There are multiple variations of preparation techniques for acute slices. The precise method should depend on the purpose of the study and the area of interest. Initially most laboratories used a tissue chopper for cutting the tissue into thin slices; later, vibratomes were adopted in many laboratories because they appeared to make slices with less mechanical damage. Efforts have been made to determine which cutting parameters (with the vibratome) are most important for preservation of fine tissue details (preservation is essential, for example, for carrying out recordings from subcellular compartments such as presynaptic terminals (Geiger et al., 2002)). Likewise the storage of slices is an important issue. In our laboratory, for experiments under submerged conditions, slices are placed on a nylon grid, which is fixed at the surface of ACSF in a homemade Perspex incubation chamber (at room temperature). The incubation chamber contains 50 ml of gassed (95% O2/5% CO2) ACSF (static bath). For recordings, slices are individually transferred to a submersion-style recording chamber. For interface chamber experiments, we have found the best tissue preservation when slices are maintained in an interface chamber on a piece of lens paper, which facilitates transfer of the slice to the recording chamber. A period of 2 hours allows recovery of swollen neurons and mitochondria.
Slice recordings can be performed in an interface chamber or in a submerged chamber. Alternatively a grease gap chamber can be used in which slices set over a separation sealed with Vaseline (Avsar and Empson, 2004), and recordings can be performed by measuring the potential difference between the two chambers. This procedure permits recording of different types of epileptiform activity and easy analysis of drug effects on such activity. Interface chambers are available in two basic variations: (1) the Oslotype chamber, developed in Per Andersen's laboratory from the original design of McIllwain, who used slices to study brain metabolism (Andersen, 1975); and (2) The Haas-type chamber (Haas et al., 1979). We prefer the Haas chamber, where the slices set on the floor of the chamber, providing mechanical stability. An always-important issue is the temperature at which the tissue is maintained because seizurelike events are difficult to induce at temperatures below 33° C. Another important issue is the composition of the ACSF, which is used to perfuse the in vitro preparations. Our laboratory uses ACSF containing (inmM): NaCl 124, KCl 3, MgSO2 1.8, CaCl2 1.6, Na^PO4 1.25, NaHCO3 26, and glucose 10. The solution is equilibrated with carbogen gas (95% O2/5% CO2) to maintain a pH of 7.4; the osmolarity of the solution should be about 290mOsm. Ca2+, Mg2, and K+ concentrations must be carefully adjusted because small variations in concentrations of these ions have marked effects on neuronal activity. The extracellular Ca2+ concentration ([Ca2+]o) in vivo is about 1.2mM which is mimicked in slices by 1.6 mM. The reason for this difference is the chelating effect of HCO3- on Ca2+, which precipitates about 25% of the free [Ca2+]o. The potassium concentration in the brain is 3mM (Lux et al., 1986), but many researchers use higher baseline concentrations following the original recipes of Mclllwain. The Mg2+ concentration in CSF, as measured from the ventricles, is somewhere between 0.9 and 1.2mM; it may be higher in the interstitial space. We use a baseline Mg2+ concentration of 1.6 mM in our experiments. Reduction of extracellular Mg2+ concentration from 1.6 to 1.2mM has facilitating effects on synaptic transmission (Hamon et al., 1987), and further lowering to 0.9 mM is sufficient to induce epileptiform activity in slices from rat pups (Gloveli et al., 1995).
An issue in preparing slices is the choice of anesthesia (and depth of anesthesia chosen before the brain is removed). Pups are frequently anesthetized by putting them on dry ice. This procedure exploits the anesthetic effect of high carbon dioxide levels combined with hypothermia. Many laboratories use gas anesthetics, such as ether or halothane. This choice gives reliable anesthesia with a reasonable therapeutic gap between deep and irreversible levels. It should be noted that very deep anesthesia leads to circulation arrest and hypoxia and causes cessation of protein synthesis (slices prepared in this way often do not exhibit long-term plasticity). Gas anesthesia works well in both sexes. Barbiturate anesthesia has the drawback that females often require more anesthetic to obtain an adequately deep state, but frequently they are put down so deeply that they die before decapitation. Ketamine anesthesia interferes with N-methyl-D-aspartate (NMDA) receptors, an effect not always desirable in studies on seizures.
Some laboratories perfuse the animal with a low calcium medium in which sucrose is substituted for sodium to reduce cell death. We have not found that this modification normally enhances tissue viability. However, in cases where preparation time may be prolonged, as in chronically epileptic animals and human tissue, we have found that immersing the slices in sucrose ACSF together with alpha-tocopherol improves tissue preservation (Gabriel et al., 2004).
The following procedures are employed to prepare entorhinal cortex-hippocampal slices (Dreier and Heinemann, 1991). Rats (150-200g) are anesthetized with ether and decapitated. The skull is opened, the dura cut, and the whole brain rapidly (within less than a minute) removed. The brain is submerged in cold (4° C) ACSF. The cerebellum is removed, and the brain is divided into two hemispheres. Each hemisphere is placed on its medial surface and divided into rostral and caudal sections by a transverse cut in a plane approximately parallel to the main axis of the hippocampus. The cut plane of the caudal piece is fixed to the plate of a vibratome (using cyanoacrylate glue), and five to eight horizontal slices are cut at a thickness of 400 mm. These slices contain the temporal cortex area, the perirhinal cortex, the entorhinal cortex, the subiculum, the DG, and the ventral hippocampus. Slices are placed separately on lens paper and stored in oxygenated ACSF in either an incubation chamber (experiments under submerged conditions) at room temperature or in an interface chamber (experiments under interface conditions) at 35° C. Slices are allowed to recover under these conditions for at least 60 to 90 minutes before starting the experiments.
To test the viability of our slices, we monitor the extracellular field potential in the medial entorhinal cortex layer III/IV or hippocampal area CA1 following electrical stimulation (single pulse duration of 100 |ms, pulse amplitude of 5-15 V) using bipolar platinum wires placed in the lateral entorhinal cortex or in stratum radiatum of area CA1, respectively. Slices are accepted for investigation if the extracellular field potential is at least 2mV in amplitude. To validate epileptiform activity, we use standard ion-sensitive microelectrodes to measure changes in the extracellular field potential and the extracellular potassium concentration simultaneously. Ion-selective microelectrodes are prepared using double-barreled theta glass (Lux and Neher, 1973). One barrel is filled with 154mM NaCl and serves as the reference. The other barrel is silanized (5% trimethyl-1-chlorosilane in 95% CCl4) and filled with a potassium ionophore cocktail (A60031, Fluka). To compute the molar extracellular potassium concentrations from the recorded potential values, a modified Nernst equation is utilized (Heinemann and Arens, 1992).
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