Novel Transmitters II: Design and Analysis of a Nitric Oxide Synapse

Introduction: Correlation, Necessity and Sufficiency

• The difficulties of proving that a gas could serve as a transmitter highlighted a more general problem in science: proving cause and effect.

• We will briefly discuss this here, but you will see this recur multiple times in this course, and in science courses more generally.

• What do we mean, scientifically, when we say that A causes B?

• The implication of the statement is that whenever A occurs, and only when A occurs, then B occurs.

• To take a specific example, think of the statement "long exposure to the sun without shade can cause heatstroke".

• If one wished to treat this as a scientific statement, one would have to observe that:
  • (Correlation) On hot, sunny days, more people are admitted to the hospital with heatstroke.
  • (Necessity) People who carried an umbrella and used it to shade themselves whenever they were out in the sun do not get heatstroke.
  • (Sufficiency) Healthy people exposed to the sun develop heatstroke.

• Note the importance of the necessity and sufficiency components. If you only have the first statement, the correlative statement, you have not established causality.

• For example, you may observe that "Incidence of heat stroke admission to the hospital is always associated with softening of the pavement."

• But the correlation between these two observations does not imply that people having heat stroke somehow cause the pavement to become soft, or that softening of the pavement somehow induces heat stroke in people.

• Rather, both may be caused by another factor. In this example, it might be the presence of hot, direct sunlight with no shade over an extended period of time.

• Thus, it is important to remember, if you are doing science, correlation does not imply causality.

• Until you have established that A is necessary to cause B, and that A alone is sufficient to cause B, you have not proven causality scientifically.

• Here is a table that provides a shorthand way to remember what needs to be done, with concrete examples drawn from neural circuit analysis:

Correlation	A observed at the same time as B	Whenever neuron A fires action potentials, neuron B also fires action potentials
Necessity	If A is prevented from occurring, B no longer occurs	If neuron A is hyperpolarized, neuron B no longer fires
Sufficiency	If A is induced to occur, then B occurs	Depolarizing neuron A so that it fires action potentials induces neuron B to fire action potentials

• Another way of showing necessity is by using an occlusion experiment. Let us take the example of neuron A firing neuron B. Assume that you want to show that neuron A releases acetylcholine (ACh), and you have measured the amount of transmitter that A releases when it is fired at a fixed rate (say 10 Hz). Say that A induces B to fire at a rate of 5 Hz.
  • If you apply that same amount of ACh to neuron B, you get the same firing rate in B (5 Hz).
  • While still applying the ACh to neuron B, you now fire A at the usual firing rate (10 Hz).
  • You observe no change in the firing rate of B (i.e., it still fires at 5 Hz, or maybe just a slightly higher frequency, say 5.5 Hz)
  • This result suggests that neuron A works through ACh, since it appears to be using the same "pathway" to activate neuron B.
  • Since neuron A can't activate neuron B more intensely while you apply ACh, this suggests that its only way of acting is via ACh. This implies that if ACh was blocked, neuron A could not activate neuron B. Thus, ACh is necessary for neuron A to excite neuron B.

• Note that the same approach (of sufficiency and necessity) could be used to show that a particular molecule is crucial for some normal process. For example, "binding of trkA receptors by nerve growth factor (NGF) is essential for normal sympathetic innervation". To apply this analysis, one would show that
  • In growth cones of sympathetic neurons during development, both trkA receptors and NGF are present (Correlation);
  • If knockout mice are created that do not have trkA receptors, or do not produce NGF, then growth cones of sympathetic neurons are not observed or malfunction, and sympathetic innervation is absent (Necessity);
  • In knockout mice that do not produce NGF, which ordinarily show serious defects in sympathetic innervation, if exogenous NGF is applied, then growth cones are normal, and sympathetic targets show normal levels of innervation, i.e., the mouse's sympathetic innervation can be "rescued" by providing exogenous NGF (Sufficiency).

