Signal transfer has an important role in the human body: without communication between cells, many processes such as action or perception could not take place. Rapid communication between cells largely goes via the nervous system. Through a mechanism in which a difference in voltage is created by diffusion and conduction of ions, nerve cells can transmit electrical signals through their outgoing processes, which are called axons. The electrical signals are transmitted at a certain velocity: the conduction velocity. There are two possible methods for measuring this velocity. The most commonly used method is the bipolar electrode stimulation method and a new method is the optogenetics method. During this research, we looked at the advantages and disadvantages of both methods in order to gain insight into which method is more accurate. Therefore, we measured the conduction velocity in the axon between the cortex and thalamus using both methods in a brain slice of a mouse. The analysis of both methodologies showed that the velocity can be measured more accurately with the optogenetics method, which is interesting for follow-up studies.

Introduction

The research that we carried out provides insight into the advantages and disadvantages of two possible methods to measure the conduction velocity through axons. Based on the results, an answer will be provided to the following research question: which method is more accurate to measure the conduction velocity in the axon between the thalamus and the cortex: the bipolar electrode stimulation method, or the optogenetics method? Based on previous studies, it was expected that optogenetics would be more accurate than the bipolar electrode method.^[1] The measurements are conducted on prepared brain slices of approximately 4 week old mice.

A more efficient method provides more accurate results, and allows future research to be carried out more accurately. With more information, the nerve system can be visualized better. In this study, we looked at the specific connection between the thalamus and the layer V cells in the cortex. It is a very long range connection, for which little research has yet been done.

The nerve cell

Nerve cells, also called neurons, are located in the central or peripheral nervous system. The central nervous system consists of the brain and spinal cord. In addition to the cell body, nerve cells consist of a large number of processes that go throughout the body. These processes, also called axons, form an important system to deliver impulses between neurons. The axons transfer the impulses to other cells via dendrites (see Figure 1a). At the end of an axon, a synapse is located. Here, the impulse is transmitted to the next neuron. ^[2]

 

Fig.1. (a) Structure of a nerve cell: the cell body with axons and dendrites^[3] (b) A bundle of axons in which the myelin sheath wrapped around the axons is colored blue (the axon itself is not visible).

Fig. 2. (a) Image of a thalamocortical slide with the bipolar electrode and a glass pipette. (b) Imaging of a thalamocortical slide with a beam of optogenetics. This is a test measurement: for the real measurements the light is placed further away from the pipette. (c) confocal image of labeled axons, somata and dendrites of layer V neurons (expression by the mCherry-ChR2 protein). (d) schematic mapping of the patch-clamp technique ^[4]

Signal transfer

Signal transfer and impulse conduction is a fundamental electrochemical process. There is a high concentration of K⁺ (potassium) ions inside the cell and a high concentration of Na⁺ (sodium) ions outside the cell. Because the concentrations of ions inside and outside the neuron differ, a potential difference (membrane potential) arises: –70 mV at rest. When a stimulus reaches the neuron, it depolarizes: Na⁺ ions flow into the neuron via Na⁺ channels. When the trigger threshold of –50 mV is reached, complete depolarization occurs: the potential changes to + 30 mV. Nearby Na⁺ channels open, therefore the impulse is passed on. The K⁺ channels then open, causing the membrane potential to drop. If the trigger threshold is not reached, the impulse will not be transmitted.^[1]

In addition to the signal transmission within the cell, the electrical impulse is also passed on to nearby cells. This is done via the synapse at the end of the axon. There is a synapse between two nerve cells. When the threshold is reached, the cell releases chemical neurotransmitters in the synapse. The neurotransmitters are responsible for the transmission or cancellation of the impulse in the next cell.^[5]

Increase in conduction velocity by myelin

Most axons are surrounded by a myelin sheath: a fatty substance (see Figure 1a and b). Due to this membrane insulation, the conduction velocity through a myelinated axon is considerably higher than in an unmyelinated axon.^[6]

The myelin sheath is interrupted by the nodes of Ranvier: axonal microdomains without myelin.

