Brain slices temperature

M., and Voigt, K. Temperature dependent burst discharges in magnocellu-lar neurons of the paraventricular and supraoptic hypothalamic nuclei recorded in brain slice preparations of the rat. In ... 

H]mepyramine from H,-receptors is very slow and negligible over a 120 min period [114]. This slow dissociation at low temperatures has meant that [3H]mepyramine has been of great utility for the autoradiographic localization of H,-receptors in brain slices [ 115-117]. [,25I]Iodobolpyramine has a similar low dissociation rate at 25 °C and has also been used for autoradiographic studies [98]. 

After the animal is sacrificed, the skull should be opened quickly and the brain removed into chilled aCSF (4-8°C). The low temperature slows down the metabolic rate of cells and reduces their energy consumption. These effects allow cells to survive the ischemic period during brain slice preparation. The brain also becomes more firm when the temperature is lowered, facilitating the dissection and slicing of the brain. 

The preparation time should be short, a prolonged ischemic penod is associated with a fall in intracellular ATP and reduction in the recovery of population spike (6-8) Although most investigators think diat the brain slice preparation should be accomplished in <5 min, another group found that there was no significant change in the amplitude of the population spike even with 30 mm postmortem delay at room temperature (9). 

Reid, K. H., Schurr, A., and West, C. A. (1987) Effects of duration of hypoxia, temperature and aCSF potassium concentration on probability of recovery of CAl synaptic function in the in vitro rat hippocampal slice, in Brain Slices Fundamentals, Applications and Implications (Schurr, A., Teyler, T. J, and Tseng, M. T., eds.), Karger, Basel, pp. 143-146. 

The semilogarithmic plot is resolved into components by the method of subtraction, and the half-time (fi/2) of each component is determined graphically. The rate of efflux of labeled solute (k, %/min) is calculated from k = 0.693/fi/2 x 100 (Cutler et al., 1971). This method reveals a fast component for amino acids, as well as other compounds of different molecular weight. It has a ty2 of 2-3 mm in tissue slices for all components studied and probably represents the washout of adherent medium. The slow component is the loss of isotope from the tissue as a whole it is different for different substances and is sensitive to temperature changes The apparent first order kinetics of the slow component does not preclude the possibility that the amino acids are lost from different tissue compartments at different rates. This does, however, suggest that the loss from one major compartment to another may be rate-limiting for clearance from the whole tissue (Cutler et al., 1971). In brain slices, the slow exponential loss of amino acids is linear throughout 40 min of superfusion, and at the end of this time, 60-70% of the labeled amino acids are recovered in the effluent (Cutler et al., 1971)... 

The whole procedure, from the time the animals brain is removed until the time the slices are placed in ACSF, usually requires 2.5—5 min. However, the timing does not appear to be crucial since viable slices can also be obtained from the hippocampus in the second half of the brain, some 5-7 min after the first set of slices are prepared. When judged by the quality of intracellular and extracellular responses obtained from pyramidal cells in the CAl region, it makes no obvious difference whether the ACSF temperature during preparation is 4, 20, or 36°C. The environment in the recording chamber is usually kept at 33-37°C. At this temperature slices can be maintained for some 10-15 hr. Others, using thicker brain slices of the olfactory cortex, have worked at a chamber temperature of 25°C (Harvey et ai, 1974 Scholfield, 1978a,6 Nicoll et ai, 1980). 

The saturable system of crossing the cells was also observed for brain slices (cut in two different directions). The process was ATP dependent (inhibited by DNP and decreased temperature) because absorbed intracellular riboflavin is rapidly converted into FMN and in turn into FAD. Additionally, neither the absorption of FAD nor FMN is possible (Speetor 1980a, 1980b). 

For crab brain slicing, setting the cryostat box temperature to —25 C instead of the commonly used —20°C encourages better slicing. 

Brain slices were transferred and anchored to a recording chamber with a nylon grid. A peristaltic pump continually perfused the slices with ACSF (bubbled with carbogen) at a rate of 1-2 mL/min. The recording temperature was controlled at either 26 or 35 °C with an inline heating system. Slices were visualized with an upright microscope equipped with DIG optics and were displayed on a monitor using a CCD camera. 

Tissue sectioning Set cryostat at -16 C and slice brain or desired tissue into sections 16—20 pm thin. Mount sections on poly-L-lysine coated or positively charged slides. Allow sections to dry overnight at room temperature. Tissue at this stage (fresh frozen, unfixed) should be stored at -20 C. 

A slice 5 mm thick is cut from fixed brain tissue and washed for several hours in distilled water to remove excess formaldehyde. The slice is immersed overnight in Tris-buffered saline (TBS, pH 9.0) and then placed in a plastic jar containing 200 ml of TBS. The jar is placed in a microwave oven for 10-15 min at full power (700 W), divided into two cycles of 5 or 7.5 min to check the fluid level. The temperature is controlled using the temperature probe of the oven. It takes 3 min to reach a temperature of 90°C. 

Mitani A., Kadoya F., and Kataoka K. (1991) Temperature dependence of hypoxia-induced calcium accumulation in gerbil hippocampal slices. Brain Res. 562, 159-163. 

Voss LJ, Sleigh JW (2010) Stability of brain neocortical slice seizure-like activity during low-magnesium exposure measurement and effect of artificial cerebrospinal fluid temperature. J Neurosci Methods 192 214-218... 

EtsSn- Intoxication and effect on P phospholipides and body temperature (3) effect on amino acid metabolism in brain cortex slices (99) effect on platelet shape and aggregation (UOTf, 104 7), effect on platelet morphology (2781), effect on oxidation of glucose and pyruvate in brain cortex slices (916) effect on brain glucose metabolism in vivo and vitro (9I8), effect on retention of potassitmi and amino acid in brain cortex slices (917)-... 

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