Neuropeptide Modulation Enables Biphasic Internetwork Coordination via a Dual-Network Neuron

Abstract

Linked rhythmic behaviors, such as respiration/locomotion or swallowing/chewing, often require coordination for proper function. Despite its prevalence, the cellular mechanisms controlling coordination of the underlying neural networks remain undetermined in most systems. We use the stomatogastric nervous system of the crab Cancer borealis to investigate mechanisms of internetwork coordination, due to its small, well-characterized feeding-related networks (gastric mill [chewing, ∼0.1 Hz]; pyloric [filtering food, ∼1 Hz]). Here, we investigate coordination between these networks during the Gly¹-SIFamide neuropeptide modulatory state. Gly¹-SIFamide activates a unique triphasic gastric mill rhythm in which the typically pyloric-only LPG neuron generates dual pyloric-plus gastric mill-timed oscillations. Additionally, the pyloric rhythm exhibits shorter cycles during gastric mill rhythm-timed LPG bursts, and longer cycles during IC, or IC plus LG gastric mill neuron bursts. Photoinactivation revealed that LPG is necessary to shorten pyloric cycle period, likely through its rectified electrical coupling to pyloric pacemaker neurons. Hyperpolarizing current injections demonstrated that although LG bursting enables IC bursts, only gastric mill rhythm bursts in IC are necessary to prolong the pyloric cycle period. Surprisingly, LPG photoinactivation also eliminated prolonged pyloric cycles, without changing IC firing frequency or gastric mill burst duration, suggesting that pyloric cycles are prolonged via IC synaptic inhibition of LPG, which indirectly slows the pyloric pacemakers via electrical coupling. Thus, the same dual-network neuron directly conveys excitation from its endogenous bursting and indirectly funnels synaptic inhibition to enable one network to alternately decrease and increase the cycle period of a related network.

Keywords: central pattern generator; internetwork; neuromodulation; neuropeptide; rectification; rhythmic.

Visual Overview

Figure 1.

Gly¹-SIFamide elicited a variable pyloric rhythm. A, Schematic of the isolated stomatogastric nervous system (STNS) of the crab, Cancer borealis. The STNS consists of the paired commissural ganglia (CoG), the single oesophageal ganglion (OG), and the single STG, plus motor and connecting nerves. The STG consists of neuropil and the cell bodies of both the gastric mill and pyloric network neurons. The line breaks on the ion and son indicate where they were cut to isolate the STG network (Saline:ions/sons cut). Two compartments were created with a Vaseline wall across the dish and separately superfused. Gly¹-SIFamide (5 µM) was bath applied selectively to the posterior compartment containing the STG. B, Schematic of the pyloric and gastric mill connectome. Resistor symbols indicate electrical coupling, diode symbols indicate rectification of electrical coupling, and ball and stick symbols indicate chemical inhibition. C, Representative electrophysiological traces of the pyloric (pdn, mvn, lpgn) and gastric mill (mvn, dgn, and lgn) networks in an example experiment in Saline:Intact STNS (Ci), Saline:ions/sons cut (Cii), and bath-applied Gly¹-SIFamide (5 µM; Ciii) conditions. The instantaneous pyloric cycle period is plotted at the top of each set of traces. All conditions are from the same experiment. The colored boxes indicate three levels of pyloric cycle period, overlapping with three distinct phases of the Gly¹-SIFamide gastric mill rhythm (blue, LPG activity; pink, IC/LG activity; gray, baseline Gly¹-SIFamide modulation when no gastric mill neurons are active; see also Fig. 2). D, The CV of the pyloric cycle period is plotted for three experimental conditions: (1) in saline prior to isolation of the STG network (Saline:Intact STNS, n = 19), (2) in saline with ions/sons cut to isolate the STG network from descending modulatory inputs (Saline:ions/sons cut, n = 12) and (3) during 5 µM Gly¹-SIFamide bath application (SIFamide, n = 19). Each dot represents individual experiments and lines connecting across the experimental conditions depict data points from the same preparation. The different n-value for ions/sons cut is due to the lack of a pyloric rhythm in seven preparations in this condition. In the absence of modulatory inputs, the pyloric rhythm sometimes shuts off (Zhang et al., 2009; Hamood et al., 2015). ***p < 0.001, one-way RM ANOVA, Holm–Sidak post hoc. Neurons: AB, anterior burster; AM, anterior median; DG, dorsal gastric; GM, gastric mill; IC, inferior cardiac; Int1, Interneuron 1; LG, lateral gastric; LP, lateral pyloric; LPG, lateral posterior gastric; MG, medial gastric; PD, pyloric dilator; PY, pyloric; VD, ventricular dilator. Nerves: dgn, dorsal gastric nerve; ion, inferior oesophageal nerve; lgn, lateral gastric nerve; lpgn, lateral posterior gastric nerve; lvn, lateral ventricular nerve; mvn, median ventricular nerve; pdn, pyloric dilator nerve; son, superior oesophageal nerve; stn, stomatogastric nerve.

