Effects of GABA and CsCl on the Passive Membrane Properties of Hippocampal Pyramidal Neurons Abstract: Passive membrane properties of a neuron are transient and can be changed by manipulating the solution the neuron is in. In this lab, our goals are to measure passive membrane properties to calculate the time constant, to manipulate the passive properties by adding GABA to our saline solution and to measure the contribution of HCN channels to the overall resting conductance. In the first experiment, we determined the tau of the EPSP to be 76.65 msec. For the second experiment, after the addition of the inhibitory neurotransmitter GABA, the resistance of the cell decreased due to the opening of chloride channels which caused the potential difference to decrease. Furthermore, decreasing the resistance also decreased the time constant because the membrane responded faster to the stimulus. For the third experiment, the addition of CsCl, a blocker of the HCN channel, increased the resistance of the cell and thus also increased the potential difference. Introduction: Neurons are electrically excitable cells and behave like functional RC circuits. This property is what allows them to transmit action potentials, the electrochemical signals of the nervous system. Their membranes contain components analogous to the resistors, capacitors, pathways for current to flow and batteries in a circuit. Because neurons are analogous to circuits, we can use Ohm’s law, V=IR, to understand the electrical behavior of neurons. The voltage gated ion channels in the membrane allow the neuron to propagate action potentials. A membrane potential, or voltage, is created by the ion transporters in the membrane which maintain the ionic concentrations inside and outside the cell. The ion channels in biological membranes also act as resistors and the membrane itself acts as capacitors because it is an insulator that separates the intracellular and extracellular fluids. When a current is applied to a neuron, the current first charges up the capacitance and then changes the voltage. When the voltage reaches a steady state, that voltage is determined only by the current and the resistance and not by the capacitance. Furthermore, the resistance and capacitance of a neuron can be used to determine the time constant (tau), which is the time required to charge the capacitor (neuron) in a circuit to 66% of its final voltage or to discharge it to 33% of its initial voltage. The equation to determine tau from capacitance and resistance is: τ = RC where τ is the time constant, R is the resistance and C is the capacitance The objective of the first experiment is to make electrophysiological measurements of the membrane properties after the injection EPSC of 10pA and see how these properties affect the speed of voltage changes. The purpose is to compare the EPSC with the EPSP and see the
difference in voltage change due to resistance. My hypothesis is that the voltage change in the EPSP will be slower than the EPSC because the neuron has capacitance and resistance so the current first has to charge up the membrane and then the resistance, due to ion channels, reduces the current flow which slows down the change in voltage. For the second part of the experiment where we’re manipulating the membrane properties, we will make measurements of the neuron in normal saline solution and then GABA solution to see the effect of GABA on membrane resistance. The purpose is to see how adding GABA, an inhibitory neurotransmitter that opens chloride channels and thus decreases resistance, changes the time constant. In this experiment, the independent variable would be the solution and the dependent variable would be the voltage. My hypothesis is that the addition of GABA will decrease tau because resistance is decreased and the equation τ=RC, shows that the time constant and resistance are proportional, which further supports my hypothesis. Lastly, we are going to add a solution of 5 mM CsCl to our sample and then measure its effect on the conductance of our cell. CsCl is a blocker of HCN channels, which are activated by hyperpolarization. Some of these channels are open at the resting potential and thus affect our resting membrane potential. The purpose is to see how the blockage of HCN channels will affect the passive membrane properties of the neuron. The independent variable for this experiment would be the CsCl solution and dependent variable would be conductance. My hypothesis is that adding CsCl will decrease the overall conductance of the cell because CsCl is blocking channels which will increase resistance. Methods: Experiment 1) We recorded and made electrophysiological measurements of neurons in hippocampal pyramidal neurons from Long­Evans rats through patch clamping. Setting up the experiment, we placed the brain slice of the rat in a saline solution that is similar to cerebral spinal fluid called artificial cerebrospinal fluid (ACSF). Because brain tissue is sensitive to anoxia, a gas consisting of 95% oxygen and 5% carbon dioxide will be constantly bubbled through the ACSF. Next, we filled our pipette with a salt solution that mimics the ionic concentrations inside the neuron and then finally patched clamp our neuron. To make our measurements, we first had to set the capacitance compensation and bridge balance controls to offset the capacitance and resistance due to the electrode. We then used a positive step pulse of 250 ms and injected current until we got a voltage response between 2­5mV in amplitude. Finally, we simulated an EPSP response by injecting a preformulated stimulus EPSC of 10pA . Experiment 2) For experiment 2, we once again set the capacitance compensation and bridge balance controls and then measured the membrane time constant using the same 250 ms step pulse. Next, we injected a 50 Hz train of simulated EPSCs that mimic real glutamatergic EPSCs in hippocampal neurons to elicit a train of EPSPs. We adjusted the current amplitude until we got a voltage response between 2­5 mV in amplitude. Moving on, we changed the normal saline solution to the 100 µM GABA solution and waited for 5 minutes so the solution could equilibrate. After 5 minutes, we injected the same train of EPSCs used in normal saline and made a recording of the EPSPs. Finally, we delivered the same 250 ms step pulse with the same amount of current used in the normal saline solution.