Action potentials arise in the nodes, causing the signal to \”jump\”, which promotes the conduction velocity. ^[7]

Layer V cells

In our experiments we determined the conduction velocity along axons between the cortex and the thalamus; two areas of the brain. The cerebral cortex of a mammal consists of different regions: the neocortex, the archicortex and the paleocortex.^[8] The cortex is important for processing sensory stimuli and it includes the brain structures that create and memorize observations and experiences. The neocortex is the largest area and consists of six different layers, layer V being one of the inner pyramid layers.^[9] Layers IV, V and VI contain neurons that are directly in contact with the thalamus.^[10]

Description of the methods

For both methods of measuring, a pulse is created to determine the conduction velocity in the axon. Based on the distance covered and the latency (time between stimulation and response) the average speed can be derived.

Bipolar electrode stimulation (method 1):

In this commonly used method, the bipolar electrode provides an electrical stimulus that creates an electric impulse. Under the fluorescence microscope the bipolar electrode is placed on a bundle of axons and sends a current surge to create the impulse (see Figure 2a).

The axon is then followed, and a healthy cell is searched for. This cell is then patched using the patch clamp technique. In this process, a glass pipette is closely connected to the cell membrane. A segment of the cell membrane is then opened by suction, and an extracellular solution is transferred from the pipette into the cell to keep it healthy (see Figure 2d).

Subsequently, the voltage in the axon can be measured with a recording electrode in the pipette. This excitatory postsynaptic potential (EPSP) is a response to the initiated current.

It is important that there is a high resistance between the cell and the pipette (>gigaohm). With a lower resistance, too much leakage of the current would occur, which would quickly result in poor measurements. ^[11][12]

Optogenetics (method 2):

This relatively new method uses the channelrhodopsin gene and the mCherry fluorescent protein.

First, the channelrhodopsin gene, obtained from an algae, and the mCherry fluorescent protein gene are connected and built into a virus. The virus is inserted into the brain of a mouse, where it infects all brain cells. Placing both genes within the brain cells. The two genes are then ‘cut loose’ from the virus DNA by the Cre-recombinase enzyme and translocated into the DNA of the mouse, allowing the virus and the gene to be expressed. In our research, we used mice from the Rbp4-Cre strain. Mice from this strain are genetically modified so that the Cre-recombinase protein is only built into the layer V cells.

After cutting, the mCherry protein emits light when a red light is shone onto the cells. This allows the position of the layer with V cells to become visible under the fluorescence microscope (see Figure 2c).^[13]

After cutting the genes lose, a blue light can create a signal; light with a wavelength of 470 nm aimed at the channelrhodopsin causes the cells to depolarize.

Materials and Methods

In our study, we used brain slices from mice. The mouse was anesthetized and quickly decapitated, after which the brain was removed and cut into slices with a cutting device called vibratome. The slices were placed in an extracellular fluid enriched with oxygen, to prevent the neurons from dying.

With both methods, a signal is given at a point on the axon, creating an impulse at site S1. Optogenetics makes use of blue light, while with the bipolar method an artificial stimulus is emitted (see Figures 2a and 2b).

The impulse is then passed through the axon and measured at the cell body of the next cell with the pipette, at spot S0. For both methods, the impulse lasts 10 ms. The artificial impulse of the bipolar electrode method has a strength of 10 volts. In both cases, S0 is determined first, after which the corresponding S1 is determined.

Determining S0: healthy cell

The pipette is placed on S0 using the patch-clamp technique. With this technique it is important that the patched cell is healthy; if the cell is not optimally healthy, the contact with the pipette can weaken the cell, making it impossible to take a proper measurement. The healthy cell is selected by appearance under the microscope; a healthy cell is round in shape and is not brightly coloured; unhealthy cells have an abnormal shape and are clearly visible.

If a cell without defects is detected, the pipette is placed against the cell membrane and the membrane is ruptured by applying suction.

