Fig 1.
Pyloric and gastric mill networks in the Cancer borealis STG respond differently to temperature perturbation.
(A) Main connectivity in the STG and innervation by the CoG projection neuron MCN1 (yellow). Blue circles represent pyloric neurons, and green circles represent gastric mill neurons. The pyloric and gastric mill CPGs receive excitatory input from MCN1, which innervates the STG via the ion and stn. MCN1 elicits a distinct version of the gastric mill rhythm that includes rhythmic bursting in LG, driven by the release of the peptide CabTRP Ia, electrical coupling between the MCN1 axon terminal and LG, and presynaptic inhibition of the MCN1 terminal [12]. CabTRP Ia activates the modulator-activated inward current IMI [14]. Bursting in LG also requires reciprocal inhibition with Int1. The pyloric circuit is pacemaker driven (AB) and modulated by MCN1. In most experiments, MCN1 influence was controlled by decentralizing the STG (arrow) and extracellular stimulation of the MCN1 axon in the remaining ion (“stim”). The yellow area indicates that temperature perturbations only affected the STG. (B) Example extracellular nerve recordings showing the spontaneous pyloric and gastric mill rhythms at low (T = 10°C) and elevated temperature (T = 13°C). Three extracellular recordings are shown. Top: dorsal gastric nerve dgn showing the activity of the dorsal gastric (DG) neuron. DG is a functional antagonist of LG. Middle: lateral gastric nerve lgn, showing the activity of LG. Bottom: lateral ventricular nerve lvn, showing the pyloric rhythm. The pyloric rhythm is triphasic and consists of the alternating activities of the PD neurons and the lateral pyloric (LP) and pyloric constrictor (PY) neurons. At 13°C, LG and DG activities cease, and the gastric mill rhythm terminates. Recordings are from the same preparation.
Fig 2.
The MCN1 gastric mill rhythm terminates at elevated temperature.
(A) Intracellular recording of LG during continuous extracellular MCN1 stimulation with 6 Hz at 10°C (top), 13°C (middle), and 10°C (bottom, post-control). LG was rhythmically active at 10°C (top). Rhythmic LG activity ceased at 13°C (middle) but could be restored by changing the temperature back to 10°C (bottom). Vertical scale bars, 10 mV. (B) Representation of LG spike activity for all preparations tested (N = 10) at 10°C (top), 13°C (middle), and 10°C post-control (bottom). Each trace shows 100 s during continuous MCN1 stimulation, with each vertical line representing an action potential in LG. Grey traces (trace 4) correspond to recordings shown in A. (C) Analysis of number of LG bursts/100 s (top) and LG spikes/burst (bottom) for all preparations tested (N = 10). Temperature was changed at 1°C/min. One-way repeated measures analysis of variance (RM ANOVA), F(2,18) = 280.503 (top), and F(2,18) = 76.963 (bottom), p < 0.001, Holm-Sidak post hoc test with p < 0.01 significance level. (D) Number of LG bursts/100 s (top) and LG spikes/burst (bottom) with slow temperature change (1°C/h). N = 6, Wilcoxon signed rank test, Z = 2.264, p = 0.031 for LG bursts/100 s and paired t-test, p < 0.01 for LG spikes/burst. (E) Change in number of LG spikes/burst plotted as a function of temperature from 8°C to 16°C during continuous temperature increase (~1°C/min). The different colors represent the LG response to two different MCN1 stimulation frequencies (4 Hz and 7 Hz). Stimulations were performed in the same preparation. Regression slope 4 Hz = −9.81*LG spikes/burst; 7 Hz = −7.52*LG spikes/burst, slopes significantly different from 0 with p < 0.001.
Fig 3.
LG membrane properties are affected by temperature change.
(A) Left: LG resting membrane potential at 10°C and 13°C for all tested preparations. Black circles represent individual experiments; colored circles are means ± standard deviation (SD). LG membrane potential hyperpolarized significantly at 13°C (N = 13, paired t-test, p < 0.001). Right: Decrease in LG resting membrane potential as a function of temperature from 9°C to 13°C during continuous temperature increase (1°C/min) in one preparation. (B) LG spike amplitude at 10°C and 13°C for all tested preparations. LG spike amplitude was significantly smaller at 13°C (N = 13, paired t-test, p < 0.001). (C) Left: Overlay of MCN1 ePSPs in LG at 10°C and 13°C. Right: Mean ePSP amplitudes at 10°C and 13°C for all preparations tested. Mean ePSP amplitude decreased significantly at 13°C (N = 13, paired t-test, p < 0.001). (D) Change in LG input resistance at 10°C and 13°C. (E) Membrane potential deflections of LG at 10°C and 13°C during de- and hyperpolarization (+3 to −3 nA, 1 nA steps are shown, 10 s duration). Measurements are from the same preparation. (F) Change in LG membrane potential as a function of current injection at 10°C and 13°C. Averages and standard error of the mean (SEM) of five experiments are shown. The starting membrane potential was set to −70 mV in all experiments. Note the difference in slope between 10°C and 13°C. Voltage deflections for all current levels were significantly smaller at 13°C (N = 5, paired t-test, p < 0.05).
Fig 4.
Changes in leak conductance are sufficient to terminate and rescue the rhythm.
