Long-term synaptic plasticity, specifically, long-term potentiation and long-term depression have been investigated widely along the transverse orientation of the hippocampus. It has also been looked at along the longitudinal orientation. However, when it comes to the longitudinal orientation, very little attention has been given to the longitudinal CA1 axis of the hippocampus with respect to synaptic plasticity.
A previous publication from our lab have shown that the CA1 pyramidal neurons of the hippocampus form associational connections that could support synaptic plasticity. For further understanding of this protocol, it is recommended that you refer to the previous publication with the given reference below. Inject urethane to anesthetize the mouse.
Place the fully anesthetized mouse on a heating pad set to 37 degrees Celsius. Apply eye gel to moisten the eyes. Fix the skull of the mouse firmly using the eye clamp.
The eye clamp gives the freedom of mobility for the experimenter during surgical procedures. And it can be particularly useful for experiments involving the auditory cortex, as well. However, much care must be taken to get the exact stereotaxic coordinates.
Separate the subcutaneous tissue and muscles at the end of the interparietal and occipital bone of the mouse with the scalpel. Blot the dura matter dry with cotton swab. Then you puncture the cisterna magna to drain the cerebrospinal fluid.
Attach a cotton swab to keep draining the cerebrospinal fluid. Cut and remove the skin on the scalp and keep the exposed region dry. Mark the points corresponding to the hippocampal region using the aid of a veniyar caliper.
Make an incision in the skull above the dorsal region of the hippocampus along the marked points using a scalpel or high speed drill while observing under a microscope. Keep the exposed brain tissue moist by applying physiological saline or an inert oil. Fix and position the stimulation and recording electrodes firmly in the stereotaxic holder.
Adjust the stimulation and recording electrodes above CA1 dorsal hippocampus. Use the mouse brain in stereotaxic coordinate as a guide in locating the coordinate for dorsal longitudinal CA1 hippocampal region. For recordings using multichannel electrodes, first locate your stereotaxic coordinates for the CA1 hippocampal region using the first channel.
Position the remaining electrodes such that the angle of stimulation and recording electrodes are in the range of 30 degrees to 60 degrees in relation to the midline from bregma point. This angle corresponds to the longitudinal orientation of the CA1 hippocampal region. Turn on the recording system.
Open the neural software for recording and data acquisition. Lower the recording and stimulation electrodes slowly using the micromanipulator until it just touches the surface of the brain. Observe an increase in the impedance just when the electrode touches the brain surface.
This can be observed in the red channel shown on the screen. Slowly lower the electrodes to the approximate depth corresponding to chosen stereotaxic coordinates for the CA1 hippocampus. Give stimulation til a stable evoke-filled excitatory post-synaptic potential is observed.
Perform an input-output curve to detect maximum stimulus intensity. Use the input-output curve to set the baseline intensity and to set the stimulus intensity for evoking high frequency stimulation or low frequency stimulation. Record the local full potential for an hour after high frequency stimulation or low frequency stimulation.
Export data and analyze using software of choice. Poor 400 ml of already-prepared slicing solution into a separate flask and oxygenate for approximately 20 minutes. Poor 200 ml of the oxygenated slicing solution.
Cover in para film and transfer to a 80 degrees freezer for approximately 20 minutes to make a slush. Poor the remaining 200 ml of slicing solution in a brain slice holding chamber and keep in water bot of 32 degrees Celsius with continuous bubbling. Bring out chilled slicing solution and poor approximately 50 ml in a beaker.
Decapitate the anesthetized mouse. Cut the skin covering the skull of the mouse and cut the skull piece along the midline to the occipital bone. Open the skull with blunt forceps.
Gently scoop out the brain with a spatula and place in the chilled slicing solution in the beaker. Separate the two brain hemispheres along the midline with a scalpel blade. Isolate the hippocampus by gently detaching it from the cortex with a spatula.
Cut out the septal and temporal end of the isolated hippocampus. Apply a small amount of glue to the slicing plate. For longitudinal CA1 hippocampal slice, attach the CA3 region of the hippocampus to the slicing plate with the glue.
For transverse slice, which will serve as control, attach the ventral end of the hippocampus to the slicing plate of the vibratome. Place it in the slicing chamber. Set the vibratome parameters.
Slice the attached hippocampus with the vibratome. A good longitudinal CA1 hippocampal brain slice will have one layer of CA1, and two layers of dentate gyrus. Also, the stratum oriens and the stratum radiatum of the CA1 region will be present.
Alternatively, a transverse hippocampal brain slice will have the dentate gyrus CA3 and CA1 regions in tact. Transfer the brain slices to the brain slice holding chamber in the water bot. Incubate for 20 minutes at a temperature of 32 degrees Celsius and bring to room temperature for recovery.
Continuously superfuse the ACS serf into the recording chamber at the speed of 2 ml per minute. Transfer the brain slice into the recording chamber, adjust the position of the brain slice with blunt forceps and hold it in place with a hap. Turn on the software for data acquisition.
Longitudinal brain slice. For longitudinal slices, position the stimulation and recording electrode in the stratum oriens. Place the stimulation electrode on either the septal or temporal side of the brain slice with recording from the same layer.
Alternatively, position the stimulation and recording electrode in the stratum radiatum, place the stimulation electrode in either the septal or temporal side of the brain slice. For transverse slices as control, position the stimulation electrode on the CA3 Schaffer collateral pathway region, and the recording electrode on the CA1 region. Turn on the isolator stimulus generator and give stimulation.
Adjust the recording electrode depth til a stable evoke excitatory post-synaptic-filled potential is observed. Perform an input-output curve to detect maximum stimulus intensity. Use the input-output curve to set the stimulus intensity for baseline recording evoking high frequency stimulation or low frequency stimulation.
Refer to the attached PDF for parameters in inducing lone-term potentiation or long-term depression. Evoked excitatory post-synaptic potential increased in response to high frequency stimulation in vivo. This shows that longitudinal hippocampal CA1 supports long-term potentiation.
For longitudinal hippocampal CA1 brain slices, increase in response to high frequency stimulation was observed at both septal and temporal side of stratum oriens and stratum radiatum. This shows that the synaptic plasticity induced along the longitudinal hippocampal CA1 region is not linear or direction specific. Using already established protocols for long-term depression, no long-term depression was induced both in vivo and in vitro.
As a conclusion, we have shown you how to investigate long-term synaptic plasticity in the longitudinal hippocampal CA1 region both in vivo and in vitro. More details of the protocol can be found in the PDF accompanying this video to assist you in investigating long-term synaptic plasticity along the longitudinal hippocampal CA1 region in your own labs.
