Lab 4: Isolation and Recording from Leech (Hirudo verbana) Neurons

Annie Young NBio 301 AA February 19, 2013

Introduction: The medicinal leech provides a convenient system for understanding neuronal activity because of its large and easily accessible ganglia, and as such have been often used as a model organism in neurophysiology (Elliot and Kutschera, 2011). The ganglia are spaced along the length of the leechs ventral blood sinus and are connected by a bundle of axons called the connectiv e. For this lab our goal was to extract a ganglia from the medicinal leech species Hirudo verbana, insert a microelectrode into a cell or cells in that ganglia, and record electrical responses in the neuron(s). The ganglia of medicinal leeches contain many types of cells, including Retzius cells and mechanosensory neurons (T-cells, P-cells, and N-cells). It was not, however, our goal to record from any specific type of neuron. By recording from medicinal leech neurons, we hoped to better understand the electrical properties of live neural cells. Methods: Our methods can be found in the NBIO 301 Course Manual, pages 142-158. Alterations made to the method of dissection (to reflect a change of procedure from snail to leech) can be found in a separate lab handout. Results: Initial Recording: We successfully recorded from two cells in the isolated ganglia. The Cell 1 had no spontaneous activity initially and had a resting potential of about -50 mV (see Figure 1). For the first 10 seconds of recording there was no change in the spontaneous activity of the cell. We were able

to induce action potentials by applying a -0.36 nA current to the cell for 1.575 s. A representative action potential from this group of induced action potentials had no overshoot, a -7.31 mV undershoot, and an overall amplitude of 40.65 mV. We did not observe initial segment spikes or synaptic activity.

Induced Spontaneous Activity in a Medicinal Leech Neuron

Current (nA)

Voltage (mV)

Time (min:sec)
Fig. 1. Induced spontaneous activity in a medicinal leech neuron. Prior to application of current, there is little to no spontaneous activity observed. After a -0.36 nA current is applied for 1.575 s (arrows indicate beginning and end of applied current), spontaneous action potentials are observed. Star indicates the representative action potential used for amplitude calculations (no overshoot, -7.31 mV undershoot, and 40.65 mV overall amplitude). The star also indicates an action potential produced as a result of anode break excitation. This image begins when we had been recording intracellularly for roughly 2 s. This recording was taken in Cell 1.

Short Pulse Experiment: Our next set of recordings was the result of stimulating the neuron in short pulses. These were recorded from Cell 2. In order to find threshold, we delivered short pulses 10 ms long and steadily increased the pulse height until an action potential was triggered. At this point we would have continued to narrow down the threshold until we saw an action potential after a

long delay after the stimulus, but due to the unbalanced bridge and time constraints we had difficulty finding this data. What we can conclude from our data is that threshold is less than -24 mV, because this is the voltage of the short delay we did observe prior to the action potential. The action potential shown in Figure 2 had no overshoot and an overall amplitude of about 47.54 mV. The undershoot (and consequently exact overall amplitude) could not be obtained because of the proximity of the action potential to the bridge spike. Duration could similarly not be obtained.

Finding Threshold in a Medicinal Leech Neuron

Current (nA)

Voltage (mv)

Time (sec)
Fig. 2. Finding threshold in a medicine leech neuron. To find threshold, we applied short pulses of increasing amperage until an action potential was observed. Unfortunately the bridge was poorly balanced, which made it difficult to analyze our data. Threshold also seemed to shift as we recorded, possibly because the cell was in distress. Above the large spikes are artifacts of the bridge being unbalanced, while the smaller spike to the right indicates an action potential that occurred with an input of 3.03 nA. This recording was taken in Cell 2.

Summated Short Pulses: Our next set of recordings involved a series of short pulses that caused a summation of voltage within the cell. These were recorded on Cell 2. We repeated 8 pulses, with durations of 5 ms and a repeat rate of 100 Hz. Some amperages led to threshold while others did not (see Figures 3 and 4). A current of 1.77 nA appeared to be the lowest applied current that could stimulate

an action potential in 8 pulses or less, though this varied over the course of the recording because our cell was beginning to fade. A representative action potential generated this way (shown in Figure 4) had an overshoot of 42.62 mV, no visible undershoot (though this is probably because the sixth pulse obscured hyperpolarization), and an overall amplitude of about 109.15 mV.

Sub-threshold Summation of Voltage in a Leech Neuron

Current (nA)

Voltage (mv)

Time (s)

Fig. 3. Sub-threshold summation of voltage in a leech neuron. Current was applied in 8 repeated pulses with durations of 5 ms and amperage of 1.26 nA at 100 Hz. The voltages sum, but do not lead to an action potential and must reach a peak that is sub-threshold. This recording was taken in Cell 2.

