Brain Slice Preparation:
Brain slice preparations are becoming increasingly popular among neurobiologists for the study of the mammalian central nervous system (CNS) in general and synaptic phenomena in particular. This technique is widely used because it has many advantages over in vivo methods. It provides precise control over experimental conditions, such as temperature, pH, and drug concentration. It also allows the examination of metabolic parameters and electrophysiological properties without contamination from anesthetics, muscle relaxants, or intrinsic regulatory substances. The stability of electrophysiological recording is greatly improved as the heart beat and respiration of the experimental animal are eliminated. The cells being studied can be located, identified, and accessed easily. Use of the brain slice has greatly increased our knowledge of the mammalian central nervous system (CNS). This technique is continuously improving and will remain valuable for a long time. While the hippocampal slice is probably the single most used such preparation, slices of olfactory cortex, neocortex, cerebellum, hypothalamus, caudate nucleus, amygdala and other brain areas have also been studied in the past decade or so.
Brain slices are being used because they offer certain advantages over in vivo approaches to the study of the CNS. These are:
- Rapid preparation, using relatively inexpensive animals (mouse, rat, guinea pig) where anesthetics are not necessary.
- Mechanical stability of the preparation, due to lack of heart eat and respiration pulsations, which permits intracellular recordings for long periods.
- Simple control over the preparation's condition, where pO2, pC02, pH and temperature can be maintained as desired;
- Direct visualization of the slice structure, allowing the accurate placement of both recording and stimulating electrodes in the desired sites.
- Slices have no blood brain barrier and thus their extracellular space is accessible to the perfusion medium and its content (ions, transmitters, drugs).
- While simplified, the brain slice preparation maintains structural integrity, unlike cell cultures or tissue homogenates.
Some of the limitations of these preparations are:
- Lack of certain inputs and outputs normally existing in the intact brain.
- Certain portions of the sliced tissue, especially the top and bottom surfaces of the slice, are damaged by the slicing action itself.
- The life span of a brain slice is limited and the tissue gets "older" at a much faster rate than the whole animal.
- the effects of decapitation ischemia on the viability of the slice are not well understood.
- Since blood-borne factors may be missing from the artificial bathing medium of the brain slice, they cannot benefit the preparation and thus the optimal composition of the bathing solution is not yet established.
Preparation of slices:
In general, rodents are the animals of choice for the preparation of brain slices. Of those, the rat and the guinea pig are the most used. After decapitation, the brain is removed rapidly from the skull and rinsed with cold artificial cerebro-spinal fluid (aCSF) which has been equilibrated with 95% O2/5% CO-2 gas mixture.
Artificial cerebrospinal fluid (aCSF) is commonly used when sampling from brain interstitial fluid. This solution closely matches the electrolyte concentrations of CSF. It is prepared from high purity water and analytical grade reagents. It is microfiltered and sterile. Final Ion Concentrations (in mM): Na 150; K 3.0; Ca 1.4; Mg 0.8; P 1.0; Cl 155.
The detailed protocol is what will be done as part of this experiment. All other actions will be in the brain slice animated simulator.
Use of brain slices:
The brain slice preparation is an excellent tool in the study of the fundamentals of neurophysiology at the cellular and simple circuit levels.
While the neuronal membrane properties can be studied using intracellular recording, including the study of ion channels and putative neurotransmitters, the study of synaptic activity can be performed with extracellular recordings and specific stimuli. The nature of different synaptic connections has been studied by evoking excitatory and inhibitory postsynaptic potentials (EPSPs and IPSPs). For such studies, other electrophysiological techniques such as voltage clamp and iontophoresis can be very useful. Slice preparations, especially the hippocampus and cerebellum, are being used extensively in plasticity studies exploring long-term potentiation (LTP), adaptation and kindling. Lab studies can be performed either entirely with the slice preparation or by inducing these physiological changes in the intact animal and then studying the affected brain area as a slice. Endocrinological studies can profit by adopting the in vitro slice approach.
The brain slice preparation is now used as a system for the study of brain metabolism, pharmacology of drugs in the CNS, and as a model for numerous pathological situations.
Through this experiment you will be able to:
- Understand various processes and techniques in preparing slices of brain tissue.
- Set-up the experimental table and preparation procedures in neurophysiological experiments.
- Understand the step-by-step procedure including most details for preparing slices of rat brain tissue.
- Understand methods prior to a patch-clamp experiment.