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Tension sensitivity of the heart pacemaker neurons in the isopod crustacean Ligia pallasii

Akira Sakurai^,* and Jerrel L. Wilkens

Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada
Present address: Department of Biology, Georgia State University, Atlanta, GA 30303, USA

View larger version (44K): [in a new window]   Fig. 1. The Ligia heart and the experimental setup. (A) Dorsal view of Ligia exotica showing the location of the tubular heart. The dorsal carapaces were partly removed to view the heart. (B) A schematic drawing of an opened heart viewed from the ventral side. CGN, cardiac ganglion neuron; HM, heart muscle; O, ostium. (C) A schematic drawing of the experimental setup for transversely stretching an opened heart. FS, fixed stage; HM, heart muscle; M, microelectrode; PS, pericardial septum; T, tergite; US, unfixed stage. After dissection (see Materials and methods), the lateral rims of the tergites remained attached to the heart via pericardial septum. The tergites were pinned on two separate Sylgard stages. One stage was pinned onto the bottom of the experimental chamber and the other stage was connected to a servomotor via a steel wire. By displacing the unfixed stage (arrow), one can apply stretch to the heart muscle. The ganglionic bursting activity was monitored by recording the CG-evoked excitatory junctional potentials (EJPs) intracellularly from the heart muscle. The inset shows a single burst of two EJPs recorded from the ostial muscle (calibration: 20 mV, 0.1 s).

View larger version (18K): [in a new window]   Fig. 2. Effects of long steady stretches on the heartbeat rhythm. (Ai—iv) In each record: top trace, membrane potential recording from the heart muscle showing bursts of excitatory junctional potentials (EJPs); middle trace, displacement of the unfixed stage, showing a 10 s stretch; bottom trace, a plot of instantaneous frequency of the EJP burst. The amplitude of displacement is shown on top of each record. Asterisks indicate slow muscle depolarization without EJPs. The records were obtained from a single preparation. The width of the opened heart during the 1.1 mm stretch (Aiv) was 2.9 mm, which is 38% larger than the calculated diastlic dimension (2.1 mm). The 1.1 mm stretch was approximately 2.5x larger than the amplitude of the circumferential dimension change in a given heart. (B) Steady-state burst frequency during the stretch (duration, 10-30 s) was plotted against amount of extension of the opened heart. Values for the burst frequency were normalized to the free-run frequency, and the change in width of the opened heart was normalized to the estimated circumferential dimension change in a beating heart tube (see Materials and methods). The horizontal bar in the graph shows a range of the changes in opened heart width (±0.5) that is equivalent to the circumferential dimension change in a beating heart. The control position is set at the median value between the maximum and minimum circumferential dimensions. Regression lines were calculated from data with the heart width less than the diastolic dimension (normalized heart width change <0.5, y=0.02x+0.99, r2=0.03) and from data with the heart width larger than the diastolic dimension (normalized heart width change >0.5, y=-0.29x+1.08, r2=0.53). Data points were obtained from 11 preparations.

View larger version (22K): [in a new window]   Fig. 5. Membrane potential responses of the cardiac ganglion (CG) neuron to stretches. (A) A schematic drawing of the experimental set-up for applying transverse stretches to the anterior half of the opened heart. After dissection, the lateral rims of only the thoracic tergites remained attached to the heart via pericardial septum. The heart wall in the posterior half was pinned extensively onto the fixed stage. (B—D) Membrane potential recordings from the CG neuron. In each record: upper trace, intracellular record from the cell body of the 6th CG neuron (Cell-6); lower trace, applied stretches shown by the amount of displacement of the unfixed stage. In B, stretch pulses (duration, 1.0 s; amplitude, 0.8 mm) were applied repetitively at 0.2 Hz. In C and D, the unfixed stage was moved in sinusoidal waveforms at 0.2 Hz (C) and 0.5 Hz (D).

View larger version (21K): [in a new window]   Fig. 3. Effects of brief stretches on the heartbeat rhythm. (A) Example records showing phase advance (i) or phase delay (ii) of the ganglionic burst cycle induced by a brief stretch (duration, 200 ms; amplitude, 0.7 mm). In each record: upper trace, membrane potential of the heart muscle showing rhythmical bursts of excitatory junctional potentials (EJPs); lower trace, applied stretch shown by displacement of the unfixed stage. A brief stretch was given at an earlier phase (i) or at a later phase in the burst cycle (ii). Dots above each record indicate the expected timing of the EJP bursts in the absence of perturbation. The 0.7 mm stretch was approximately 1.4x larger than the estimated amplitude of the circumferential dimension change in a given heart. (B) The relationship between changes in the burst interval and phases of the burst cycle in which the brief stretches were presented. The control burst interval was determined as the average interval of 10 bursts prior to the stretch. Data were obtained from three preparations, and each symbol represents data from one specimen. Free-run burst frequencies were 1.5 Hz (circle), 1.1 Hz (square) and 0.9 Hz (triangle). The stretches were given at a duration of 200 ms (circles and triangles) or 300 ms (squares). The amplitudes of the stretches (circles, 0.7 mm; squares, 0.5 mm; triangles, 0.8 mm) were approximately 1.4x larger than the estimated amplitude of the circumferential dimension change in the given hearts.