• Similarly, to argue that a particular compound is a neurotransmitter, one would need to show that
  • It is present whenever transmission occurs across a particular synapse; its levels may track the intensity of activity in a particular pathway (Correlation);
  • If a specific antagonist is applied that blocks its actions at its receptors or its targets, transmission at the synapse is blocked (Necessity);
  • If the compound is applied exogenously at levels comparable to those measured physiologically, it has the same effects as the endogenous compounds; also, if there is a very specific agonist, applying it mimics the effects of the endogenous compound (Sufficiency).

• In general, in designing experiments and in writing grants, having a clear understanding of how to move from correlation, which is just careful observation of the system, to causality, which allows you to make the claim that you understand the mechanism underlying a phenomenon, is crucial for success as a scientist.

Analysis of a Nitrergic Synapse

Here is the simulation that you began to study in the previous class:

• Question 1: Nitric oxide has been suggested as a neurotransmitter that could pass information from the postsynaptic to the presynaptic cell. Is that what is happening here? In a scientific context, if you want to test whether A causes B, you will often start by trying to show correlation (A and B occur together), sufficiency (if A then B), and necessity (if not A then not B). In this case, we want to ask if nitric oxide release (A) causes the changes we're seeing in spike shape presynaptically (B) (note that this is discussed in greater depth above).
  • Let's assume that we've run an experiment showing that nitric oxide is released from the postsynaptic cell (visualized by the indicator DAR-4M) by activation of the synapse with 50 Hz stimulation of the presynaptic cell for 1 minute. This experiment would show correlation between nitric oxide release and the change in spike half-height width we see with this kind of stimulation.
  • We next might try to test sufficiency: is release of nitric oxide sufficient to cause the changes in spike shape that we've observed? We could do this by adding nitric oxide directly and seeing if that causes similar effects, but nitric oxide is a toxic gas that can be difficult to work with. Instead, we can add a compound that releases nitric oxide in a biological environment. One compound that does this is the drug sodium nitroprusside (SNP), which is a short-acting vasodilator used to treat malignant hypertension and decompensated heart failure. By adding SNP to the solution bathing the neurons, we are able to generate nitric oxide near the neurons.
  • Is nitric oxide release sufficient to cause the presynaptic spike shape changes we've observed? Reset the simulation, and then add 100 µM sodium nitroprusside as a pretreatment. Measure the resulting spike half-height width, and compare it to the spike half-height width without the sodium nitroprusside but with 60 seconds of 50 Hz presynaptic prestimulation 10 minutes before the measurement (you took this measurement in Question 4 of the previous unit, but you can easily do so again). Are the two half-height widths similar? Does this support the hypothesis of sufficiency?

• Question 2: To test necessity, we need to show that without nitric oxide release, prestimulation does not cause a change in spike shape. One type of experiment that can be used to test this is an occlusion experiment. The idea of an occlusion experiment is that if cause A1 (e.g. nitric oxide release) and cause A2 (e.g. prestimulation) produce a similar effect B (e.g. change in presynaptic spike shape) through different pathways, then their maximal effects should be approximately linearly additive. If A1 and A2 share a common pathway, however, and if A1 saturates that pathway, then adding A2 should have little or no effect (A1 has "occluded" the effect of A2); so if the net effect is a small percentage change, that would not be approximately linear additive.
  • Is an increase in extracellular nitric oxide necessary for the change in presynaptic spike shape? Reset the stimulation. Measure the spike half-height width in a single experiment with 60 seconds of 50 Hz presynaptic prestimulation 10 minutes earlier; then add 100 µM sodium nitroprusside and repeat the experiment. Compare the half-height width to the one you found when you just applied the sodium nitroprusside in Question 1. Does this suggest that the effects on the presynaptic neuron due to activating the synapse, or applying a drug that releases nitric oxide (sodium nitroprusside), share a common mechanism? Do the results support the hypothesis that nitric oxide is necessary for the effect on presynaptic spike half-height width?

