Different proctolin neurons elicit distinct motor patterns from a multifunctional neuronal network

Abstract

Distinct motor patterns are selected from a multifunctional neuronal network by activation of different modulatory projection neurons. Subsets of these projection neurons can contain the same neuromodulator(s), yet little is known about the relative influence of such neurons on network activity. We have addressed this issue in the stomatogastric nervous system of the crab Cancer borealis. Within this system, there is a neuronal network in the stomatogastric ganglion (STG) that produces many versions of the pyloric and gastric mill rhythms. These different rhythms result from activation of different projection neurons that innervate the STG from neighboring ganglia and modulate STG network activity. Three pairs of these projection neurons contain the neuropeptide proctolin. These include the previously identified modulatory proctolin neuron and modulatory commissural neuron 1 (MCN1) and the newly identified modulatory commissural neuron 7 (MCN7). We document here that each of these neurons contains a unique complement of cotransmitters and that each of these neurons elicits a distinct version of the pyloric motor pattern. Moreover, only one of them (MCN1) also elicits a gastric mill rhythm. The MCN7-elicited pyloric rhythm includes a pivotal switch by one STG network neuron from playing a minor to a major role in motor pattern generation. Therefore, modulatory neurons that share a peptide transmitter can elicit distinct motor patterns from a common target network.

Fig. 1.

Schematics of the stomatogastric nervous system, including somata location and axonal pathways of the proctolin projection neurons MPN, MCN1, and MCN7. A, There is a pair of MPN somata located either in the OG or in the nerve posterior to this ganglion. Each MPN projects an axon through eachson to the CoG and two axonal branches through thestn to the STG. It also projects an axonal branch from each son through the peripheral nervedpon. For clarity, the complete projection of only one MPN is shown. B, There is a single MCN1 and MCN7 in each CoG. Each MCN1 projects an axon through the ion andstn to the STG. Each MCN7 projects an axon through theson and stn to the STG. For clarity, the complete projection of only one MCN1 and one MCN7 is shown. Ganglia;CoG, commissural ganglion; OG, esophageal ganglion; STG, stomatogastric ganglion; nerves:dgn, dorsal gastric nerve; dpon, dorsal posterior esophageal nerve; ion, inferior esophageal nerve; lgn, lateral gastric nerve; lvn, lateral ventricular nerve; mvn, medial ventricular nerve; pdn, pyloric dilator nerve; son, superior esophageal nerve; stn, stomatogastric nerve; neurons: MCN1, modulatory commissural neuron 1;MCN7, modulatory commissural neuron 7;MPN, modulatory proctolin neuron. Anterior is toward thetop, and posterior is toward thebottom.

Fig. 2.

Anatomy and projection pathway of MCN7.A, Confocal image of a LY intracellular fill of MCN7 in the CoG. Anterior is toward the top, and medial is toward the right. A thin neurite projects from the MCN7 soma. It expands into a larger neurite, from which the fine processes arise. The neurite diameter is reduced again to form the axon, which leaves the CoG via the son. The image is a confocal composite of 24 optical sections collected at ∼2 μm intervals.B, Overlaid oscilloscope traces were triggered by MCN7 action potentials elicited by injection of depolarizing current into its soma. Constant latency action potentials were recorded in theson and stn (arrows), followed by a constant latency postsynaptic potential in the IC neuron, an STG network neuron.

Fig. 3.

The CoG and STG arbors of MCN1 contain proctolin, GABA, and CabTRP Ia. Confocal images of the MCN1 arbor in the CoG and STG demonstrate the MCN1 transmitter complement in each ganglion. In all panels, green represents LY, redrepresents antibody labeling, and yellow represents double labeling. A–C, Intracellular LY fill of MCN1 in the CoG. D–F, Intracellular LY fill of MCN1 in the STG. Intracellular fills are paired with proctolin immunocytochemistry (A, D), GABA immunocytochemistry (B, E), and CabTRP Ia immunocytochemistry (C, F). All panels show composite confocal images of eight optical sections taken at ∼0.5 μm intervals, with the exception of B, which is a composite of four optical sections. Note: In many of the images shown here, there appears to be incomplete colocalization of dye and immunoreactivity. This is because nearly all modulator immunoreactivity in the neuropil is confined to varicose swellings present on the small-diameter terminal branches and is not always found within processes that connect these varicosities (Christie et al., 1997c).

Fig. 4.

MPN contains proctolin (Nusbaum and Marder, 1989a) and GABA, whereas MCN7 contains proctolin but not GABA. In all panels,green represents LY, red represents antibody labeling, and yellow represents double labeling. A, Low-magnification view of an intracellular LY fill of MPN in the on. The arrowpoints to MPN soma. B, Same preparation processed for GABA immunocytochemistry. Note that the LY-filled soma (arrow) double labels for GABA immunoreactivity. Thearrowhead indicates second MPN, which was not filled with LY but exhibits GABA immunoreactivity. A andB were collected simultaneously and are composite confocal images of 15 optical sections, each section being ∼2 μm thick. Scale bar is for both images. C, MCN7 is proctolin-immunoreactive. C1, High-magnification view of a region of a CoG neuropil from an intracellular LY fill of MCN7 paired with proctolin immunoctyochemistry. C2, High-magnification image of a region of the son.C1 and C2 are from the same preparation and are composite confocal images of 16 and 8 optical sections, respectively, taken at 0.5 μm intervals. D, MCN7 is not GABA-immunoreactive. D1, High-magnification confocal image of a region of CoG neuropil from an intracellular LY fill of MCN7 paired with GABA immunocytochemistry. D2, High-magnification image of a region of the son from the same preparation as D1. Confocal images are composites of eight optical sections taken at ∼0.5 μm intervals. Scale bar inD1 is for C1 and D1. Scale bar in D2 is for C2 andD2.