Figure 2.

Pyloric cycle periods occurred at three different levels during the Gly¹-SIFamide gastric mill rhythm. A, The pyloric rhythm occurs with a shorter cycle period during LPG activity (blue box), with a longer cycle period during LG:IC activity (pink box) and an intermediate level in the absence of any gastric mill-timed bursting (SIFbaseline; gray box). Representative traces show an example rhythm during Gly¹-SIFamide bath application, with instantaneous pyloric cycle period plotted at the top. Dotted line across instantaneous cycle period plot indicates SIFbaseline cycle period. B, The average pyloric cycle period is plotted for no gastric mill neuron activity (SIFbaseline; gray box) and during activity of each combination of 1, 2, 3, and 4 gastric mill neurons (n = 19). Each dot represents the average pyloric cycle period during that particular neuron activity across a 20 min analysis window during Gly¹-SIFamide steady state in a single experiment. Black bars indicate the mean pyloric cycle period across all experiments during that neuronal activity. Numbers at the bottom of the graph depict the total number of pyloric cycles contributing to the averages for that neuronal activity. Dotted line represents average pyloric cycle period during SIFbaseline (gray box). Blue box highlights neuronal activity combinations that include LPG and pink box highlights combinations that include IC.

Figure 3.

IC or LG hyperpolarization eliminated longer cycle periods and decreased pyloric cycle period variability. A, Extracellular and intracellular recordings monitor the pyloric rhythm (pdn) and gastric mill-timed bursting in the IC and LG neurons in control (Pre; Ai, Bi; top), during IC (Aii) or LG (Bii) hyperpolarization, and after hyperpolarization (Post; Aiii, Biii), all during Gly¹-SIFamide bath application. A and B are from different experiments. Pyloric cycle period on top of the extracellular pdn recording plots the instantaneous pyloric cycle period during each condition. Note the absence of longer pyloric cycle periods during both LG and IC hyperpolarization (pink boxes indicate LG:IC activity, gray box indicates LG activity without an IC gastric mill burst (Ai). IC gastric mill bursts are identified by brackets above the IC recordings. Downward filled arrowheads indicate hyperpolarizing current injection (Aii,Bii). Downward white arrowheads above instantaneous pyloric cycle period indicate the SIFbaseline cycle period (dashed line).

Figure 4.

IC hyperpolarization decreased pyloric cycle period variability without altering LG activity. A, The average CV of pyloric cycle period across 200 s windows is plotted for IC (Ai, n = 6), LG (Aii, n = 4), DG (Aiii, n = 4), or all three neurons (Aiv, n = 6) before (Pre), during (Hype), and after (Post) hyperpolarization. B, The number of IC gastric mill bursts (burst duration >0.45 s) occurring before (Pre), during (Hype), and after (Post) LG hyperpolarization is plotted (n = 4). C, LG burst duration (Ci), firing frequency (Cii), and number of spikes per burst (Civ) before (Pre), during (Hype), and after (Post) IC hyperpolarization are plotted (n = 6). For all graphs, data points represent individual experiments connected by lines across conditions. Gray bars indicate the average across experiments. *p < 0.05, one-way RM ANOVA, Holm–Sidak post hoc test.

Figure 5.

