The animals were maintained in accordance with the established standards of animal care and procedures of the Institutes of Brain Science and State Key Laboratory of Medical Neurobiology of Fudan University, Shanghai, China.
1. Solution Preparation
NOTE: See the Table of Materials.
2. Preparation of Acute Hippocampal Slices
NOTE: See the Table of Materials.
3. Modifications of the Carbogenation for the aCSF Recycling of Small Reservoirs
4. Recording Synaptic Responses in a Submersion Slice Chamber
NOTE: See the Table of Materials.
5. Cleaning the Setup and Hints
NOTE: See below for general tips.
In the protocol section, we described the preparation of acute hippocampal slices from the ventral and intermediate part of the hippocampal formation (Figure 1) of male C57BL/6 mice and male Wistar rats (5-8 weeks). The position of the hemispheres on the slicer platform helps to keep them stable and removes the need of stabilization with agar or agarose. The perfusion system itself is based on a peristaltic pump operated on high rotation to give the required flow rate of the liquid. Thus, the rapid aging of standard tubes within the pump created a substantial problem that we overcame by using non-silicon tubes (ID: 1.02 mm). The negative pressure for the outflow is also created by two parallel-connected tubes that have an inner diameter of 2.79 mm. These two tubes are connected to a cannula in the recording chamber to allow for the aspiration of the liquid (Figure 4A). The carbogenation rate of the aCSF in the reservoir must be stable over many hours, which can be difficult to achieve using ventilation stones or tubes with small pores. Since the outflow-carbogenation does not rely on small pores, the carbogen flow rate over time remains stable (Figure 2). If the experiments are conducted with a small aCSF reservoir, the outflow-carbogenation helps to achieve an equilibrium of the dissolved oxygen (Figure 3). In addition, it minimizes the variability of the oxygen level between the experiments based on an altered inflow/outflow tube position, the number of active pores at the carbogen bubble tubes, and the liquid recycling rate.
The slice is placed on lens paper to allow some liquid exchange from below (Figure 4A). Since the slice chamber can be mounted on an upright microscope that is equipped with a 40X objective, the position of the electrodes can be well controlled (Figure 4B and Figure 5). This further reduces the variability of the experiments in day-to-day work.
To determine the stimulation strength required for the baseline recording of field potential, the dependency of the field potential size upon the stimulation strength must be determined by recording the fEPSPs at different stimulation strengths (Figure 5). The measurement of the input-output characteristic creates some stress on the slice at higher stimulation strengths. Thus, another approach is to search for a stimulation strength that just evokes a positive population spike component in the fEPSP-decay (black arrows in Figure 5)26.
After recording a stable baseline for at least 30 min, drug administration can begin. Here, we presented an example of the effects of an eEF2K inhibitor on synaptic transmission. Inhibition of the eEF2K promotes synaptic transmission (Figure 6A), an effect that was attenuated by the inhibition of p38 MAPK (Figure 6B)20.
Activity-dependent synaptic plasticity can be induced by a variety of stimulation paradigms10. Here, we presented representative experiments of synaptic plasticity that are based on a continuous sequence of stimuli at 100 Hz and on repeated stimulations at 1 Hz over 15 min. The resulting modulation of the synaptic transmission has been depicted in Figure 7 and Figure 8. In addition, it is shown in Figure 7 and Figure 8 that the stabilization of the oxygen level of small buffer reservoirs by the outflow-carbogenation (white circles, relative oxygen level at 24-40 min in Figure 3) improved the LTP and LTD induction in comparison to experiments with reservoir-carbogenation only (gray circles, oxygen level at 7-24 min). We did not test the combination of outflow-carbogenation and reservoir-carbogenation (7-24 min) because we were interested in creating an experimental system with lower and simpler carbogen use. The reservoir-carbogenation with no recycling (up to 7 min) represents the baseline level of oxygen in the aCSF reservoir.
Figure 1: Positioning the brain hemispheres to obtain transverse slices of the hippocampus using a vibratome. (A) The hippocampal formation is indicated in a lateral view of the right hemisphere, in green, within a reconstructed mouse brain model. (B) A ventral view of the hippocampal formation. (C) The rostral-to-caudal view demonstrates a section of the ventral and intermediate part of the hippocampal formation for the right hemisphere at the horizontal plane. Since different regions along the septotemporal axis of the hippocampus have different degrees of innervation and function31,32, a different cutting angle is required for transverse slices of the dorsal hippocampus, for example. Scale bar = 3 mm (horizontal white lines). (D) The photo shows the hemispheres of a rat brain glued on a vibratome platform. Scale bar = 6 mm. (E) The sketch depicts the angle for trimming the dorsal edge of the cortex (upper schema) of the separated right hemisphere (dotted black line) and the rotation (red arrow) to glue the brain on the created surface on the slicing platform. Please click here to view a larger version of this figure.