After a cell has been patched, a series of stimuli is used to check whether the cell is healthy. If this is the case, a series of action potentials arise after the impulse (see Figure 3a).

Determining S1: spot on axon

When the cell is patched on S0 in the cell body, S1 is determined. With the bipolar electrode method, a bundle of axons is followed from the cell (see Figure 3b). The electrode is placed over the bundle of axons, as far away from the cell body as possible.

Using the optogenetics method, one single axon can be followed instead of a bundle. Because of the mCherry protein that is incorporated by the virus, the axons light up red under the microscope when light, with the corresponding wavelength, is shined on. The axon is followed as far away from the cell body as possible.

Synapse

The latency is measured between S1 and S0. However, the synapse is located between the axon and the cell body. Therefore, the measured latency time between the initiation of the impulse and measurement, includes the time needed for the impulse to bridge the synaptic cleft: the synapse time. Due to the synapse time, the actual conduction velocity can be approximated less accurately, because the measured latency time is extended. To calculate a conduction velocity without synapse time, a second measurement between S0 and S2 has been performed if possible (see Figure 3b). S0 is the cell body, and S1 and S2 are spots on the same axon, which runs from the cell body. There is a certain distance between S1 and S2.

Fig. 3. (a) Example of a graph used to check whether there is a strong contact between the pipette and the cell body. Only when action potential trains like this can be recorded, the cell is healthy and there is good contact. (b) Schematic drawing of a cell body with axon, where S0, S1 and S2 indicate different points of stimulation. (c) Example of a graph using the bipolar electrode method. The delay time indicates the time until the EPSP starts. The small peak indicates the effect of the electrical stimulus. (d) Example of a graph of the optogenetics method. This method does not use an electrical stimulus so there is only a large peak and no disturbance of the minimum.

The conduction velocity through the axon is then calculated by dividing the distance between S1 and S2 (in meters) by the difference in latency time between the measurement of S1 and S2 (in seconds). If two measurements can be carried out with one cell, the synapse time can be filtered out. This enables a better approximation of the conduction velocity.

Determining the latency time and distance

The used distances and latency times are determined with graphs and images made with the programs Axograph (version X), GraphPad Prism and ImageJ. The graph, as for example in Figure 3c, first shows a minimum, after which the graph rises to the maximum. The minimum is the lowest point of the graph and is manually determined with Axograph. The minimum shows when the EPSP starts, at this point the signal arrives at the cell body. The maximum shows when the EPSP is at its peak. To determine the latency time, the time is determined from the moment the impulse is created (start of the graph), to the minimum in the graph. The graphs of the bipolar electrode and the optogenetics method differ. The bipolar method makes use of an artificial electrical signal. Since the biological impulse is also an electrical signal, a problem arises.

The electrical stimulus arrives earlier than the biological impulse. Therefore the actual minimum of the graph is difficult to determine, since the minimum is overshadowed by the artificial electrical stimulus.

Results

The distance between the cell body and place of stimulation is plotted against the latency time in a graph,

(see Figures 4a and b). The conduction velocity through the axon can be deduced from these graphs: the slope of the linear regression curve is equal to the conduction velocity. Ideally, with both methods, two measurements are done on the same cell in different places on the axon. In that case, the conduction velocity is determined without the synapse time. However, with the bipolar electrode it was not possible to stimulate one axon at two positions. Therefore the conduction velocity is determined by plotting the distance with corresponding latency time of different axons against each other in a graph. The slope of the trendline through these points represents the mean conduction velocity. The conduction velocity measured with the bipolar electrode stimulation method is 0.528 m/s. With the optogenetics method, it was possible to create an impulse at two positions. The latency time of position 2 is longer than the latency time of position 1, measured closer to the cell body: the minimum of position 2 is therefore later than position 1 (see Figure 4d). The conduction velocity resulting from this measurement is 0.357 m/s. Both methods require a different approach, which resulted in a difference in conduction velocity. To determine with which method the conduction velocity can be measured more accurately, the bipolar electrode method and optogenetics are compared on several important criteria (see Table 1).

