Electrophysiological Characterization of Hippocampal Synaptic Transmission

Sixteen naïve mice (8 weeks of age) were used for recording fEPSPs evoked by stimulation of the afferent PP and MF pathways. Mice were anesthetized with Isoflurane (Baxter, Deerfield, IL, USA) and decapitated. Brains were quickly and carefully dissected and chilled in oxygenated ice-cold sucrose based cutting medium containing (in mM) 200 Sucrose, 50 NaHCO₃, 10 Glucose, 2.5 NaH₂PO₄, 1 MgCl₂ and 2 CaCl₂. Either HEC or coronal slices at 350 μm thickness were prepared from each brain with a VT 1200S vibratome (Leica). Live slices were maintained in oxygenated artificial cerebral spinal fluid (aCSF) containing (in mM) 130 NaCl, 3 KCl, 1.25 NaH₂PO₄, 25 NaHCO₃, 10 Glucose, 1 MgCl₂ and 2 CaCl₂, and kept in a 34–35° water-bath for at least 1 h before being transferred to the recording chamber. One brain typically yields 3–4 HEC slices, from which the hippocampal circuitry (including DG, CA1 and CA3) can be clearly identified under a dissecting microscope.
Field EPSPs evoked by PP stimulation were recorded in sl-m of CA1 (Figure 1B) with an Axopatch 1D amplifier (Axon Instruments, Union City, CA, USA) and the Clampex 9.2 data acquisition program (Molecular Devices, Sunnyvale, CA, USA), as previously reported (Schwarzbach et al., 2006 (link); Johnson et al., 2014 (link)). Stimulation was applied at the distal end of sl-m via a concentric and bipolar tungsten electrode (Frederick Haer Corporation, Bowdoin, ME, USA). The recording electrode for evoked field potentials were pulled from borosilicate glass (World Precision Instruments, Sarasota, FL, USA) with a tip resistance of 2–6 MΩ when filled with aCSF. The recording electrode was also placed in sl-m, proximal to the stimulation electrode such that orthodromic responses were produced at the recording electrode. Both electrodes targeted the median one-third of sl-m dorsoventrally, with a minimal distance of 600 μm between the two electrodes. A razor blade cut (Figure 1B, blue arrow) was made at the proximal point of CA1 to prevent activation of CA pyramidal cells disynaptically from area CA3 or trisynaptically from DG and CA3, as performed by other groups (Colbert and Levy, 1992 (link); Empson and Heinemann, 1995 (link)). For CA3 field recording (Figure 1B), the stimulating electrode was placed in the lateral part of the suprapyramidal blade of gcl. The recording electrode was located at sl of subregion CA3a/b, with at least 600 μm between recording and stimulating electrodes. All extracellular recording experiments were performed at room temperature, in an interface chamber (Scientific Systems Inc., State College, PA, USA) with an aCSF flow rate of 2.0 ml/min. Field potentials were recorded with single stimuli (100 μs in duration) ranging from 50 μA to 1000 μA in 50 μA increments to generate input/output (I/O) curves. The inter-stimulus interval for these field potentials was 8 s. Responses were quantified as the slope of the early, pseudo-linear portion of the response, and comparisons were made at the stimulus strength which gave an approximately half-maximal response, as determined by the I/O curve recorded in each slice. To check for release probabilies, 10 pairs of stimuli were also delivered, with a paired pulse inter-stimulus interval of 75 ms. From each animal, 2–3 slices were recorded.
To identify different components of the response in the recordings, chemical reagents were sequentially applied to the slices as needed. (2R)-amino-5-phosphonovaleric acid (APV, 50 μM; Abcam, Cambridge, MA, USA) together with 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 6 μM; Abcam) were used to block excitatory responses in the PP. To block the inhibitory component of the responses, bicuculline methiodide (BMI, 30 μM; Abcam) was applied. To selectively block MF-pyramidal cell transmission (Uchigashima et al., 2007 (link)), (1R, 2R)-3-[(1S)-1-amino-2-hydroxy-2-oxoethyl] cyclopropane-1,2-dicarboxylic acid (DCG-IV, 2 μM; Tocris Bioscience, Avonmouth, Bristol, BS11 9QD United Kingdom), a group II-specific agonist for metabotropic glutamate receptors was added to the superfusing aCSF. To isolate stimulation and/or system artifacts, we applied tetrodotoxin (TTX, a sodium channel blocker; 0.4 μM; Abcam) to block all biological responses.
For comparison of the evoked fEPSPs between HEC and coronal slices, we included only the stable baseline periods of the recordings, i.e., responses collected during repeated stimulation at an inter-stimulus interval of 30 s, and after the response to the bath applied reagents had reached a steady value. All of the traces acquired during the stable baseline period for each recording in a given condition were averaged together, and shown as a single waveform with Clampfit 9.2 program (Molecular Devices, Sunnyvale, CA, USA). The waveforms of all conditions were merged together to show differences in an identical slice.
Field potentials were analyzed by measuring the slope of the EPSP over the linear region of the initial portion of the response, and using stable responses acquired during the last 5 min (10 traces) of recording for a given condition. For each slice, comparisons were done using 10 individual slope measurements for a given slice under the different measurement conditions. For group analysis, the responses for each slice in the control condition and in the test condition, were normalized by the average value for each slice in the control condition. A one-way analysis of variance (ANOVA) with Bonferroni Multiple Comparison Test was conducted for the data from recording in CA1 sl-m. For the MF two-group analysis, a Student’s t test was performed. P values shown in the text were generated from group comparisons. EPSP slopes shown as Mean ± SEM.