Figure 2: Elements of the carbogenation system for aCSF reservoirs below 50 mL. (A) Standard carbogenation systems using (a) perforated tubes as a carbogen bubble device; a tube was perforated by multiple punctures with a 250 µm-diameter stainless steel wire. (B) Outflow-carbogenation by (b) a 3-way tube connector (inner diameter: 2 mm) or (C) a vent unit that prevents liquid back flow with a hydrophobic filter. The middle arm of the 3-way tube connector is connected to a carbogen tank after a manometer (gas pressure: <1 atm) and a flow meter. The remaining arms are connected within the aCSF outflow tube. (D) The carbogen flow rate is controlled by flow meters. During the experiments, the aCSF reservoirs are kept at 28-30 °C. Please click here to view a larger version of this figure.
Figure 3: The oxygen levels in 40 mL of aCSF at different conditions. The oxygen level during the reservoir-carbogenation (res.-carb.) and aCSF recycling decreased constantly (gray-filled circles, n = 3). However, the outflow-carbogenation (outflow-carb.) during aCSF recycling reduced the drop of the oxygen level, which reached equilibrium at baseline levels (white-filled circles, n = 3). Baseline levels were measured with res.-carb. without aCSF recycling. The vertical dotted gray lines indicate the switch to different carbogenation protocols. The effects of res.-carb with recycling (7-24 min) and outflow-carb. with recycling (white circles, 24-40 min) on the induction of LTP and LTD are depicted in Figure 7 and Figure 8. The data are presented as the mean ± SEM. Please click here to view a larger version of this figure.
Figure 4: Components of outflow-carbogenation for experiments in a submersion slice chamber. (A) Presentation of the submersion slice chamber. The U-shaped platinum wire with the nylon fibers holding a hippocampal slice is depicted. The silver wires were wrapped around a small cannula, creating a coil to increase the area of the silver wire ending. The stimulation reference wire was placed in the outflow chamber, and the recording reference was in the main chamber. To keep the reference electrode potential stable at different liquid levels, the recording reference wire can be insulated by a plastic tube, leaving only the "underwater" part of the wire exposed to the buffer. (B) The bright-field image shows a representative acute hippocampal slice and the electrodes. CA1, CA3: Cornu Ammonis 1, 3; DG: dentate gyrus; sub: subiculum; EC: entorhinal cortex. (C) The schematic highlights the main components of the outflow-carbogenation, without an indication of flow meters for the carbogen and the peristaltic pump. A low-pass filter can be placed just in front of the inline heater to reduce the pulsation of the aCSF flow. Please click here to view a larger version of this figure.
Figure 5: Dependency of field potential shape upon stimulation strength and the position of the electrodes. (A) Representative examples of field potentials, evoked at different distances from the stratum pyramidale (str. pyr.), and stimulation strengths are depicted. The horizontal black arrows indicate the positive source component of the population spike in the field potential decay. The positive source component of the population spike remained approximately at the middle of the initial fEPSP-rise phase at all tested "in-line" electrode positions, except the one very close to the cell body layer (e). (B) The position of the positive source component of the population spike varied greatly due to the misalignment of the stimulation and recording electrodes. Lac.: stratum lacunosum moleculare; Rad.: stratum radiatum; Pyr.: stratum pyramidale; Rec.: recording electrode; Stim.: stimulation electrode. Scale bar = 100 µm. Please click here to view a larger version of this figure.
Figure 6: Representative example of the effects of an eEF2K inhibitor on hippocampal synaptic transmission using the outflow-carbogenation, with the recycling of a small aCSF reservoir20. After recording fEPSPs at 1 min intervals for 20 min, the eEF2K inhibitor A-484954 was added directly to the aCSF reservoir (horizontal line). A rapid increase of the synaptic transmission was detectable (white-filled circles). If the drug was not applied, the fEPSP-slope remained stable over the time of the recording (gray-filled circles). (B) Application of the p38 MAPK inhibitor (gray horizontal line) prevented the eEF2K inhibito- induced enhancement of field potentials. The data are presented as the mean ± SEM. Please click here to view a larger version of this figure.
Figure 7: Representative recordings of activity-dependent potentiation of synaptic transmission using outflow-carbogenation (outflow-carb.; white circles) and reservoir-carbogenation (res.-carb.; black circles), with the recycling of a small aCSF reservoir. The potentiation was induced after time point 0 by three times 100 Hz/s trains (Tet.: tetanization, high-frequency stimulation). The data are presented as the mean ± SEM. Bracket: statistical comparison of groups per time point; unpaired T-test, not assuming consistent standard deviation. *p <0.05. Please click here to view a larger version of this figure.
