Neuroscientists study the function of the brain by investigating how neurons in the brain communicate.Here. Changes in the electrical activity of a single neuron are evoked upon application of an experimentally controlled conductance using the dynamic clamp technique. First thin wrap brain slices are cut and stored in an incubation chamber filled with artificial cerebral spinal fluid.
A slice is then transferred to a patch clamp recording rig. A neuron is selected and then patched with a recording electrode using a dynamic clamp computer connected to the recording amplifier. An NMDA receptor conductance is applied into the cell.
The resulting recordings show a burst of action potentials suggesting that activation of NMDA receptors is one cellular mechanism through which dopaminergic neurons can generate bursts. The main advantage of this technique over current methods like direct current injection or pharmacological treatment, is that we are able to precisely determine the effects of activation of an experimentally defined receptor on the electrical activity of a living neuron. After quickly extracting the brain from an anesthetized Bragg dolly rat cut 240 micrometer horizontal brain slices using a vibrating microtome.
As the slices are cut, use a transfer pipette to place them in a 32 degree Celsius incubation chamber filled with artificial cerebral spinal fluid until it is time to perform recording experiments. The intracellular recording rig consists of a microscope with objectives set up for gradient contrast imaging, A monitor, a profusion system, a multi clamp 700 B amplifier, an analog to digital converter, a micro manipulator and head stage. A Mac Pro computer runs the data acquisition software, Axo Graph X and multi clamp commander Software adjacent to the intracellular recording rig and Mac Pro computer is a Sun Microsystems computer running real-time Linux, along with its analog to digital converter, which will be used with the dynamic clamp technique.
The intracellular recording rig should perfuse an artificial cerebral spinal fluid at approximately 35 degrees Celsius. If the slice to be recorded was horizontally prepared, use a transfer pipette to place it in a Petri dish with artificial cerebral spinal fluid and using a cutting tool bisect the slice along the midline. Next, to perform electrophysiological recordings, use a pipette to transfer the slice to the intracellular recording rig.
Using the 40 x objective, visualize the target neuron here, an individual substantial nigra dopaminergic neuron is seen. Note the elliptically shaped soma with two extending dendrites. Using an electronic P 97 micro pipette puller pull electrodes with a tip resistance of four to 10 mega ohms.
Next, using a microfill syringe needle, fill an electrode with internal solution. Insert the filled electrode into the half cell, connected to the head stage of the amplifier, using a syringe that is connected to the half cell by a small length of tubing. Apply a small amount of positive pressure to the recording electrode.
Under visual guidance, lower the electrode onto the target neuron. A dimple will be seen on the surface of the cell. Apply a small amount of negative pressure to create a giga seal onto the desired neuron.
Apply suction by mouth to rupture the seal. This constitutes a whole cell recording. After the seal has been ruptured, use the multi clamp commander software interface to place the amplifier in the current clamp mode in which no current can be injected.
To apply an NMDA conductance into this cell, begin by executing RTXI software on the dynamic clamp computer. Then load a custom written model containing an NMDA receptor into memory. Once the model has loaded into memory RTX, I will calculate the current to be injected into the cell in real time using the equation shown here where G-N-M-D-A is the desired conductance in nanos, Semens set to zero nanos.
Semens by default MG is the magnesium concentration. E-N-M-D-A is the reversal potential for the NMDA receptor set to zero millivolts, and VM is the membrane potential of the cell measured from the amplifier in millivolts. Now with the standard BNC cable, connect the output from the dynamic clamp computer to the command input of the amplifier with an analog to digital converter.
Place the amplifier in the standard current clamp mode ic using the multi clamp commander software interface. RTXI has been set up to receive the desired conductance GFT from a voltage signal from the Axo Graph X data acquisition software. Here we execute a simple AXO Graph X protocol in which a one second 40 nano Siemens conductance step is applied to the cell.
A burst of action potentials is evoked in response to the conductance step using the setup described in this video, a whole cell somatic recording from a dopaminergic neuron in the substantial nire pars compacta was performed. Dopaminergic cells typically fire spontaneously at low rates with a pacemaker like pattern. A burst of action potentials can be evoked by phasic application of an NMDA receptor conductance with dynamic clamp.
After watching this video, you should have a good understanding of how to apply an NMDA conductance into a living dopaminergic neuron in an isolated brain slice. However, this technique can be used for any cell type that can be recorded using standard electrophysiological techniques. Similarly, the dynamic clamp can be extended to mimic the electrical effects, but wide range Of receptor types.
We hope that this video has been useful in demonstrating what the dynamic clamp technique is and how we use it in our lab. So thanks for watching.