• Question 3: We will now try to determine the mechanisms that induce the presynaptic action potentials to change shape after the synapse is activated.
  • Is the change in spike half-height width caused primarily by a change in the rising time (upstroke) or the falling time (downstroke) of the action potential? Reset the simulation, and measure (a) the time from half of the maximum rise to the peak of the action potential and (b) the time from the peak of the action potential to half of the maximum fall in the presynaptic neuron. Check to be sure that your sum matches the half-height width that you measured in Question 4 of the previous unit. Add 60 seconds of prestimulation of the presynaptic neuron at 50 Hz with a 10 minute delay before the experiment, and repeat these measurements of the presynaptic neuron during the experiment. Is the change greater in the rising time or the falling time of the spike? Based on your measurements, would you hypothesize that control of voltage-dependent sodium channels or of voltage-dependent potassium channels dominate the change in the half-height width of the presynaptic action potential?

• Question 4: To distinguish the hypothesis that the primary action of nitric oxide on the presynaptic neuron is via its actions on voltage-gated sodium channels versus the voltage-gated potassium channels, it would be useful to block one class of these channels, and see if that could affect the response of the presynaptic neuron. Voltage gated potassium channels have several different subtypes, and one of the subtypes that is present in this cell is the Kv3 subtype. The drug tetraethylammonium (TEA) will block many potassium channels in higher concentrations, but in lower concentrations it can be used to selectively block Kv3 channels.
  • Using Question 1 as a template, design (write down!) an experiment using 3 mM TEA to test if blocking Kv3 channels is sufficient to cause the change in presynaptic spike shape seen with prestimulation of the presynaptic neuron. Describe your control and experimental conditions. Run the experiment using the simulation, and record your results. Do your results support the hypothesis? Explain.

• Question 5: Using Question 2 as a template, design (write down!) an occlusion experiment using 3 mM TEA to test if deactivating Kv3 channels is necessary for the change in presynaptic spike shape seen with prestimulation of the presynaptic neuron. Describe your control and experiment. Run your experiment with the simulation, record your results, and describe and explain what you observed.

• Question 6: We've now seen that previous presynaptic stimulation can affect both the presynaptic and the postsynaptic action potential. Can it also affect the synaptic strength? As you know from previous problem sets, the size of the postsynaptic potential (PSP) can provide useful information about synaptic strength. These are difficult to see when they trigger an action potential, however, because the action potential can obscure them. There are many different ways to prevent the action potential. For example, one could apply a drug like saxitoxin (STX) to reduce the excitability of a cell, or use voltage clamp. Because we already have a current clamp available, however, we will inject a hyperpolarizing current into the postsynaptic neuron to prevent the postsynaptic potential from inducing an action potential.
  • Reset the simulation. Set the number of presynaptic pulses to zero. Set the number of postsynaptic pulses to 1, the stimulus current to -0.4 nA with a delay of 10 ms and a duration of 150 ms. Set the simulation duration to 200 ms and run the simulation. You should see a single large hyperpolarizing response in the postsynaptic neuron.
  • Now, keeping the settings for the postsynaptic neuron in place, set the number of presynaptic pulses to 2 and the stimulus delay to 60 ms. Once you run the simulation, you should see two PSPs superimposed on the hyperpolarizing step (but no postsynaptic action potentials).
  • Is the PSP size influenced by prestimulation of the presynaptic neuron? Measure the heights of the two PSPs with and without 60 seconds of prestimulation of the presynaptic neuron at 50 Hz. By measuring the heights, we mean measure the voltage from the bottom of the hyperpolarizing pulse to the peak of the postsynaptic potential and report the difference in voltage between those two points. Compare the heights of the PSPs with and without pre stimulation. What do you observe? Explain.

• Question 7: Is nitric oxide release sufficient to cause the change seen in PSP size? Use Question 1 as a template to design an experiment (write down both the control and the experimental conditions!), run your experiment, and record your results. What do you observe? Is the hypothesis supported? Explain.

• Question 8: Is an increase in nitric oxide necessary for the change seen in PSP size? Use Question 2 as a template to design an experiment (write down both the control and experimental conditions!), run your experiment, and record your results. What do you observe? Is the hypothesis supported? Explain.