Experiment 3) For the last experiment, we patched clamp another neuron in normal saline solution and injected currents of ­5pA, ­10pA, and ­15pA. Next, we changed the normal saline solution out with a solution of 5 mM CsCl and once again waited for 5 minutes for the solution to equilibrate. Finally, we injected currents of ­5pA, ­10pA, and ­15pA and made our recordings. Results: From our data of the three experiments we performed, we were able to show that increasing resistance due to the closing or blocking of ion channels increases the voltage response and decreasing resistance by opening channels decreases the voltage response. Furthermore, we were also able to show that increasing resistance also increases the time constant. In experiment 1, our goal was to see how passive membrane properties affected the speed of voltage changes and we did this by measuring the membrane time constant. In the first graph where we’re looking at a recording of the change in potential difference over time after the delivery of a positive step pulse of 250 ms, we calculated tau to be 76.65 ms. Also, since our current input was 60 pA and our voltage response was around 3 mV, using Ohm’s law, we calculated the input resistance to be 50 mega Ohms. Figure 1: For the second part of experiment 1, we wanted to compare the time constant of the EPSC with the time constant of the EPSP. From the graph, it is obvious that it takes longer for the voltage to change in the EPSP, the red graph, since tau is larger.
Figure 2: In the second experiment, we compared the voltage responses to the 250 ms current pulses in both normal saline and GABA solutions to see the effect of GABA on passive membrane properties. From the graph below, we can see that the voltage response is smaller in the GABA solution than the response in normal saline.
Figure 3: For the second part of experiment 2, we compared the subthreshold EPSP responses in normal and GABA solutions after the injection of identical current amplitudes. Figure 4 shows that there is temporal summation in normal solution but not in the GABA solution. Furthermore, the figure also shows that the time constant is smaller in the GABA solution.
Figure 4: For experiment 3, we wanted to compare the voltage responses in normal and CsCl solutions. Figure 5 shows us the voltage responses after injection of currents of ­5, ­10 and ­15 pA in normal saline and graph 6 shows the responses in CsCl solution using the same currents. The last graph is the result of combining the 2 recordings in the different solutions at 10 pA. Figure 7 shows that the change in voltage of the CsCl solution is larger than the one in normal saline solution. Furthermore, the addition of CsCl causes a change in the overall resting conductance of the cell since it is blocking channels. Figure 7 shows that the HCN channels contribute about 7mV to the resting membrane potential since the blockage of HCN channels due to CsCl increases the resting membrane potential by about 7 mV.
Figure 5: Figure 6:
Figure 7: Discussion: The fundamental concept of these experiments is to see how passive membrane properties affect the voltage response and the speed of voltage changes. The hypotheses are that increasing resistance will increase tau and the voltage response and vice versa. Looking at our data, it can clearly be seen that an increase in membrane resistance does cause an increase in voltage response and time constant. The speed of membrane voltage changes is determined by the capacitance and resistance of the membrane according to the equation τ=RC. Therefore, if there is no resistance, the change in voltage will be instantaneous. Applying this concept to experiment 1, we can see from figure 2 that the change in voltage is not instantaneous so there must be resistance. This resistance is the cause of the larger tau in the EPSP. Since resistance and tau are proportional, an increase in membrane resistance will also cause an increase in tau. This is due to that fact that the membrane can’t respond as fast when there’s resistance. Additionally, the EPSP is also much more slowly rising than the EPSC that elicited it because the current first had to charge up the membrane. The one major problem in our experiments is the resting membrane potentials in figures 1­4. The resting potential of most neurons is around ­65 mV but in these figures, they are between ­10 and ­5mV. This might be due to an error in offset voltage. However, the only thing that matters is the voltage response and the response doesn’t change no matter what voltage we set the resting membrane potential to be so our data is still accurate.
