This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.
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.
In 1951, the first-reported acute brain slice experiments were conducted1. In 1971, after successful in vitro recordings from the piriform cortex2,3 and the discovery that hippocampal neurons are interconnected transversely along the septotemporal axis of the hippocampus4, one of the first in vitro recordings of hippocampal neuronal activity was achieved5. The similarity of the neurophysiological or neurostructural parameters of neurons under in vivo and in vitro conditions are still the subject of some debate6, but in 1975, Schwartzkroin7 indicated that the basal properties of neurons are maintained in vitro and that high-frequency stimulation (i.e., tetanization) of afferents in the hippocampal formation induces a long-lasting facilitation of synaptic potentials8. Electrophysiological recording of neuronal activity in vitro greatly expanded the study of the cellular mechanisms of activity-dependent synaptic plasticity9,10, which had been discovered in 1973 by Bliss et al.11 in in vivo experiments with rabbits.
The study of neuronal activity or signaling pathways in brain slices, and especially in acute hippocampal slices, is now a standard tool. However, surprisingly, in vitro experiments have yet to be standardized, as evidenced by the multiple approaches that still exist for the preparation and maintenance of acute hippocampal slices. Reid et al. (1988)12 reviewed the methodological challenges for the maintenance of acute brain slices in different types of slice chambers and the choices of bathing medium, pH, temperature, and oxygen level. These parameters are still difficult to control in the recording chamber due to the custom-made elements of in vitro slice-recording setups. Publications can be found that might help to overcome some of the methodological challenges and that describe new types of submersion slice chambers, such as an interstitial 3D microperfusion system13, a chamber with enhanced laminar flow and oxygen supply14, a system with computerized temperature control15, and a multi-chamber recording system16. Since these chambers are not easy to build, most scientists rely on commercially available slice chambers. These chambers can be mounted on a microscope system, thus allowing for the combination of electrophysiology and fluorescence imaging17,18,19. Since these chambers keep the brain slices submerged in artificial cerebrospinal fluid (aCSF), a high flow rate of the buffer solution needs to be maintained, increasing the expense of drug application. To this end, we have incorporated a recycling perfusion system with outflow-carbogenation that provides sufficient stability for the long-term recording of field potentials in a submersion slice chamber using a relatively small aCSF volume. In addition, we summarized how the use of this experimental carbogenation/perfusion system affects the outcome of activity-dependent synaptic plasticity10 and how inhibition of eukaryotic elongation factor-2 kinase (eEF2K) modulates synaptic transmission20.
Although interface slice chambers exhibit more robust synaptic responses25,26,27,28, submersion chambers provide additional convenience for patch-clamp recording and fluorescence imaging. Thus, we have described several aspects of field potential recordings in acute hippocampal slices using a commercial submersion slice chamber that can easily be extended to the imaging of fluorescence probes i…
The authors have nothing to disclose.
W.W. conducted, analyzed, and designed the experiments and wrote the manuscript. D.X. and C.P. assisted in figure preparation and conducted the experiments. This work was supported by NSFC (31320103906) and 111 Project (B16013) to T.B.
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 |