Fig. 5.

MPN and MCN1 elicit distinct STG motor patterns.Left, During saline superfusion and without any neuronal stimulation, there was an ongoing pyloric rhythm (mvn,pdn). There was no gastric mill rhythm (dgn, lgn). V_m: MPN, −72 mV; MCN1, −66 mV. Middle, When MPN was stimulated (9 Hz), there was an increase in the activity of IC and VD (mvn). MPN did not elicit a gastric mill rhythm (dgn, lgn). Right, When MCN1 was stimulated (11 Hz), a gastric mill rhythm was elicited, as is evident from the alternating bursting in the DG (dgn) and LG (lgn) neurons and the modified VD neuron pattern (mvn). This pattern is the MCN1-elicited gastric mill rhythm (Blitz and Nusbaum, 1997). MCN1 stimulation also caused increased activity in the IC and VD neurons.

Fig. 6.

MCN7 elicits a distinct STG motor pattern. Before MCN7 stimulation, there was an ongoing pyloric rhythm (IC, mvn, pdn) and no gastric mill rhythm (lgn, dgn). When MCN7 was stimulated (23 Hz), the IC neuron was strongly excited, and the LG and DG neurons were activated. There was also a decrease in the average pyloric cycle frequency, which is most evident in the decreased frequency of the rhythmic PD neuron bursts (pdn). Note that the IC and LG neurons were coactive, whereas the DG neuron activity was less closely correlated to the activity of the other neurons. Most hyperpolarized V_m: MCN7, −65 mV; IC, −62 mV.

Fig. 7.

MCN7 excitation of the IC neuron causes a decreased pyloric cycle frequency. A, MCN7 stimulation (21 Hz) excited IC and caused a decrease in pyloric frequency, evident in the periodic decrease in frequency of PD neuron bursts. Note the extended period of hyperpolarization in PD during each extended IC burst. PD is one of the pyloric pacemaker neurons. B, When IC was hyperpolarized with constant-amplitude negative current and MCN7 was again stimulated (21 Hz), there was no decrease in pyloric cycle frequency. Current injections were via explicitly unbalanced bridge circuits.

Fig. 8.

Selective stimulation of MPN, MCN1, or MCN7 elicits different pyloric motor patterns. Left, The mean ± SD phase of burst onset and offset for the PD, IC, and VD neurons during a pyloric cycle is plotted during MPN, MCN1, and MCN7 stimulation. One pyloric cycle was arbitrarily designated as beginning with the onset of a PD neuron burst (phase = 0) and ending with the onset of the next PD burst (phase = 1). Pyloric cycles during MCN1 stimulation were divided into those occurring during either the retraction (DG neuron burst) or the protraction (LG neuron burst) half of the gastric mill rhythm. Pyloric cycles during MCN7 stimulation were divided into short- and long-duration cycles. Short-duration cycles are those that are shorter than the mean + 1 SD of the control pyloric cycle period before stimulation. Long-duration cycles are those longer than 1 SD above the mean control pyloric cycle period before stimulation. For each of the five conditions shown, 10 cycles per preparation were measured from four preparations. During MCN1-DG phase pyloric cycles, the IC neuron was not always active. Thus there were only 18 total measurements for this parameter. During MCN7 long-duration cycles and MCN1-LG phase cycles, there are only 10 total measurements each of VD phase onset and offset, because the VD neuron was often silent during these patterns. For details of analysis, see Materials and Methods. Right, For each parameter (e.g., PD neuron burst offset, IC neuron burst onset) a one-way ANOVA was conducted followed by multiple comparisons using the Tukeyt test. Asterisks represent significant differences. For IC neuron burst onset and offset, all significant differences represent p < 0.001. For PD and VD phase, significant differences range from p < 0.05 to p < 0.001.

Fig. 9.

Summary of the distinct motor patterns elicited from the crab STG network by selective activation of the proctolin neurons MCN1, MPN, and MCN7. Top, MCN1 contains the transmitters CabTRP Ia, GABA, and proctolin, and it elicits a gastropyloric motor pattern. MPN contains proctolin and GABA but not CabTRP Ia. It elicits a particular pyloric rhythm and no gastric mill rhythm. MCN7 contains proctolin but not GABA and CabTRP Ia. MCN7 elicits a distinct pyloric motor pattern, which is dominated by IC neuron bursts. During this motor pattern, IC neuron activity regulates the pyloric frequency. Bottom, Schematic representation of the motor patterns elicited by MCN1, MPN, and MCN7.Boxes represent bursts of action potentials in an individual neuron.