Summation of Voltage in a Leech Neuron Leading to an Action Potential

Current (nA)

Voltage (mV)

Time (s)
Fig. 4. Summation of voltage in a leech neuron leading to an action potential. Current was applied in 8 repeated pulses with durations of 5 ms and amperage of 1.77 nA at 100 Hz. The voltages summed, and threshold was reached after the fifth pulse leading to an action potential. This action potential had an overshoot of 42.62 mV, no visible undershoot (though this is probably because the sixth pulse obscures hyperpolarization), and an overall amplitude of 109.15 mV. This recording was taken in Cell 2.

Responses to Long Pulses: The last set of measurements we took were responses to long pulses of current. These recordings were done on Cell 1. We began by depolarizing the cell for 1 s. We stepped up the applied current and plotted the number of action potentials produced by different current amperages (see Figure 5). A linear fit of this plot provided an equation y = 3.2x - 0.2 (see Figure

6). We then depolarized the cell with an applied current of -0.5 nA over several seconds in order to observe and measure the resistance-capacitance (RC) curve (see Figure 7). By measuring the voltage reached compared to the resting potential, resistance was found to be 50.22 M. By finding the time from the end of the pulse to when the curve reached 63% of resting potential, the time constant was found to be 2.5 ms. Using and the resistance, the capacitance was found to be 49.78 pF. Anode break excitation, when the cell produces an action potential following a long hyperpolarization, was observed in Cell 1. An example of anode break excitation can be seen in Figure 1, with the star indicating the action potential produced as a result of anode break excitation.

Production of Action Potentials with Varying Depolarization in a Medicinal Leech Neuron

Current (nA)

Voltage (mV)

Time (seconds)
Fig. 5. Production of action potentials with varying depolarization in a medicinal leech neuron. This recording shows applied currents 1 s in length and with increasing amperage being applied to the neuron. Note that the number of action potentials produced increases with increasing amperage (see Figure 6). Also note two of the stimuli last for more than 1 s this is due to operator error.

Number of Action Potentials Produced by Varying Applied Current

7 6 Number of Action Potentials 5 4 3 2 1 0 -1 0 0.5 1 1.5 2 2.5 Applied Current (nA) y = 3.2x - 0.2

Fig. 6. Number of action potentials produced by varying applied current. We depolarized the cell for 1 s and observed the number of action potentials produced during that time with different applied currents. A linear fit of the data produced the equation y=3.2x - 0.2.

Response of a Medicinal Leech Neuron to a Long Hyperpolarizing Pulse

Current (nA)

Voltage (mV)

Time (minutes:seconds)
Fig. 6. Response of a medicinal leech neuron to a long hyperpolarizing pulse. We hyperpolarized a leech neuron using a -0.5 nA applied current over a few seconds. The R value was found to be 50.22 M, the time constant was found to be 2.5 ms, and the capacitance C was found to be 49.78 pF. The arrow indicates the time that the curve reaches 63% of its maximum value, which was used to find .

Discussion: Impalement of a neuron with a microelectrode might have the effect of slightly depolarizing the cell if it tears a small hole and sodium ions are allowed to enter. This may cause some transient spontaneous activity which should either dissipate (if the issue resolves) or grow worse (if it does not) as time goes on. Real properties of the cell are likely to remain constant over time, while properties that are the result of injury or which are being affected by injury will likely change. Although we did not have good data for the short pulse section of the lab, we expected to see the membrane potential hang at threshold after the stimulus before firing (or not firing) an action potential. This is because at threshold the inward and outward flow of ions is equal and opposite, so with no further stimulus the cell will remain at threshold until a random opening or closing of a channel either depolarizes the cell and causes an action potential or hyperpolarizes it and prevents one. A train of pulses can stimulate an action potential when a single magnitude does not because of the capacitance of the membrane. The capacitance causes the membrane potential to decrease slowly after the stimulus is removed. This means that if pulses are repeated at high enough frequency the voltages will sum. If enough pulses are applied, they can bring the membrane voltage to threshold even if a single pulse of the same magnitude would not have. This is important for biological systems because it means that a neuron that does not respond to a single input may respond to repeated, high frequency inputs (Poirazi, 2003).

The increase in frequency of action potential firing with increased applied current is relevant to sensory transduction because it allows for the coding of intensity of stimulus even using the allor-none action potential. The action potentials amplitude cannot increase, but its frequency can. This frequency coding can also be used to determine stimulus identity, but the patterns of bursting help the brain translate between the two (Stopfer et al. 2003). Anode-break excitation occurs because the hyperpolarization opens all or many more of the inactivation gates on sodium voltage-gated channels. This lowers the cells threshold, so when the hyperpolarizing stimulus is removed, the cell fires an action potential.

References: Elliot, J. M., and U. Kutschera. "Medicinal Leeches: Historical Use, Ecology, Genetics and Conservation." Freshwater Reviews. 4: 21-41. 2011. Poirazi, P. et al. Arithmetic of Subthreshold Synaptic Summation in a Model CA1 Pyramidal Cell. Neuron. 37 (6): 977-987. 2003. Stopfer, M. et al. Intensity versus Identity Coding in an Olfactory System. Neuron. 39: 9911004. 2003.