Bio 301, Smith College Lab 8: Crayfish Swimmeret System
Reference: Tschuluun, N, WM Hall, and B Mulloney (2009) State-changes in the swimmeret system: a neural circuit that drives locomotion. J. Exp. Biol. 212: 3605-3611.
Some of the methods used in these experiments may be unfamiliar to you. Among these are:
Measuring input resistance
The input resistance of a neuron reflects the extent to which membrane channels are open. A low resistance (high conductance) implies open channels, while high resistance implies closed channels. In practice, to measure input resistance, packets of charges (current pulses) are injected through a microelectrode, and the potential that results is measured. If the injected charges produce a big change in potential, few charges have leaked out across the membrane and the input resistance is high. If the injected charges produce a small change in potential, charges have leaked across the membrane and the input resistance is low (open channels have let the charges escape). Measuring input resistance while applying a transmitter shows whether the transmitter opens or closes channels.
Figure 6A shows a test for input resistance. At three points in the record (red arrows), trains of electrical pulses were applied to the neuron. Hyperpolarizing responses to the pulses appear like rectangular smears because the slow time-scale cannot show individual pulses. When the responses are placed next to each other (right), it is clear that the second response, which follows the application of octopamine (OA), is smaller than the first response. Octopamine has opened channels, reducing the input resistance. The third response is slightly larger as the octopamine diffuses away.
Discontinuous Current Clamp (DCC)
Although measuring input resistance is best accomplished with two microelectrodes, one to inject current pulses and one to measure the resulting potential, it is also possible to make the measurement with a single electrode. An electronic switch rapidly switches the wire to the electrode between a stimulator delivering the current pulses and an amplifier to measure the change in potential. If this is done frequently enough, and the artifacts at each switchover are small (which requires a low capacitance electrode), the method works as well as using two electrodes. "Discontinuous" refers to the switching back and forth (neither the stimulation nor the recording are continuous). "Current clamp" refers to injecting a constant level of current regardless of the resistance that is encountered.
Discontinuous Single-Electrode Voltage Clamp (dSEVC)
Single-electrode voltage clamping uses the same discontinuous system to switch a single electrode rapidly between measuring voltage and supplying current. The electrode does double-duty, playing the role of two electrodes in clamping a neuron at a particular voltage and measuring the currents flowing through channels that are open. In this paper, the authors achieve a sampling rate of 5 KHz, which means that the switching occurs fast enough to measure current samples five times per millisecond. This is fast enough to record the slow inward and outward currents flowing through the channels opened by the application of transmitters.
The motoneurons respond to transmitter with slow changes in their membrane potential, but they also make spikes in response to depolarization. To focus just on the slow changes, the authors use software to filter out high-frequency components of the recordings, allowing only low fequencies to pass through ("low pass" filters). Their cut-off frequency is 4 cycles/second, high enough to show oscillations associated with CPG cycles, but far too low to show spikes. All of the membrane current records in this paper are low-pass filtered.