The overall goal of this procedure is to perform patch clamp recordings of up to 12 cells in whole cell mode. This is accomplished by first marking the position of each cell of interest and assigning a pipette to each cell. The second step is to locate the tip of each pipette and automatically move the pipettes next to their assigned cells.
Next, approach each cell with one pipette at a time. To establish tight seals with the cell membrane, the final step is to rupture the membrane of the cells and perform whole cell recordings. Ultimately, computer assisted multi electrode patch clamp recording is used to show the network properties of small neuronal circuits with high resolution.
The main advantage of this technique over existing methods like manual patch clamp is that the number of connections that can be observed in a network grows with the square of the number of neurons that are being recorded. Visual demonstration of this technique is important because the patch clamp steps can be difficult to learn because of the complexity and the speed at which they need to be performed. Begin this procedure by placing a brain slice with the region of interest in the center of the microscope's displacement range.
Next, identify the cells of interest. Then save the position of the cells based on the microscope coordinate system. The graphical interface will display the selected cells as well as the micro pipettes for a global overview, and software will add a marker on the cell position that will be overlaid on the images.
Additionally, an image of the cell is captured for future reference. After selecting the cells of interest, assign the pipettes that will be used to record the individual cells by setting the assignment controls in the graphical interface. Visualize a preview of the final configuration by selecting the checkbox show final positions.
Now prepare the pipettes with intracellular solution and place them onto their holders. Place the head stages in their fixations, but do not slide them forward. To avoid touching the bath with the pipette tip, enable the positive pressure control and select all pipettes.
To ensure that the tips will remain clean, gently slide each head stage in place. Next, position the microscope to a central position with the focus of two millimeters above the slice by pressing the R two button while holding button A.Use the corresponding position for each manipulator as stored from the previous experiment by pressing the L two button while holding button A.At this point, there should either be a distinctive shadow of the pipette in view or a small movement along the pipettes axis should be enough to observe it. Compensation for the slight differences in pipette shape has to be performed manually.
Next, bring the pipette tip into focus. Then place the tip on the red central dot of the video display. Without moving the microscope, inform the software that the pipette is at the center of the display by pressing the Z button while holding button C of the controller.
After the individual pipette tip is located, send that pipette backwards so that the following one can be located by pressing the L one button while holding button a. Once all pipette tips have been precisely located and each pipette is attributed to a cell. Automatically position the pipettes close to their respective cells by simply right clicking the center of the group of cells in the graphical representation window and selecting the option cluster on the pop-up menu.
In the cluster options window, select all the pipettes that you want to position at this time and click go with the checked pipettes. Repeat this procedure for each cluster of cells of interest. To perform the final approach, specify the position of the pipettes relative to the cells as 200 microns away from the cell in the axis of the pipette and 200 microns above it in the vertical direction, which is enough to keep the pipette tip outside the tissue.
Then wait until the positioning of the pipettes is finished. Then move the microscope towards the pipette by pressing the R one button while holding button C.Recalibrate each pipette position by focusing the microscope on its tip anywhere in the video display and pressing the B button while holding button C.A square grid should briefly appear indicating the identified position of the pipette. If the position is not correct, position the tip on the central dot of the video display and press the Z button while holding button C of the controller.
Now confirm that the cell of interest for the current pipette is correctly marked, actively marked. Otherwise, move the microscope to match the cell of interest with the central red dot mark the cell by pressing the Y button while holding button C.Then press the L two button while holding button C.To position the pipette 200 microns away from its assigned cell, the microscope will automatically move to the corresponding position. Adjust the pipette position so it matches the red dot.
Then adjust the pipette offset by pressing the R one button. While holding button x. Activate the test pulse by pressing the L one button while holding button x.
After that, slowly move the pipette to its attributed cell. Upon observing the formation of a dimple on the surface of the cell's membrane, apply a brief pulse of negative pressure by pressing the Y button while holding button Z to allow the applied pressure to reach the cell. A holding potential of about negative 65 millivolts should be established at this point by pressing the L two button while holding button x.
Once a giga seal is formed for each cell, start rupturing the membranes by applying negative pressure. Next, use the stimulation acquisition system to perform recording. Apply pulses or trains of pulses on individual cells one at a time, and observe the responses of the remaining cells to map connectivity among these recorded cells.
Once recordings are finished, recede the pipettes slowly from the tissue by right clicking the table radio button. Select recede pipettes 500 microns to have the pipettes receded a short distance along their axes. Then observe the drift in the potential of the cells to recede pipettes all the way back.
Set the positioning speed to fast and repeat the same operation, but choose the all option. Remove the used pipettes by gently sliding out the head stages and unscrewing the pipettes from the holders. Here is an example of networks of direct synaptic connections mapped in individual experiments.
These three connectivity diagrams are sets of 12 parametal cells recorded in layer five with respective inters somatic distances. And here is the connectivity diagram superimposed on morphological staining of recorded parametal cells. This figure shows the common neighbor effect.
Blue columns indicate the pairs of neurons that are simultaneously connected to at least one other neuron in the sampled network. Exhibit A significantly increased probability of being interconnected. Here shows the pairs of neurons sharing multiple common neighbors occur more often than expected by chance in the sampled networks, the connection probability within pairs of neurons increases as a function of the number of common neighbors shared by the payer.
These properties in turn give rise to small world clustered networks. This figure shows the quantification of the recruitment of Martin nty cells. This is a graphical representation of a parametal cell in red that forms synapses onto a Martin nty cell in blue, which in turn forms synapsis on a second parametal cell.
In black supra threshold stimulation of the parametal cell leads to recruitment of the Martin nty cell. Through the integration of facilitating excitatory post-synaptic potentials, the recruited Martin nty cell then inhibits the second parametal cell. This diagram represents the stimulation of increasing numbers of patch clamped parametal cells and the effects on another parametal cell.
Here are the averaged inhibitory postsynaptic potentials recorded from a parametal cell as a function of the number of other nearby parametal cells that are stimulated. The amplitude of DYS synaptic inhibition in a local circuit tends to saturate when nine or more parametal cells are stimulated and the fraction of cells receiving DYS synaptic inhibition quickly rises to one as increasing numbers of parametal cells are stimulated. While attempting this procedure, it's important to keep the tips of the pipettes clean and minimize tissue distortion by avoiding blood vessels and other cells during the approach phase.
Following this procedure, additional methods can be employed such as photo stimulation to address additional questions such as the innovation of the patched clamp cells by another cell type. After watching this video, you should have a good understanding of how to accelerate and perform the patch clamping procedure for multiple neurons.