Fig. 4. (a) Graph of the bipolar electrode stimulation method. For different positions, the distance from the bipolar electrode to the glass pipette is plotted against the latency time. The trendline through these points is a measure of conduction velocity. (b) Graph of the optogenetics method. For two positions on one axon, the distance is plotted against the latency time. The directional coefficient of the line between these points is a measure of the conduction velocity. (c) Graph of the EPSP made with the bipolar electrode method. (d) EPSP graphs of position 1 and 2 from the measurement with the optogenetics method. The graphs are superimposed to show a clear difference in latency time.

	Bipolar electrode stimulation	Optogenetics
Stimulation of the chosen axon	–	+
Determining the distance between the place of stimulation and recording	–	+/–
Stimulation of single layer V cells	–	+
Possibility of second measurement with the same cell	–	+/–
Analyze graph	–	+
Applying patch-clamp technique	–	–
Virus injection necessary	+	–

Table 1: Assessment on key criteria of bipolar electrode stimulation and optogenetics: + means that the statement left is a characteristic of the method. – means that it is not and +/- means that it is not stated with certainty

Discussion

The adjacent criteria in the table are distinctive for the degree of accuracy with which the conduction velocity can be measured with the methods. The measurements consisted of several steps, as explained in the ‘Material and Methods’ section. While performing the measurements we encountered some difficulties on some points, which made it impossible to perform the measurement or to analyse the results.

By comparing the two methods per criterion, it can be assessed which method is more accurate.

With the bipolar electrode method, a bundle of axons is followed (see Figure 2a); the single axons are not clearly visible under the microscope. In addition, the electrode consists of two separate electrodes and is very large in size, relative to the axon; the cross section of the electrode is 500 µm and of an axon 0.1 – 2 µm. Due to its size, the electrode can only be placed over a bundle of axons, possibly stimulating several axons contacting the same cell body. It is therefore possible that the impulse measured by the pipette differs from the actual latency time when the impulse of only one axon is measured. The latency time and the potency of the measured impulse may differ, for example.

Due to the two electrodes and the size of the electrode, it is not possible to correctly determine the distance between S0 and S1. After all, it is unknown which axon within the bundle is stimulated. Hence, the distance is estimated: S1 is selected in the middle between both ends of the electrode (see Figure 2a). The difference in distance from one end of the electrode to the nucleus and the distance from the other end to the nucleus is 20%. This leads to a very large error, which results in a large measurement uncertainty.

Furthermore, it is difficult to perform two measurements with the same cell body using the bipolar electrode stimulation method. The entire electrode has to be moved, during which the brain slice can be easily displaced and contact with the cell might be lost.

Compared to the bipolar electrode stimulation method, the optogenetics method is less uncertain with respect to these criteria. Since the axons light up under the microscope because of the mCherry protein (see Figure 2c), the single axons are clearly visible. Therefore one single axon can be stimulated. Nevertheless, during our measurements it was not possible to optimally focus the blue light, by which the channelrhodopsin gene is activated. As a result, the blue light spot in Figure 2b is relatively large (100 µm). It is possible, however, to focus the light and increase the power by using a laser. We would recommend this method in future studies. While using a smaller light source, the distance between S1 and the nucleus can be determined better, since the area in which the signal is created becomes smaller. Because this error is measured in single micrometers, instead of hundreds of micrometers as with the bipolar electrode method, the optogenetics method is more accurate than the bipolar electrode stimulation method in this respect.

Additionally, a second measurement is more likely to be accomplished with the optogenetics method. The light source simply needs to be moved; therefore no contact with the cell body is lost. Because of the mCherry protein, the axon is easily followed, which makes it easier to select S2.