Experiment 2 further supports the hypothesis that decreasing resistance would decrease the voltage response. Figures 3 and 4 effectively show that the voltage response is much lower in the GABA solution. GABA is an inhibitory neurotransmitter that opens up chloride channels in the membrane. Therefore, the addition of GABA would cause a decrease in membrane resistance since there are more open channels allowing current to flow through. This decrease in resistance will also decrease the voltage response according to ohm’s law, V=IR. Since the current stays the same, changing resistance will also change voltage. Figure 3 supports the hypothesis because the change in voltage in normal solution is a little over 2 mV but the change in GABA solution is only around 0.4 mV. Figure 4 shows a similar trend with the voltage response in normal solution being bigger than the response in GABA solution. However, figure 4 is different from figure 3 because it shows temporal summation in normal saline. This difference is due to the injection of different kinds of current in figures 3 and 4. Figure 4 is the result of the injection of a train of EPSCs and it is clear that the voltage is slowly rising in normal saline with each current injection but the voltage response stays the same in GABA solution. This is due to the fact that there is less membrane resistance in the GABA solution. Because GABA opens up channels, it decreases membrane resistance which decreases tau because the membrane can respond faster to the injected current. In normal saline, the time constant is longer due to the higher resistance so it therefore takes longer for the membrane to repolarize. Because of this, there is more effective temporal summation in normal saline since more current is injected before the membrane is able to go back to resting potential. This experiment also shows that a neuron with a higher resistance would be more sensitive to small amplitude synaptic inputs. Since current isn’t changing, an increase in resistance will also result in an increase in voltage so if you’re injecting a small current, you would want a membrane with higher resistance to be able to see a larger voltage response. However, the subthreshold response in the GABA solution better reflects the timing of the simulated input since the membrane in GABA less resistance. Therefore, it has a smaller tau and can respond faster to the injected current. From the analysis of the data in experiment 2, I would agree that there is a trade off between sensitivity and temporal accuracy. Figure 4 shows that a higher sensitivity would mean less temporal accuracy since the membrane can’t respond as quickly in the case of the membrane in normal solution. Inversely, figure 4 also shows that an increase in temporal accuracy, in the case of the neuron in GABA solution, results in a decrease in sensitivity due to the lower resistance and therefore smaller voltage response. Experiment 3 supports the hypothesis that an increase in resistance causes a greater change in voltage. From figures 5, 6, and 7, it is evident that the addition of CsCl increases the voltage response. This is due to the fact that CsCl blocks HCN channels and a very small fraction of potassium channels. Blockage of these channels increases the membrane resistance and therefore increases the change in voltage according to Ohm’s law since we injected the same current in both the normal and CsCl solutions. A unique property about HCN channels is that they’re activated by hyperpolarization. This is the reason why we injected negative current in this
experiment. Looking at figure 7 where the recordings of the neuron at ­10pA in normal and CsCl solutions are compared, we can see a dip in the recording at the offset of the current injection in normal saline solution but not in the CsCl solution. The dip goes away in the CsCl recording because some channels are blocked. Furthermore, because CsCl also blocks a very small portion of potassium channels, the addition of CsCl tends to depolarize the cell a little because you’re preventing some potassium ions from flowing out so the resting membrane potential in the CsCl is higher than in normal saline. Conclusion: This lab not only taught me how to patch clamp neurons to make electrophysiological measurements, but it also taught me the relationship between resistance and voltage response. I’ve learned that an increase in membrane resistance due to the blockage of channels will lead to an increase in voltage response. This is apparent in experiment 3. Inversely, I’ve also learned that a decrease in membrane resistance due to the opening of channels, leads to a decrease in voltage response. This is shown in experiment 2. I’ve also learned that an EPSP is more slowly rising than the current that elicited it because of capacitance and this is shown in experiment 1.