LPG photoinactivation decreased pyloric cycle period variability in Gly¹-SIFamide. Ai, In Gly¹-SIFamide with LPG neurons intact, there are shorter cycle periods during LPG gastric mill-timed bursts (blue box), compared with SIFbaseline (dashed line) and longer cycle periods during IC activity (pink box). Aii, After both LPG neurons were photoinactivated (see Materials and Methods), pyloric cycle period variability appeared lower. Pink box highlights an IC gastric mill burst (group of pyloric-timed bursts >0.45 s duration). The small units in the lpgn recording are PY neurons. B, The pyloric cycle period CV is plotted for a 20 min window of steady-state activity in the LPG:Intact (blue dots) and LPG:Kill (black dots) conditions (n = 9). Each pair of dots plus their connecting lines represents a single preparation. Gray bars indicate the average CV across experiments in each condition. ***p < 0.001, paired t test. C, Overlaid histograms (bin size: 3 ms) of all pyloric cycle periods across 20 min windows during steady-state Gly¹-SIFamide application in the LPG:Intact (blue) and LPG:Kill (gray) conditions (n = 9). Each individual cycle period was normalized to the average SIFbaseline of the same preparation. Bin count of each condition was normalized to the peak count of that condition to facilitate comparison of histograms. Brackets on top of histogram indicate shorter pyloric cycle periods, SIFbaseline (arrow), and longer pyloric cycle periods.

Figure 6.

IC firing frequency was not affected by LPG photoinactivation. A, Representative traces illustrate differences in IC gastric mill-timed activity (brackets on top of mvn recording) before (Ai, LPG:Intact) and after (Aii, LPG:Kill) photoinactivation of the two LPG neurons. Pyloric-timed IC bursts within each IC gastric mill burst were identified if there was a gap between IC action potentials that coincided with a PD neuron burst (brackets below mvn recording). B, IC gastric mill-timed activity was quantified across 20 min windows during steady-state Gly¹-SIFamide bath application, including number of bursts (Bi), burst duration (Bii), number of spikes/burst (Biii), and firing frequency (Biv). C, IC pyloric-timed activity within each gastric mill-timed burst was also quantified, including number of bursts (Ci) burst duration (Cii), number of spikes/burst (Ciii), and firing frequency (Civ). Blue dots indicate LPG:Intact condition and black dots indicate LPG:Kill condition. All dots represent the average within an experiment with lines connecting the two conditions in each experiment. Gray bars indicate averages across experiments. *p < 0.05, paired t test.

Figure 7.

The same IC firing frequency did not alter pyloric cycle period in the absence of LPG neurons. Instantaneous pyloric cycle period is plotted against IC firing frequency in the LPG:Intact (blue dots) and LPG:Kill (gray dots) during 20 min windows. Each graph plots the data from a single experiment and each dot represents the instantaneous pyloric cycle period and the IC firing frequency within its pyloric-timed burst during the same pyloric cycle. Only cycle periods overlapping with IC gastric mill bursts (IC bursts >0.45 s; Fig. 6) are plotted.

Figure 8.

During the Gly¹-SIFamide modulatory state, gastric mill bursting in LPG decreases pyloric cycle period, while gastric mill bursting in the IC neuron requires LPG to prolong the pyloric cycle period. In saline (left, black traces and circuitry), LPG is active only in pyloric time, coincident with the AB/PD pyloric pacemaker neurons. Gly¹-SIFamide (right, green bar), elicits a unique gastric mill rhythm (Blitz et al., 2019; Fahoum and Blitz 2021, 2024), during which the pyloric cycle period varies between a baseline level due to Gly¹-SIFamide modulation (gray), shorter periods which occur during LPG gastric mill-timed slow bursting (blue), and longer periods which occur during IC gastric mill bursting (pink). Gastric mill bursting in LPG (blue box, G, gastric mill burst) likely allows sufficient current though the rectifying electrical synapses (diode symbol) to the AB and PD neurons to decrease the cycle period of the pyloric rhythm (blue). IC gastric mill bursting (pink burst; G, gastric mill burst) is necessary for rhythmic increases in cycle period of the pyloric rhythm (pink). However, this inhibition appears to act primarily via IC chemical synaptic inhibition of LPG (thick pink stick/ball), with a possible weak contribution via chemical inhibition of the AB and/or PD neuron (thin pink line/ball). The IC→LPG inhibitory synapse is strengthened during Gly¹-SIFamide modulation (compare thin line/ball in saline with thick line/ball in Gly¹-SIFamide; Fahoum and Blitz, 2023).