Recording CA3-CA1 Synaptic Responses in a Rat Hippocampal Slice

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

1. Recording of CA3-CA1 Synaptic Responses

NOTE: The electrophysiology set-up used for field potential recording is shown in Figure 1A. A Faraday cage is strongly recommended if the electrical interference is beyond the control after the proper grounding of the electrical settings. Many different types of submerged and interface chambers are commercially available. However, interface chambers are preferred as slices exhibit more robust synaptic responses in them.

1. Positioning of electrodes​
   1. Turn on the electrical apparatus (stimulators and amplifiers) to be used. Mount and secure the stimulating and recording electrodes in the plexiglass holders of the micromanipulators.  
      NOTE: We use monopolar, lacquer-coated, stainless-steel electrodes of 5 MΩ resistance for both stimulating and recording purposes.
   2. Before use, insert these electrodes inside the pulled glass capillaries and secure with epoxy glue, exposing only small portion of the electrode tip (Figure 1E). This gives strength to the otherwise slender electrodes and helps to secure them firmly in the electrode holders.
   3. Guided under the microscope, position the stimulating electrode(s) in the stratum radiatum of the CA1 region to stimulate the Schaffer collateral fibers and the recording electrode in the apical dendritic region of CA1 to record field-EPSP (fEPSP) responses.  
      NOTE: Approaching the liquid surface above the slice with the electrodes gives a sound that helps to locate the surface of the slice quickly (provided, the amplifier is connected to a loudspeaker).
   4. In synaptic tagging and capture experiments, according to the need of the experiment, position two or three stimulating electrodes (S1, S2, or S3) on either side of the recording electrode to stimulate two or more independent but overlapping inputs. Position the stimulating and recording electrodes about 200 µm apart.
   5. If necessary, locate another recording electrode in the stratum pyramidale layer for recording population spike (Figure 2A). When both electrodes have touched the slice, using the acquisition software, give a test stimulation to ensure a proper fEPSP signal.  
      NOTE: We use biphasic, constant-current pulses (impulse duration 0.1 msec/half-wave) for test stimulation.
   6. Once a proper fEPSP signal is obtained, carefully lower the electrodes about 200 µm deep using fine movement knobs of the manipulators. Allow 20 min for the slices to recover.
2. Input-output relation​
   1. Determine the input-output relation (afferent stimulation vs fEPSP slope) for each input by measuring the slope value at a range of current intensities. Perform this between 20 µA to 100 µA. Then set the stimulation intensity for each input to obtain 40% of the maximum fEPSP slope. Keep this constant throughout the experiment.
   2. After 15-20 min, start recording the baseline. Monitor the fEPSP slope closely during this period and reset the stimulus intensity if the slope fluctuates more than 10% from the set value and start a new baseline. Record at least 30 min or 1 hr stable baseline before proceeding.  
      NOTE: For the test or the baseline stimulation, we use four sweeps of 0.2 Hz biphasic, constant current pulses (0.1 msec per polarity) given every 5 min. An average slope of these four responses is then considered as one repeat. The signals are filtered and amplified by a differential amplifier, digitized using an analog-to-digital converter and monitored online with custom-made software.

Figure 1. Electrophysiology set-up for field-potential recordings consisting of (A) stimulators (b) a differential amplifier (c) an analog-to-digital converter (d) Oscilloscope (e) computer with acquisition software (f) Vibration-resistant table-top (g) microscope with >4x magnification (h) interface brain-slice chamber (i) a perfusion system for ACSF and carbogen supply (j) temperature controller (k) an illumination source (l) manipulators with electrode holders. (B) Interface brain-slice chamber. (C) & (D) Hippocampal slices in the interface chamber. (E) Stainless steel electrode sealed in a glass capillary. 

Figure 2. (A) Schematic representation of a transverse hippocampal slice and electrode location for field-potential recording: In this representation, two stimulating electrodes (S1 and S2) are positioned in the stratum radiatum of the CA1 region to stimulate two independent but overlapping synaptic inputs onto CA1 pyramidal neurons. Two extracellular recording electrodes, one to record field-EPSP (excitatory post-synaptic potential) from the apical dendritic compartment and another to record somatic population spike from the pyramidal cell bodies, are located in the stratum radiatum and stratum pyramidale respectively. CA1- cornu ammonis region 1, CA3- cornu ammonis region 3, DG- dentate gyrus, SC- Schaffer collateral fibers, S1- stimulating electrode 1, S2-stimulating electrode 2. (B) Weak before strong paradigm to study STC: Weak tetanization (WTET) is applied to S1 (open circles) for inducing early-LTP followed by strong tetanization (STET) of S2 (filled circles) at 30 min to induce late-LTP. The early-LTP in S1 gets reinforced to late-LTP showing tagging and capture interaction (n = 6). (C) Weak before strong paradigm to study cross-tagging: Early-LTP is induced by WTET in S1 (open circles) followed by the induction of late-LTD in S2 (filled circles) using SLFS after 30 min. In S1, the early-LTP is transformed to late-LTP lasting 6 hr showing cross-tagging and capture (n = 6). Single arrow represents weak tetanization applied for inducing early-LTP. Triplet of arrows represents strong tetanization for inducing late-LTP. The broken arrow represents the time point at which SLFS was applied to the representative synaptic input to induce late-LTD. Error bars indicate SEM.