Figure 8: Dependency of activity-dependent depression of synaptic transmission upon stimulation strength and carbogenation. (A) Using reservoir-carbogenation (res.-carb.) with the recycling of a large aCSF volume, a 1 Hz stimulation for 15 min at 80% of maximal fEPSP-slope evoked a strong depression of synaptic transmission (white circles). However, as the representative fEPSP traces before (black trace) and after (red trace at the 110th min) LTD induction indicate, the presynaptic fiber volley (vertical black arrow) was greatly reduced. This is an example indicating that electrochemical processes at the stimulation electrode could harm the afferents, causing a depression that does not rely partially on LTD-related mechanisms. Reducing the stimulation strength to 50% of the fEPSP-slope maximum induced a depression of synaptic transmission to a lower degree, but without changing the presynaptic fiber volley (gray-filled circles). (B) LTD induction did not evoke a lasting depression of fEPSP-slopes in the experiments with the reservoir-carbogenation and the recycling of a small aCSF reservoir (res.-carb.; gray-filled squares). (C) The outflow-carbogenation with recycling of a small aCSF reservoir improved the outcome of the LTD induction. The inserts present representative fEPSPs before (black traces) and after (110th min, red traces) LTD induction. For the representative fEPSPs, the vertical scale bars indicate 1 mV, and the horizontal scale bars correspond to 2 ms. The data were obtained from acute hippocampal slices (350 µm) of 1 to 2 month old C57/BL 6 mice and are presented as the mean ± SEM. Brackets: statistical comparison of groups per time point; unpaired T-test, not assuming consistent standard deviation. *p <0.05, ns: non-significant. Please click here to view a larger version of this figure.
Reagents required | |||
NaCl | Sinopharm Chemical Reagent, China | 10019318 | |
KCl | Sinopharm Chemical Reagent, China | 10016318 | |
KH2PO4 | Sinopharm Chemical Reagent, China | 10017618 | |
MgCl2·6H2O | Sinopharm Chemical Reagent, China | 10012818 | |
CaCl2 | Sinopharm Chemical Reagent, China | 10005861 | |
NaHCO3 | Sinopharm Chemical Reagent, China | 10018960 | |
Glucose | Sinopharm Chemical Reagent, China | 10010518 | |
NaH2PO4 | Sinopharm Chemical Reagent, China | 20040718 | |
HEPES | Sigma | H3375 | |
Sodium pyruvate | Sigma | A4043 | |
MgSO4 | Sinopharm Chemical Reagent, China | 20025118 | |
NaOH | Sinopharm Chemical Reagent, China | 10019718 | |
Tools and materials for dissection | |||
Decapitators | Harvard apparatus | 55-0012 | for rat decapitation |
Bandage Scissors | SCHREIBER | 12-4227 | for mouse decapitation |
double-edge blade | Flying Eagle, China | 74-C | |
IRIS Scissors | RWD, China | S12003-09 | |
Bone Rongeurs | RWD, China | S22002-14 | |
Spoon | Hammacher | HSN 152-13 | |
dental cement spatula | Hammacher | HSN 016-15 | |
dental double end excavator | Blacksmith Surgical, USA | BS-415-017 | |
Vibrating Microtome | Leica, Germany | VT1200S | |
surgical blade | RWD, China | S31023-02 | |
surgical holder | RWD, China | S32007-14 | |
Electrophysiology equipment and materials | |||
Vertical Pipette Puller | Narishige, Japan | PC-10 | |
Vibration isolation table | Meirits, Japan | ADZ-A0806 | |
submerged type recording chamber | Warner Instruments | RC-26GLP | |
thermostatic water bath | Zhongcheng Yiqi,China | HH-1 | |
4 Axis Micromanipulator | Sutter, USA | MP-285, MP-225 | |
Platinum Wire | World Precision Instruments | PTP406 | |
Amplifier | Molecular Devices, USA | Multiclamp 700B | |
Data Acquisition System | Molecular Devices, USA | Digidata 1440A | |
Anaysis software | Molecular Devices, USA | Clampex 10.2 | |
Fluorescence Microscope | Nikon, Japan | FN1 | |
LED light source | Lumen Dynamics Group, Canada | X-cite 120LED | |
micropipettes | Harvard apparatus | GC150TF | extracelluar recording |
borosilicate micropipettes | Sutter, USA | BF150-86 | patch clamp |
tungsten electrode | A-M Systems, USA | 575500 | |
peristaltic pump | Longer, China | BT00-300T | |
tubes for peristaltic pump | ISMATEC, Wertheim, Germany | SC0309 | 1x inflow, ID: 1.02mm |
tubes for peristaltic pump | ISMATEC, Wertheim, Germany | SC0319 | 2x tubes for outflow, ID: 2.79 mm |
CCD camera | PCO, Germany | pco.edge sCMOS | |
lens cleaning paper | Kodak | ||
50 ml conical centrifuge tube | Thermo scientific | 339652 | |
Prechamber | Warner Instruments | BSC-PC | |
Inline heater | Warner Instruments | SF-28 | |
Temperature Controller | Warner Instruments | TC-324B |
Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.
Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.
Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.