The Brain Slice Technique

Reprinted from: Brain and Behavior. Raju TR, Kutty BM, Sathyaprabha TN and Shanakranarayana Rao BS (eds.), National Institute of Mental Health and Neuro Sciences, Bangalore, India. 2004:112-114.

THE BRAIN SLICE TECHNIQUE Shankaranarayana Rao BS and Raju TR The brain slice technique was first applied to metabolic studies of small tissue sections as early as 1920. With the advent of the microelectrode technique at the beginning of the 1950s it was possible to characterize the resting membrane potentials of cells contained in freshly sliced tissue and compare them to brain cells in situ. It was soon realized that such slices provided new routes for the study of synaptic phenomena. Towards the end of the 1960s the use of surviving, metabolically maintained tissues from the brain for electrophysiological and pharmacological studies was becoming an accepted, valued and widely applied technique. Brain slice technique became very popular after the advent of the whole-cell recording technique in the mid 1980s and its application to sections of brain tissue (Sticker 1997). The Brain Slice preparation and maintenance for electrophysiological recordings The in vitro brain slice preparation often is used to study processes involved in synaptic plasticity and to evaluate the role of native receptor subtypes in neurotransmission. Brain slices have been used for physiological studies since the early 1970’s, and recording from neurons in brain slices using patch pipettes gained widespread use in the late 1980s, often with the aid of video microscopy. Brain slices allow recording from semi-intact neural circuits, with the advantages of mechanical stability and control over the extracellular environment. Wholecell recording in brain slices is often combined with imaging techniques and indicator dyes to measure intracellular pH, calcium concentration, etc. It can also be combined with retrograde tracing techniques to record responses from neurons that project to a certain brain area. Brain slices are used for a wide variety of studies including synaptic plasticity and development, network oscillations, intrinsic and synaptic properties of defined neuronal populations, and many others (Dingledine, 1984; Kettnmann and Grantyn, 1992).

Depending on the experimental needs several variants of the technique have been developed and are being used. One distinction between the variants relates to how thick the slices have been cut. The thin slice technique was developed to allow visualization of individual cells in slices less than 250µm while the thick slice technique is used in experiments where connectivity and maintenance of normal dendritic structure are crucial for the study. Once prepared, slices can be kept alive in various media for hours or days. Ultimately slices can be kept in culture. Best conditions for brain removal Animal should be treated under the less stressful conditions. Thus, animals should be housed in their cages just till the beginning of the experiment. The fastest and easiest way of animal execution is by decapitation, although alternatively halothane (for anesthesia), or CO2 (suffocation) could be employed. These last two methods are less recommendable, because they take more time, and could produce some neuronal damage prior to the brain removal. Anesthesia application is not advisable, because of the possible interference of the drug in the neural physiology. Once animal is decapitated, skull should be removed with scissors (scalpel is not recommended), cutting along the sagital axis from caudal (posterior) to rostral (anterior) part, and then open the two skull pieces laterally. For shelling out the brain introduce the forceps or the scissors in the caudal part, and remove backwards the whole organ. Place it rapidly (preferably less than 60 seconds) into oxygenated ice-cold saline or artificial cerebrospinal fluid. This solution should be so cold that it contains a few ice crystals and to maintain this temperature the container should be sitting on ice. Faster dissection- better results The process of brain removal should be gently accomplished, but as fast as possible. The brain 112

should be placed in oxygenated ice-cold saline or artificial cerebrospinal fluid. There, it should be cooled for 3 minutes to 10 minutes. Next, the brain is ready for slicing or dissection. To reduce damage to the tissue, it is particularly important to keep the brain immersed in oxygenated ice-cold saline or artificial cerebrospinal fluid during the whole process. Slices should be kept at about 32-37°C in oxygenated saline or artificial cerebrospinal fluid, for at least 30 minutes before recording. Once this time is over, the remaining slices are kept at room temperature (25°C) before use, for a maximum of 5 - 10 hours. Slicing procedure Despite the many different procedures employed, the main goal is to prepare a slice of tissue where the neurons, fibers, synapses and glia that are important to the experiment are in a viable condition. The animals used in preparing slices are most often small rodents; guinea pig, rat and mouse. Young animals have some advantages for the slice preparation. Their skulls are soft, and therefore easier to remove. Since the brain is smaller, cools more rapidly (3 minutes are usually enough) when placed in ice-cold solution. About the tissue, older animals are more susceptible to anoxia, and the neuronal damage is higher as time goes. More myelination and the presence of more connective tissue may result in more damage to the cells, and their processes during slicing, and in a worse acquisition during the electrophysiological recordings. Once the tissue is removed, it is normally cooled down by placing it in an ice-cold oxygenated artificial cerebrospinal fluid (ACSF) to minimize the metabolic activity. Cutting is done in ice-cold ACSF using a vibratome. The angle and vibration frequency (usually near the maximum) of the blade should be adjusted to prevent the tissue being pushed while cutting the slices. Slicing should be accomplished in less than 10 minutes. Take carefully the brain slices using a cut and fire polished Pasteur pipette filled with the ice-cold solution. A standard slice is cut at 300-400mm thickness. Regions of the slices that are thicker than

