The overall goal of the following experiment is to examine the interaction between changes in voltage or current, such as changes in action, potential firing frequency, and the intracellular calcium concentration in supra matic nucleus neurons. This is achieved by preparing the brain slice for the recording of SMA nucleus neurons using a micro electrode filled with the calcium sensitive probe. As a second step, the micro electrode is sealed with the neuronal membrane.
Next, the neuronal membrane sealed with the micro electrode is ruptured to introduce the calcium fluorescent probe into individual neurons and to record along with the membrane electrical activity results are obtained that show measurements of voltage or current and images of calcium probe fluorescence at two wavelengths alteration in the ratio of fluorescence at these wavelengths reflects the change of intracellular calcium within the neuron. Subsequent analysis can be performed to determine the calcium concentration of the cell. The main advantage of this technique is that the changes in membrane electrical activity and intracellular calcium produced by synaptic activation can be simultaneously measured in individual supra cosmetic nucleus neurons.
Demonstrating this technique will be my colleague Dr.Robert Irwin. Begin this procedure by placing the rat brain on a circular filter paper moistened with slicing solution on a glass Petri dish, resting on ice, cut the brain corona only to remove the cerebellum and the brainstem, and cut off portions of the right and left sides of the cortex, leaving a block of brain containing the hypothalamus and carefully preserving the ventral side of the brain containing the SCN optic nerves and optic chiasm. After that, spread a thin layer of Sano acrylic glue, large enough to attach the brain and aros blocks in an ice cold microtome slicing chamber.
Transfer the ice cold brain block co side down onto the glue with the side containing the SCN positioned for cutting first and the optic nerves forming a v pointing up place the aros blocks on three other sides. Subsequently fill the chamber with ice cold slicing buffer, and continuously bubble with carbogen. The Sano acrylic glue will solidify with moisture and holds the brain and aros blocks in place.
Then cut the coronal hypothalamic slices containing the SCN at 220 to 250 micron thickness. Note that the left aros block detached during the cutting. This can happen if insufficient glue is applied.
After that, identify the slice containing the SCN. Typically, a slice is chosen where the optic chiasm is about two to four millimeters across appearing like a white band with the SCN just dorsal appearing as two small indentations in the optic chiasm on either side of the third ventricle. Transfer a slice to the 36 degrees Celsius recording chamber mounted on the stage of a microscope.
The chamber is filled with continuously flowing recording solution at two milliliters per minute. Aerated with 95%carbogen and preheated with an inline heater. Identify the SCN by observing the two translucent SCN nuclei on either side of the third ventricle, dorsal to the optic chiasm that appears like a black band when illuminated by transmitted light.
To prepare the micro electrode for recording, backfill it with the internal solution containing the calcium probe filtered with a four millimeter syringe filter via a microfill tube. Then place the micro electrode into the micro electrode holder. Next, apply a small amount of continuous positive pressure to the electrode.
Lower the micro electrode into the recording chamber with the guidance of red light illumination. Then place the micro electrode over the SCN using a low power objective. After that, switch the microscope to a high power magnification for selecting a sub region of SCN and an individual SCN neuron.
Continue to apply gentle positive pressure to the micro electrode as it is advanced onto the cell surface. Once it is on the surface, apply gentle negative pressure to form a seal with the resistance of three to 10 giga ohms. After that, adjust the membrane voltage to minus 60 millivolts.
Then apply additional negative pressure to the electrode to rupture the cell membrane in order to form the whole cell patch clamping. Upon entering the whole cell mode, the SCN neuron soma and dendrites will be filled with the fluorescent probe. Measure the membrane voltage in current clamp mode or the current in voltage clamp mode.
While the calcium probe image data is being monitored and stored, the acquired images can be viewed along with a graph of 340 nanometer and 380 nanometer ratio fluorescence data. During the experiment, a mono chronometer is used to obtain the calcium measurements quantitatively by rapidly exposing the SCN to UV light. At 340 nanometers and 380 nanometers, the excitation light passes through a UG 11 optical filter to restrict harmonic wavelengths above 400 nanometers and a 400 nanometer DCLP dichroic mirror reflects the light onto the tissue.
The emitted light passes through a 510 nanometer emission filter and is captured by the camera. To analyze the result, select a region of interest from the initial image and a background region to be converted to relative fluorescence intensity units. The region of interest data is seen in real time during the experiment.
The background is used later in subsequent data analysis. Here is a bright field image of a coronal slice of hypothalamus containing the SCN and optic chiasm. A glass micro electrode filled with internal solution containing BISRA two is sealed onto a single neuron.
Shown here are the calcium responses to a series of 50 voltage steps recorded from a voltage clamped SCN neuron. The voltage gated sodium currents were inhibited with 0.5 micromolar TTX, and this is a screen capture image of somatic and dendritic regions of interest corresponding to the calcium responses. These are the examples of calcium responses to a variable number of voltage steps.
This figure shows the simultaneous recording of membrane voltage and intracellular calcium. In an SCN neuron, the current clamp experiment was performed in the presence of 50 micromolar ritoxin. A small depolarization of the membrane potential triggered several bouts of action, potential firing with a corresponding rise and subsequent fall of intracellular calcium.
After watching this video, you should have a good understanding how to prepare healthy supra cosmetic nucleus brain slices, and to perform simultaneous electrophysiological and imaging experiments. Don't forget that working with ultraviolet light can be extremely hazardous, and precautions such as wearing proper eye protection should always be taken while performing this procedure.
