The overall goal of this procedure is to investigate local functional organization of inhibitory neuronal circuitry. The procedure begins with preparing living brain slices from the primary somatosensory cortex of mice. The next step is to visualize and record from targeted GFP expressing inhibitory cell types in the brain slice.
Following this laser scanning photo stimulation via glutamate, uncaging is used to activate small clusters of pre-synaptic neurons as excitatory or inhibitory. Post-synaptic currents are recorded in the GFP expressing inter neurons. The final step is to go through photo stimulation, data analysis, and generate quantitative maps of synaptic input to the recorded neurons.
Ultimately, laser scanning photo stimulation combined with whole cell recordings enables the construction of detailed maps of synaptic inputs to specific types of inhibitory neurons, which may allow for determining the organization and function of local inhibitory cortical circuits. The photo stimulation based technique was pioneered by Dr.Larry Katz, Ned Calloway, and further developed by Dr.Carols Waboda, my pi, Dr.Xing Z trained with Dr.Dr.Calloway, the Salk Institute, where he led many studies investigating interoral circuitry. Our group is currently extending and continuing these studies, investigating interoral circuits.
To begin this procedure, sacrifice a transgenic mouse at postnatal two to three weeks by decapitation and quickly remove the brain, place the brain in a frozen and oxygenated cutting solution. Then use the GFP goggles to visually screen the brain and check for GFP expression. Next, slice the primary somatosensory cortex into sections of 400 micrometer thickness with a vibrator.
Incubate the slices in the sucrose containing A CSF bubbled with 95%O2 and 5%CO2 at 32 degrees Celsius for 30 minutes to one hour. After that, transfer the slices to the regular A CSF at room temperature to get the laser ready for the experiment, turn on the laser cooling system and power supply. Then turn on all the hardware related to photo stimulation control, including the scanning mirror system and optical modulator and electronic shutter, and a photodiode input amplifier.
Next turn on the electrophysiological equipment such as multi clamp 700 B amplifier and micro manipulators. Electrophysiological recordings, photo stimulation and imaging are conducted in the slice perfusion chamber mounted on the motorized stage of the microscope. After that, turn on the imaging camera, the slice will be visualized in the upright microscope with infrared DIC and epi fluorescent optics through the imaging system.
Then open the MATLAB based FS software and Q capture camera software on the monitor. Before running the experiment, check the laser photo stimulation system and make sure that it is at a working state. Place a piece of paper under the four x microscope objective to observe the laser scanning pattern while the system is running.
To make the micro electrode with four to six mega OM resistance, pull a glass electrode with the pipette puller. Then fill the electrode with an internal solution containing 0.1%biotin for cell labeling and morphological identification. Next, turn on the pressurized perfusion system to pump A CSF into the chamber.
Ensure a constant fluid level of 2.0 millimeters above the slice in the chamber. An aliquot of the stock solution of MNI caged glutamate is added to 25 milliliters of circulating A CSF for a concentration of 0.2 millimolar caged glutamate. Then transfer one brain slice to the recording chamber.
Run the imaging system to check the slice quality and anatomically locate the primary somatosensory area. Next, anchor the slice with a custom made stringed platinum ring, and be careful not to put the anchor over the brain region intended for recording. Visualize the cells with a 60 x objective based on the visualization of GFP expression under A DIC fluorescent microscope.
Identify and select the inhibitory cell types. Please note that recording will be performed under visual control aided by infrared DIC. Video monitoring and switching between the DIC and fluorescent modes is necessary to confirm the GFP cell location while lowering the electrode to the target cell.
Using the conventional patch clamping technique. First, apply positive pressure to the electrode. Move it close to the cell surface to establish a discernible dimple upon the targeted cell membrane.
Then apply negative pressure quickly to form a giga seal and break in the cell while monitoring the current injection responses on the video monitor. Upon breaking in, take the images of the recorded GFP expressing intra neuron at 60 x magnification under the DIC and fluorescent modes for the online verification. Before collecting the photo stimulation data, inject hyperpolarizing and depolarizing current pulses to examine each cell's basic electrophysiological properties.
Once stable, whole cell recording is achieved with good access resistance, which is usually less than 20 mega ohms, switch the microscope objective from 60 x to four x magnification for laser scanning photo stimulation, acquire a slice image at four x magnification and use it for guiding and registering the photo stimulation sites. Set the photo stimulation and data acquisition parameters by activating the configuration switch module of phys. Use a one millisecond laser pulse of about 20 milliwatts with a stimulus interval of one second.
For photo stimulation mapping, acquire data traces of one second sampled at 10 kilohertz. Next, load the four x slice image into the mapper module of phys. Define the location of the soma and set up the photo stimulation sites of a 16 by 16 pattern with 80, micrometer by 80 micrometer.
Spacing around the cell location, which covers all the cortical layers. Then map the excitation profile of the recorded neuron by examining the spiking locations in response to photo stimulation at the current clamp mode. Our user-friendly software with the online display features facilitates the mapping experiments.
After that, map the local excitatory circuit connections by detecting the excitatory post-synaptic currents from the recorded cell With laser scanning at different locations, hold the cell at negative 70 millivolts in voltage clamp mode to detect inward excitatory currents. Then repeat the excitatory post-synaptic current maps two to three times with the photo stimulation pattern rotated or flipped. To map the local inhibitory circuit connections, detect the inhibitory post-synaptic currents from the recorded cell.
Hold the cell at close to zero millivolts in this case, negative 15 millivolts in voltage clamp mode in order to detect outward inhibitory currents. After completing all the physiological assays, remove the electrode gently from the recorded cell. Remove the brain slice from the chamber, then fix it in 4%para formaldehyde overnight, and stain the recorded cells against biotin.
In this experiment, the combination of GFP expression, intrinsic electrophysiology and morphological characteristics are used to classify the inhibitory neurons.Shown. Here are the example and a raw data map to illustrate the laser scanning photo stimulation, which allows extensive mapping of local cortical circuit connections to the recorded inhibitory neuron. Here are the examples of the color coded quantitative input maps derived from the raw traces just shown in Figure D.The amplitudes of the synaptic events detected in the recorded inter neuron are color coded and topographically superimposed upon the photo stimulation locations.
The cell body location is denoted by the red open circle in the scale bar. The warm colors indicate strong synaptic inputs and cool colors indicate weak or no connectivity to the targeted cell. Similarly, figure E presents the number of synaptic events per sampled uncaging location to the recorded neuron.
Figure F demonstrates the onset time of the first detected synaptic event to the recorded cell post photo stimulation in the scale bar. The warm colors indicate short input, latency, and cooler colors indicate longer synaptic latencies. Thus, LSPS enables the construction of detailed maps of the position, strength, latency, and number of inputs impinging onto specific types of inhibitory neurons.
Well, attending this procedure, it's important to be patient, make clean experiment condition, but neuron effectively and very carefully to change from object high magnification to low modification. Then the last use of land software very well.