(A) Top: Intracellular recording of LG during tonic MCN1 stimulation with 7 Hz at 10°C. Rhythmic activity ceased when artificial leak was added with dynamic clamp (10°C + Δleak). Bottom: Corresponding dynamic clamp current that was injected into LG. (B) Top: Intracellular recording of LG during tonic MCN1 stimulation with 7 Hz at 13°C. Rhythmic activity was recovered when artificial leak was subtracted (13°C − Δleak). Bottom: Corresponding dynamic clamp current that was injected into LG. Traces in B and C are from the same preparation. (C) Effect of artificial leak addition (10°C + Δleak) and subtraction (13°C − Δleak) on LG spike activity for all tested preparations (1 to 4). Each vertical line represents an AP in LG over 100 s of continuous MCN1 stimulation. Grey traces (4) correspond to recordings shown in A and B.
Fig 5.
In vivo gastric mill rhythms occur at elevated temperature.
(A) Extracellular recording of the lvn in an intact animal with spontaneous pyloric and gastric mill activity at 10°C (top) and 13°C (bottom) during fast temperature change (~1°C/min). As access to the motor nerves is limited in vivo, LG activity was assessed on the lvn. LG spikes are superimposed on those of the pyloric neurons (LP and PD are clearly discernable). For visualization, LG spikes are shown as vertical lines above the recording trace. (B) LG spikes/burst in vivo as a function of temperature from 9°C to 16°C during fast temperature increase (~1°C/min). Regression slope: −0.46*LG spikes/burst, slope significantly different from 0 with p < 0.049. (C) LG spike activity in vivo at 13°C for all animals tested (N = 7). Here, temperature was increased slowly (1°C/h) from 10°C to 13°C. Each trace represents one animal, and each vertical line an LG action potential.
Fig 6.
Increased MCN1 activity rescues the rhythm from temperature-induced breakdown.
(A) Extracellular recording of the ion showing the spike frequency (f) of MCN1 at 10°C and 13°C. CoGs were isolated from the STG by transecting all connecting nerves. MCN1 activity was recorded on the remaining stump of the ion that was still connected to the CoG. (B) Change in MCN1 spike frequency at 10°C and 13°C for N = 8 preparations. In each preparation, both ions were recorded (n = 16 recordings of bilateral MCN1). Black circles represent individual experiments; colored circles are means ± SD. MCN1 frequency was significantly higher at 13°C (N = 8, n = 16, paired t-test, p < 0.05). (C) Intracellular recording of LG at 10°C (top trace) and during increasing MCN1 stimulation frequency at 13°C (subsequent traces). Traces are from the same preparation. Vertical scale bars, 10 mV. Rhythmic LG activity was lost at 13°C but could be recovered by increasing MCN1 stimulation frequency. (D) Number of LG bursts/100 s (top) and LG spikes/burst (bottom) of all preparations at 10°C, 13°C, and 13°C with increased MCN1 stimulation frequency (= 13°C/rescue). Values at 10°C and 13°C/rescue were significantly different from 13°C (N = 10, Friedman RM ANOVA on ranks, χ2(2) = 15, p < 0.001, Tukey post hoc test with p < 0.01 overall significance level (top) and one-way RM ANOVA, F(2,18) = 79.199, p < 0.001, Holm-Sidak post hoc test with p < 0.01 (bottom). (E) Change in minimum MCN1 stimulation frequency needed to induce rhythmic LG activity (= “stim. threshold”) as a function of temperature. Means ± SD are shown (N = 5). Regression slope: 0.96*stim threshold, slope significantly different from 0 with p < 0.001.
Fig 7.
CabTRP Ia application rescues the rhythm from temperature-induced breakdown.
(A) Intracellular recording of LG at 10°C (top trace) and 13°C (subsequent traces) and response to 1 μm CabTRP Ia at 13°C in the absence and presence of tonic MCN1 stimulation (7 Hz). All traces are from the same preparation. Rhythmic LG activity was lost at 13°C but could be recovered in CabTRP Ia without increasing MCN1 stimulation frequency (iv). Vertical scale bars, 10 mV. (B) Change in number of LG bursts/100 s (top) and LG spikes/bursts (bottom) at 10°C, 13°C, 13°C + CabTRP Ia and after washout. N = 4, One Way RM ANOVA, F(3,9) = 50.558 (top) and F(3,9) = 124.33 (bottom), p < 0.001, Holm-Sidak post hoc test with p < 0.01 significance level.
Fig 8.
IMI counterbalances leak conductance to rescue neural oscillations in a computational model.
(A) Model output of LG membrane potential at (i) low leak and IMI conductance (gleak = 20 nS, ), (ii) increased leak (gleak = 40 nS,
), and (iii) increased IMI (gleak = 40 nS,
). Vertical scale bars, 20 mV. (B) Color map of number of bursts occurring in 100 s for 1,100 simulations as a function of leak and IMI conductances. Warmer colors represent more bursts;* highlights areas with one burst. (C) Color map of the number of LG spikes/100 s for 1,100 simulations as a function of leak and IMI conductances. Warmer colors represent more LG spikes. (B–C) Labeled areas (i–iii) indicate parameter sets corresponding to the traces shown in A.