The overall goal of the following experiment is to examine the topographical organization of synaptic and integrative properties of neurons in the medial enter RH cortex. This is achieved by preparing slices of medial enter RH cortex oriented in a dorsal ventral plane. As a second step patch clamp recordings are made mento RH cortex neurons to investigate their synaptic and integrative properties.
Next, the location of the recorded neuron is determined to assess the dorsal ventral organization of the electrophysiological measured properties. Results are obtained that show the topographical organization of the intrinsic properties of stellate cells in layer two based on the measurements of their input resistance, membrane time constant and action potential threshold, Demonstrating the procedure will be Hugh Pastel, a graduate student from my laboratory. The main advantage of this technique over existing methods such as horizontally oriented brain slices, is that the position of the recorded neuron along the dorsal ventral axis of the interal cortex can be accurately recorded.
To begin this procedure, remove the brain from the mouse and immediately place it in cold cutting. Artificial cerebral spinal fluid bubbled with carbogen. After three minutes, carefully remove the brain from the cutting A CSF using a spatula.
Gently place it in an upright position onto the filter paper that has been moistened with cutting A CSF. Then use a razor or scalpel to remove as much of the cerebellum as possible without impacting the medial enteral cortex, which is located at the cordal extreme of the cerebrum. Remove the rostral part of the cerebrum by sectioning in the coronal plane.
Next hemis sec, the brain at the vertical plane of the midline. After that, return the hemispheres to the bubbled cutting a CSF for one and a half minutes before mounting. Ensure that the cutting edge of the vibrato blade is angled at 20 degrees from horizontal On the mounting surface, make a shallow strip of super glue parallel to the vibrato blade, approximately the width of a hemisphere, and long enough to accommodate the two hemispheres end to end.
Then transfer each hemisphere from the cutting a CSF to the mounting surface by positioning the hemisphere so that its medial surface rests on the spatula and its dorsal extent faces towards the vibrate tone blade. Gently slide the hemisphere onto the strip of super glue and make sure that the medial surface of each hemisphere is parallel to the vibrato base. After that, immediately submerge the hemispheres in cold cutting.
A CSF. Maintain the temperature and car and saturation throughout the slicing procedure. With the vibram, remove the cortices from both hemispheres in the sagittal plane until the most lateral part of the medial enteral cortex in each hemisphere is identified at least 600 micrometers from the lateral surface.
The lateral extent of the medial enteral cortex is identified by the absence of the thick white band around the ventral cordal curve of the hippocampus. The non convex shape of its rostral boundary and the angular dorsal cordal corner cut 400 micron para sagittal sections from both hemispheres until the medial extent of the medial enteral cortex is reached. After each cut, immediately place the slices in carbogen saturated standard A CSF maintained in a water bath at 35 degrees Celsius and incubate them for approximately 15 minutes.
After 15 minutes, remove the slice holder from the water bath and continue bubbling with carbogen at room temperature for at least 45 minutes. Transfer a slice to the recording chamber, which is continuously bubbled with carbogen saturated standard A-C-S-F-A 35 degrees Celsius. Next, identify an approximate recording region within the medial enter RH cortex.
Then switch to a high magnification objective. Identify viable cells within this region. At this point, experiments using the conventional whole cell patch clamp to record membrane potential or membrane current from the identified neurons can be carried out.
Neuron identity can be verified from the electrophysiological properties of the recorded neuron and by including fluorescent labels within the intracellular solution. After the experiment to determine the position of a recorded neuron along the dorsal ventral axis, switch to a low magnification objective. Mark the location of interest by either including the recording electrode in an image or by stepping down the field iris diaphragm.
To leave a bright circle around the location of interest in a duplicate image image, the medial and the RH cortex region and the surrounding areas of the slice to pinpoint the recording location within the image, the duplicate can then be superimposed on the initial image of the recording location and its surroundings. Up to three separate low magnification images may be required to cover the area from a ventral recording location to the dorsal border of the medial interal cortex. These images can then be stitched together using image manipulation software.
The dorsal border of the medial enteral cortex provides a convenient landmark to measure dorsal ventral position, but the ventral border of the medial enteral cortex is not as well defined.Here. The DIC and NIAL para sagittal sections show the circular hippocampus and the lack of a paras orbicular protrusion into layer one of the lateral enteral cortex respectively. These are images of the typical slices that contain the medial enteral cortex.
The paras area is clearly revealed by missile staining. In the corresponding images, the dorsal border of the medial enteral cortex indicated by the black arrow is ventral to the group of paras orbicular cells that protrudes far out into layer one as indicated by the red arrow. Here is an example of a four times magnification composite image of a para sagittal slice.
The positions of a dorsal cell and a ventral cell are indicated by blue and green filled circles respectively. The black arrow marks the estimated dorsal border of the medial interal cortex and is extended into the deep layers by the white dotted line. The solid white line is a guide showing the contour path, along which the pixel measurement of distance from the dorsal border of the medial interal cortex to the ventrally located cell recordings from the marked dorsal and ventral stellate cells are shown here.
These recordings help to establish the identity of the cells and illustrate how electrical properties of medial enter r cortex. Layer two stellate cells differ at dorsal and ventral locations While attempting this procedure. It's important to remember to be exceptionally careful during preparation of slices as high quality slices are essential for productive electrophysiological recordings.