View larger version (22K): [in a new window]   Fig. 4. Effects of repeated brief stretches on the heartbeat rhythm. (A) Example records showing entrainment of the cardiac ganglion (CG) burst rhythm by repeated brief stretches. In each record: upper trace, membrane potential of the heart muscle showing bursts of excitatory junctional potentials (EJPs); lower trace, applied stretch shown by displacement of the unfixed stage. Brief stretches (duration, 300 ms; amplitude, 0.7 mm) were applied at 1.0 Hz (i), 1.3 Hz (ii), 1.7 Hz (iii) and 2.0 Hz (iv). (B) The relationship between the frequency of the CG burst during repeated stretches and the frequency of the stretches. Dotted lines indicate the frequency range of free-running heartbeat (1.44 Hz and 1.61 Hz). A linear regression line in the stimulus range between 1.1 Hz and 1.8 Hz is also shown (y=0.92x+0.11, r2=0.973). Data shown were obtained repeatedly from a single preparation.

View larger version (38K): [in a new window]   Fig. 6. Membrane potential responses of the cardiac ganglion (CG) neuron to stretches. (A,B) Superimposed traces of intracellular recording from Cell-6 (upper traces) in the normal saline solution (i) and in the presence of 1.0 µmol l-1 tetrodotoxin (TTX) (ii). Stretches were applied to the anterior half of the heart and are shown by the amount of displacement of the unfixed stage (lower traces). The records shown here were obtained from a single preparation. In B, the hyperpolarizing responses were shown at higher gain. Note that the stretch-induced hyperpolarization gradually decreased in amplitude during the stretch in the normal saline (Bi) whereas it stayed relatively constant in TTX (Bii). (C) Membrane potential of the CG neuron during the stretch versus amplitude of the stretch. Data were obtained repeatedly from a single preparation in the normal saline solution (open circle) and in TTX (filled circle). (D) Time latency for the rebound burst discharge after termination of stretch versus amplitude of the stretch. Stretches of various amplitudes were applied with durations of 1.5 s (triangle), 1.0 s (square) and 0.5 s (circle). Data were obtained repeatedly from a single preparation.

View larger version (15K): [in a new window]   Fig. 7. Effects of spontaneous muscle contraction on the membrane potential of the cardiac ganglion (CG) neuron. In each record: upper trace, the membrane potential activity recorded from Cell-6; lower trace, the myocardial tension recorded from the anterior half of the heart. Records A to E were obtained successively from a single preparation. (A) Control activity of the CG neuron and the heart muscle recorded in the normal saline solution. (B) 3 min after application of 10 µmol l-1 Joro spider toxin (JSTX). (C) 7 min after application of JSTX. The JSTX was applied for 17 min, and then the perfusion was switched to the saline containing both JSTX and tetrodotoxin (TTX). (D) 3 min after addition of 1.0 µmol l-1 TTX. (E) Bursting activity of the CG neuron recovered 3 min after washout of TTX.

View larger version (17K): [in a new window]   Fig. 8. Interaction between the cardiac ganglion (CG) neurons and the heart muscle. (A) A schematic drawing of the membrane potential activity in the CG neurons and the heart muscle tension. A burst discharge of the CG neurons induces tension of the heart muscle via the excitatory neuromuscular transmission (a). The muscle tension has a hyperpolarizing effect on the CG neuron (b-), which may assist the termination of the burst and enhance the after-burst hyperpolarization in the CG neuron. Relaxation of the heart muscle causes the postinhibitory rebound excitation (b+), which may advance the following burst discharge. See text for details. (B) A single neuron reflex arc formed by the CG neurons in the Ligia heart. The CG neurons function as endogenous oscillators by generating rhythmical burst discharges. They produce rhythmic motor output that is sent to the heart muscle via the neuromuscular junctions (a). They are also sensitive to the muscle tension (b), so that the heart muscle activity can entrain the ganglionic bursting activity under specific conditions such as when `a' is artificially blocked by Joro spider toxin (JSTX).