this exhibit centrally-located necrotic cells suggestive of hypoxic damage. The slices are then incubated at a temperature of around 360C for at least 1 hour. Oxygenation and normal pH are maintained by bubbling the ACSF with 95%O2 / 5%CO2. This allow the tissue to recover from the damage imposed by the preparation and adjust to the new extra cellular milieu as well as to changed metabolic activity. Composition of the artificial cerebrospinal fluid (ACSF) The artificial cerebrospinal fluid should be freshly prepared for each experiment (quantities in grams are given in brackets for 2 litre solution preparation). It contains NaCl 126 mM (147.3 g), KCl 2.5 mM (3.7 g), NaH 2PO4 1.25 mM (2.9 g), MgCl 2 1.3 mM (2.48 g), CaCl 2 2.4 mM (7.1 g), Glucose 11mM (4 g) and NaHCO3, 25 mM (4.2 g), with a pH of 7.3 when gassed with 95% O2 and 5% CO2. If you want to create some stocks, you can prepare the inorganic component (NaCl, KCl, NaH2PO4, MgCl2 and CaCl2), and just before the experiment add the Glucose and NaHCO 3 . To adjust the pH correctly, solutions should be bubbled for at least 30 minutes. This solution could be employed as extracelullar solution in the patchclamp experiment. The above mentioned composition of ACSF reflects the basic requirements for maintaining healthy slices. Depending on the experimental needs, the composition might have to be adjusted considerably. Slice Chambers For experimental use slices must be kept in an environment providing appropriate oxygenation, pH, osmolarity and temperature. In addition, depending on the techniques used, it is necessary to have excellent visual control, good mechanical access and stability. Most commonly used chambers allow superfusion of ACSF across the slice. This imposes special demands on the mechanical stability of the superfusion system. Most of the slice chambers are temparature controlled and the superfusion rate of the ACSF is 113

atleast 1ml/min. The flow rate determines the O2 and 5% CO2 escape by diffusion as the perfusate travels along the tubing supplying the chamber. Submersion chambers are normally used for wholecell patch recording, whereas interface chambers are better suited for monitoring extracellular field potentials (Sticker 1997). Temperature Although the body temperature of small rodents is around 38oC, most investigators maintain the slices at 30-35 oC in the experimental chamber. There are two reasons for this, firstly, it has been found that preparations survive longer and in a healthier state at the lower temperature. Secondly, the higher humidity resulting from warmer solutions leads to the formation of droplets on recording and stimulating electrodes. Many researchers depending on their experimental design tend to work at room temperature. Cell visualization The real advantage of the slice is its accessibility, especially the visibility of structures such as cell body layers. Thin slices of (250-300µm) allow a

greater optical resolution due to smaller effect from light scattering. The slices from younger animals have thinner myelin sheets, which also improves visibility. The cells and parts of the dendrites can be visualized using a 40 times high numerical aperture, long working distance water immersion objective. To further improve the contrast between different cells, Normaski or Hoffman optical arrangements are preferred. Electrophysiological evaluation of brain slices The assessment of electrical parameters of slices depends on the characteristics of the particular cells within the tissue and varies from brain region to brain region. The basic indicators include resting membrane potential, input resistance and amplitude of the action potential. More sensitive measures include the ability of the cells to produce a regular, rhythmic train of action potentials after the injection of a small current. Damaged neurons will often respond with a single action potential at the onset of the current pulse. Besides direct cellular parameters, amplitudes of extracellular fields reflect the synaptic action and are convenient for assessment of the overall state of a slice or at least of small regions within a slice (Sticker 1997).

Advantages and disadvantages of brain slice preparations Advantages

Disadvantages

1

Direct visualization

Loss of afferent and efferent connectivity

2

Technical accessibility

Shearing of dendritic processes and axons; Damage to neurons and glia

3

Mechanical Stability

Tissue debris around the cutting surface mixed with healthy cells

4

Ease of use

Slow release of cellular enzymes and ions from damaged cells

5

Control of extracellular medium

Altered metabolic state; the artificial conditions may alter cellular metabolism in many ways

6

Precise control over concentration of drugs

Loss of vascular and hormonal regulation

References 1. Dingledine R (1984) Brain slices, Plenum Press, New York. 2. Kettnmann H and Grantyn R (1992) Practical electrophysiological methods. Wiley-Liss Inc. New York.

3. Sticker C (1997) Slices of brain tissue. In: Neuroscience Methods, Martin R (ed.) Harwood academic publishers, Australia. pp 3-10. 114