris et al. (1994). T-bars, synaptic excitation; filled circles, synaptic inhibition; resistor symbol, electrical coupling. Nerve labels (initalics) include the following: dgn, dorsal gastric nerve; dpon, dorsal posterior oesophageal nerve; ion, inferior oesophageal nerve; lgn, lateral gastric nerve; lvn, lateral ventricular nerve; mgn, medial gastric nerve; mvn, medial ventricular nerve; son, superior oesophageal nerve; stn, stomatogastric nerve; vcn, ventral cardiac nerve. Neuron labels include the following: AM, anterior median neuron; CPN2, commissural projection neuron 2; DG, dorsal gastric neuron; GM, gastric mill neuron; Int1, interneuron 1; LG, lateral gastric neuron; MCN1, 5, 7, modulatory commissural neuron 1, 5, 7; MG, medial gastric neuron; VCN, ventral cardiac neuron.

Figure 2.

The VCN-elicited gastric mill rhythm is altered by selective CPN2 removal. A, Experimental manipulation was used to eliminate CPN2. An intra-axonal recording of CPN2 was made in the stn near the STG. During the VCN-elicited gastric mill rhythm the stn axon of the still-connected CPN2 was hyperpolarized sufficiently to reduce action potential amplitude, suppress its active propagation, and, consequently, remove the CPN2 influence on the STG circuits. B, The activity of gastric mill neurons before VCN stimulation (left), during a VCN-elicited gastric mill rhythm (middle), and while hyperpolarizing CPN2 during the gastric mill rhythm (right). Note the abbreviated LG neuron impulse bursts, lack of GM neuron activity, and enhanced activity of the DG, MG, and IC neurons. The amplitude of the IC neuron spikes is smaller than that of the VD neuron spikes (mvn).

Figure 3.

Quantification of VCN-elicited gastric mill rhythm parameters before and after the removal of CPN2 influence in the STG. Black bars represent data from gastric mill rhythms occurring when the MCN1 and CPN2 projection neurons influenced the STG (control data), whereas white bars represent data from rhythms in which CPN2 influence was removed selectively (experimental data). All control and experimental data were obtained from the same preparations. A, Removing CPN2 influence in the STG changed the impulse burst duration in several gastric mill neurons (LG, n = 8; MG, n = 7) and eliminated GM neuron bursting in seven of eight preparations. The burst durations of the DG (n = 7) and AM (n = 3) neurons were unchanged. B, The number of action potentials (“spikes”) per impulse burst also was decreased in the LG neuron (p < 0.001; n = 8) and increased in the MG neuron (n = 7) but was unchanged in the DG (n = 7) and AM (n = 3) neurons. C, The intraburst firing frequency was unchanged by CPN2 removal for most gastric mill neurons (LG, n = 8; DG, n = 7; AM, n = 3). CPN2 removal did increase MG neuron intraburst firing frequency (n = 7). D, CPN2 removal phase-advanced the LG neuron burst termination (LG, n = 8), phase-delayed the MG neuron burst termination (n = 7), and phase-advanced the onset of the DG neuron impulse burst (DG, n = 7). Except as noted, ^**p < 0.01 and ^*p < 0.05. All statistical analyses were performed on data derived within preparations (Wilcoxon signed rank test).

Figure 4.

The VCN-elicited gastric mill rhythm was altered by selective MCN1 removal. A, Experimental manipulation was used to eliminate MCN1. The influence of the single connected MCN1 was eliminated by transecting the remaining ion (see Materials and Methods). B, Gastric mill neuron activity before (left) and after (middle) VCN stimulation and after MCN1 removal (right) during the VCN-elicited gastric mill rhythm. In general, removing MCN1 activity weakened the activity levels of gastric mill neurons during the VCN-elicited gastric mill rhythm. The tonically active unit in the dgn corresponds to the activity of the anterior gastric receptor (AGR) neuron, a muscle tendon proprioceptor neuron that is spontaneously active in the isolated STNS (Combes et al., 1995).

Figure 5.

