Contribution of the Na⁺/K⁺ Pump to Rhythmic Bursting, Explored with Modeling and Dynamic Clamp Analyses

Dynamic clamping can modify or introduce any membrane current into a neuron. We introduce and modify the sodium potassium pump and persistent sodium current into a living heart interneuron of a leech. Using a dynamic lamp allows complete control with a keystroke of the dynamics and amplitude of any current introduced into a neuron in real time.

The development of real-time interactive systems such as the dynamic clamp underpins all BMI research. All aspects of the cellular studies on the electrical activity of neurons and networks can potentially benefit from the use of dynamic clamp. The main challenges of this technique are developing the real-time models in neuron and calibrating the dynamic cramp adjustments to the neuron selected for study.

For isolation of the ganglion 7 from a leech nerve cord, fill a black resin line dissecting dish with about one centimeter of chilled saline supplemented with sodium chloride, potassium chloride, calcium chloride, D-glucose and HEPES in deionized water and pin a cold anesthetized leech dorsal side up in the chamber. Use a stereo microscope at a 20X magnification with oblique light guide illumination and five millimeter spring scissors to make a longitudinal cut at least three centimeters long through the body wall in the rostral third portion of the body. Use pins to pull apart the body wall tissue to expose the internal organs and vacuum the blood to get a better view.

Isolate an individual mid-body ganglion 7. Using sharp number five forceps to help guide the cutting and to hold the sinus, use the scissors to open the sinus in which the nerve cord resides, taking care to split the sinus dorsally and ventrally. Keeping the sinus attached to each of the two bilateral nerve roots that emerge from the ganglion, cut the rostral and caudal connective nerve bundles to remove the ganglion from the body and cut the roots lateral to where they emerge from the sinus.

Use old blunted number five forceps to secure the sinus strips and loose tissue with shortened Minutien insect pins ventral side up in clear resin-lined Petri dishes. Pin the rostral and caudal connectives as far from the ganglion as possible and securely pin the roots. Increase the magnification of the stereo microscope to 40X or greater and adjust the oblique illumination such that the neuronal cell bodies can be easily observed on the ventral side of the ganglion just below the perineurium.

Using micro scissors, begin cutting the loose peroneal sheath between the roots on one side of the ganglion, continuing the cut laterally to the other side and making sure to keep the scissor blade superficial without harming the neuronal cell bodies directly beneath the sheath. Make a similar superficial cut caudally from the lateral cut along the midline and use fine number five forceps to pull one caudal lateral flap of sheath away from the ganglion at a time to allow each to be excised. When both flaps have been removed, both heart interneurons should be exposed.

Place the dish in the recording setup and superfuse the sample with saline at a five milliliter per minute flow rate at room temperature. To identify the interneurons on the recording setup, select a 50 to 100X magnification with dark field illumination below and locate an HN 7 neuron of the bilateral pair by its canonical location at the posteriolateral position in mid body ganglion 7. Next, use a micro manipulator to position a sharp micro electrode filled with two molar potassium acetate and 20 millimolar potassium chloride micro electrode very near the target cell body and use a neurophysiological electrometer to continuously observe the recorded potential.

Set this potential to zero millivolts and penetrate the neuron with the microelectrode using the manipulator to slowly drive the electrode along its long axis. Use a 100 millisecond electrometer buzz function until a negative shift in membrane potential and vigorous spiking activity is observed. Set the electrometer to at least three kilohertz in discontinuous current clamp mode to simultaneously record the membrane potential while passing the current to the neuron.

Use an oscilloscope to monitor the setting of the electrode and use a steady current injector to inject a steady current of minus 0.1 nanoamps for one to two minutes to stabilize the recording. The HN 7 neuron can be identified by its characteristic spike shape and weak bursting activity. For dynamic clamp conductance and current implementation, open a dynamic clamp software program custom built for the digital signal processing board.

And while the model is running, set the maximal pump current to 0.1 to 0.2 nanoamps and gradually increase the maximum conductance of the persistent sodium current until regular bursting ensues. Systematically co-vary these currents in 0.1 nanoamp increments for the maximum output of the pump current and one nanosiemens increments for the maximum conductance of the persistent sodium current and assess the effects of these increases on the spike frequency, inter-burst interval, burst duration and burst period. Hold the maximal conductance of the sodium current at a specific fixed value and sweep in one nanoamp increments over a range of maximal pump currents to support regular bursting activity before increasing the fixed value of the sodium conductance by one nanosiemens and sweeping over a second range of maximal pump currents.

For each implemented parameter pair, collect data containing at least eight bursts so that reliable average measures of the spike frequency, inter-burst interval, burst duration, and burst period can be made. Then collect data from several additional neurons as just demonstrated to generate a composite graph. Using this model, the outward pump current oscillates throughout the burst cycle as the internal concentration of sodium around a baseline level.

This pump current contributes to burst termination during the burst phase. The hyperpolarization produced by the pump current activates hyperpolarization activated inward current during the inter-burst interval. The real-time heart interneuron model indicates that the persistent sodium current in heart interneurons contributes to much of the sodium entry strongly affecting the internal concentration of the sodium and thus the pump current.

Because the persistent sodium current is active at relatively negative membrane potentials, it opposes the pump current during both the burst and inter-burst intervals. As illustrated, robust bursting is restored in tonically active heart interneurons by the co-addition of persistent sodium and sodium potassium pump currents with a dynamic clamp. Preliminary results indicate a strong complicated interaction between the two currents, which can be further explored using the model.

A successful experiment depends on the good desheathing of the ganglion and carefully directed driving, penetrating, and buzzing of the microelectrode. We augment the dynamic clamp by implementing a sodium dependent pump current that is calculated by estimating the intracellular concentration of an ion. Such estimates can be used to inject any ion-dependent current into a neuron.