When the bipolar electrode method is used, it is not possible to determine in advance whether the neuron that is patched is in the requested area. This can only be verified through a stimulation protocol after the cell has been patched and an impulse can be created and recorded. This is checked via the paired pulse facilitation (PPF). With this method two stimuli are released in rapid succession, after which a distinctive pattern of the postsynaptic response can be observed: the second response is larger than the first one. If the pattern corresponds to the PPF, a thalamic cell that is connected to the layer V has been patched. If it does not correspond, the cell is contacted by neurons from another cortical layer and the patching and measuring has to be repeated. As the mCherry protein only expresses in layer V cells, since the enzymes are only present in this layer, solely layer V cells light up. The layer V cells and their axon terminals can therefore be recognized prior to patching.

To determine the conduction velocity, the distance from the stimulus to the minimum of the EPSP is measured in the graph. The minimum marks the beginning of the EPSP. With the bipolar electrode method, the minimum cannot be accurately determined, since the peak of the artificial electrical stimulus coincides with the beginning of the biological impulse (see Figure 3c). This leads to an error as large as the distance between the red lines; the actual minimum lies somewhere within these lines. This results in a difference of ± 2.5 ms, which is a significant difference on a measured value of approximately 3.5 ms. With the optogenetics method there is no artificial electrical stimulus, thus a natural EPSP can be seen and the minimum of the graph can be determined more accurately.

Both methods have the disadvantage that the cell body needs to be patched. The glass pipettes are very small and cannot be used when damaged. They are damaged very quickly, so measurements often have to be taken again.

The preparation of the mouse in advance of the measurements differs greatly between the two methods. In contrast to the optogenetics method, the bipolar electrode method does not require any specific action. The mouse should be around 4 weeks old, but does not need to receive any interventions in advance. Using the optogenetics method, the mouse undergoes surgery two weeks prior to the measurement to inject the virus with the mCherry protein and the channelrhodopsin gene in the brain. An opening needs to be drilled in the scalp to allow injection of the virus. In addition, it is required that the mouse is part of the Cre family line.

During our research we looked specifically at the layer V cells. Therefore, we used mice from a specific family line, in which the mice have been genetically modified so that the Cre-recombinase enzyme is only present in layer V cells. This enzyme cuts the mCherry protein and channelrhodopsin gene loose from the virus DNA. However, there are many transgenic mouse lines, each genetically modified in a different way. Hence, there are lines where the Cre-recombinase enzyme is present in other cortical layers and brain areas. Thus, the optogenetics method is widely applicable.

 

Conclusion

By comparing the advantages and disadvantages of both methods, we conclude that the optogenetics method is a more accurate method to measure the conduction velocity in axons than the bipolar electrode stimulation method.

The only advantage of the bipolar electrode stimulation method is that the mouse does not need to be prepared. With the optogenetics, the axons can be stimulated more precisely and the analysis of the graph is better. A second measurement can be executed on the same cell, which makes it possible to determine the latency time more accurately. Finally, it can be determined whether the connection that is researched, in our case between the thalamus and the layer V cells, is indeed stimulated.

With our findings, follow-up studies can be improved, which can assist with research into, for example, brain disorders.

For a better approach of the actual conduction velocity in an axon between the thalamus and the cortex, the optogenetics method can be performed with a centered laser. When the measurements are repeated multiple times, measurement errors will be reduced. For more data, the study can be carried out on other connections whose conduction velocity is known, such as the axons between the thalamus and cortex.^[6]

Acknowledgments

We thank Prof. dr. Maarten H. P. Kole and Nora Jamann (PhD student), at the Netherlands Institute for Neuroscience in Amsterdam for making this assignment possible and for their guidance during the research. Much of the necessary information has been provided from research carried out by them. The images without reference have been taken from articles yet to be published. In addition, we would like to thank Saskia van Asselt (MSc), who works at Utrecht University, for the guidance during our research.

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About The Authors

Manon van de Pol and Nienke Metzger are high chool seniors at the Goois Lyceum in the Netherlands.

 

 

 

 

They carried out the research trough an extracurricular program at Utrecht University. After high school graduation they will both continue studies in the medical field at universities in the Netherlands.