Quantification of gastric mill rhythm parameters before and after MCN1 removal. Black bars represent control VCN-elicited gastric mill rhythms, and the white bars represent data from gastric mill rhythms in which the MCN1 influence was removed. Both data sets were obtained from the same preparations. A, Removing MCN1 reduced the impulse bursts of the LG, DG, and GM neurons (LG, n = 8; DG, n = 6; GM, n = 7), but not those of the MG (n = 2) and AM (n = 4) neurons. B, The number of spikes per burst was decreased for the LG (n = 8) and DG (n = 6) neurons, but not for the MG (n = 2) and AM (n = 3) neurons. C, The intraburst firing frequency was decreased for the LG (n = 8) and DG (n = 6) neurons by MCN1 removal but was not changed for the MG (n = 2) and AM (n = 4) neurons. D, MCN1 removal phase-advanced the termination of the LG (n = 8) and GM (n = 7) neurons. There was no effect on the MG (n = 2), DG (n = 6), and AM (n = 4) neurons. ^**p < 0.01; ^*p < 0.05. All statistical analyses were performed on data derived within preparations (Wilcoxon signed rank test). The data for those neurons for which the activity was eliminated completely by MCN1 removal (MG, n = 1 of 3 preparations; AM, n = 2 of 6 preparations) were not included in these analyses.

Figure 6.

Simultaneous MCN1 and CPN2 removal eliminates the ability of VCN stimulation to elicit the gastric mill rhythm. Left, Before the VCN sensory pathway was stimulated in the isolated STNS, the gastric mill neurons either were inactive (LG, GM) or expressed pyloric-timed activity (MG). Note also that MCN1 and CPN2 were weakly active. Middle, Gastric mill rhythm generated by VCN stimulation. Right, Selective elimination of MCN1 (ion transection) and CPN2 (axon hyperpolarization) prevented VCN activation of the gastric mill circuit. The ion recording was on the STG side of the transection.

Figure 7.

MCN1 and CPN2 exhibit gastric mill- and pyloric-timed bursts during the VCN-elicited gastric mill rhythm. MCN1 exhibited a stereotyped activity pattern that included tonic activity during each LG neuron burst and pyloric-timed bursts during each LG neuron interburst. The bottom recording (dvn) includes the activity of the paired pyloric dilator (PD) neurons, members of the pyloric circuit. During the LG interburst the silent period of MCN1 correlated with PD neuron activity. CPN2 activity during each VCN-elicited gastric mill rhythm was characterized predominantly by a tonic burst during each LG neuron burst. Most hyperpolarized membrane potential: LG, -57 mV; CPN2, -66 mV.

Figure 8.

MCN1 and CPN2 activity is sufficient to elicit a VCN-like gastric mill rhythm. A, The gastric mill rhythm was not spontaneously active, but VCN stimulation in such preparations elicited the gastric mill rhythm. B, In preparations in which both CoGs were removed, selective activation of MCN1 (ion stimulation, bars) and CPN2 (intra-axonal depolarizing current injection, bars), using their gastric mill rhythm patterns, elicited a VCN-like gastric mill rhythm (see Results for details). C, An example of an experiment similar to that described in B but with a two-step depolarizing current injection delivered to the stn axon of CPN2 to mimic better the CPN2 activity observed during the VCN-elicited gastric mill rhythm (see Results for details).

Figure 9.

Quantification of the VCN-elicited gastric mill rhythm (black bars, control rhythms) and comparison with the rhythm elicited by MCN1/CPN2 stimulation (white bars, one-step CPN2 current injection; gray bars, two-step CPN2 current injection). A, The burst durations of the gastric mill neurons were not different among the VCN-elicited (LG, n = 16; MG, AM, n = 9; DG, GM, n = 13), the one-step MCN1/CPN2-elicited (LG, DG, GM, n = 9; MG, n = 3; AM, n = 5), and the two-step MCN1/CPN2-elicited (LG, MG, n = 5; DG, GM, AM, n = 4) gastric mill rhythms. B, The numbers of spikes per burst generated by the gastric mill neurons were not different among these three rhythms, except for the DG neuron during the two-step MCN1/CPN2-elicited gastric mill relative to the control rhythm. All n values are the same as in A. C, During the control rhythm the intraburst firing frequency of the DG neuron was lower, whereas the AM neuron firing frequency was higher than during the one-step MCN1/CPN2-elicited rhythm. During the two-step MCN1/CPN2-elicited gastric mill rhythm only the DG neuron intraburst firing frequency was distinct from the control rhythm. All n values are the same as in A. D, During the one-step MCN1/CPN2-elicited gastric mill rhythm the termination of the DG neuron impulse burst and the onset of the GM burst were phase-advanced relative to the control rhythm. All of the phase relationships among the gastric mill neurons during the two-step MCN1/CPN2-elicited gastric mill rhythm were the same as during the control rhythm. All n values are the same as in A. Data were compared across preparations; therefore, data analysis was performed by using ANOVAs (Kruskal-Wallis). ^*p < 0.05; a plus symbol signifies differences between the one-step MCN1/CPN2-elicited and the VCN-elicited gastric mill rhythm; filled star signifies differences between the two-step MCN1/CPN2-elicited and the VCN-elicited gastric mill rhythm.