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A wide range of preparations have been developed for the study of the nervous system. Each strikes a different balance between fidelity to the system ultimately of interest, access for recording and the ability to control and manipulate the system to implement experimental protocols.
Ultimately it is thein vivo human brain that is of interest, it can be studied directly using non-invasive techniques such as fMRI and EEG and with more in- vasive approaches, such as ECoG, when they are indicated for clinical reasons and additional data can be obtained without significant modification to the surgical pro- tocol. For example, depth electrodes may be implanted to determine the location of seizure foci for evaluation for resection, (for examples see Truccolo et al. (2011); Van Gompel et al. (2014); Chang et al. (2012)). However, experiments that require
more invasive surgical procedures than clinically indicated or that require otherwise unnecessary pharmacological manipulation are deemed unethical and so cannot be carried out.
Pharmacological manipulation and invasive recording techniques can, how- ever, be applied to ex vivo human brain tissue. Resection of the brain is indicated for a number of neurological problems, for example, for the treatment of some cases of pharmacologically refractory focal epilepsy. The tissue removed, after appropri- ate clinical tests, is often made available for research purposes. This tissue often provides exact insights in to disease processes, rather than only being a model of them. However, the availability of such tissue is limited and clinical considerations must always take precedence over maximising the quality of the samples for research. Additionally, the tissue removed is almost always affected by a disease process and so it is unlikely to be suitable for the study of physiological processes.
Although it is the human brain that is ultimately of interest, the brains of other mammals are thought to provide a good model of human brain function and are widely used for the study of the nervous system. As with human brain, the brains of other mammals can be studiedin vivo, however, although there are strict ethical controls, where justified, more invasive in vivo surgical procedures can be carried out. Furthermore these can be implemented in both healthy animals and those affected by disease as well as in transgenic lines. The access for both recording and manipulation is, however, still limited and soin vivo preparations are unsuitable for some protocols. Equally, some protocols do not requirein vivomeasurement and so it would be unethical to use such a preparation.
Typically experiments are either carried out in vivo, in tissue slices or in cultured cells. However, an intermediate preparation is possible with the guinea pig brain. The guinea pig brain can be removed and perfused through its circle of Willis, allowing the intact brain to be viably maintained out of the skull, this is termed the isolated brain. This preparation allows improved access and pharma- cological manipulation while preserving the complete network connectivity. Access for recording is, however, still more limited than in tissue slice or cell culture.
cess. The brain of an animal is rapidly removed and, whilst cold, cut in to slices ∼ 400 µm thick. The slices are then rewarmed and immersed in an oxygenated artificial cerebrospinal fluid (aCSF) which maintains the viability of the tissue by diffusion. The cutting process severs a large proportion of the network connections, however, sections are cut that maximise connectivity within the slice and such slices exhibit spontaneous activity, indicating a significant degree of connectivity. The advantage of the acute tissue slice preparation is the degree of access to the tissue that it facilitates, both for recording from the tissue and for pharmacological, ionic, electrical and mechanical manipulation. Additionally, all experimentation is carried out once the animal is dead and so the suffering caused is minimised. For these rea- sons all experiments presented in this thesis that involved animal tissue employed this preparation.
Typically acute tissue slices are only viable on the day of preparation, how- ever, tissue from young animals can be cultured. Cultured slices are termed organ- otypics and are well suited to the study of slow processes that require direct access to the cells (Gahwiler et al., 1997). For example, cells in culture can be transfected and the effect of expression of a resulting protein can be investigated, a process that would not occur within the time scale of viability of an acute tissue slice. Unlike in cultures of separate cells, some synaptic connections between cells are preserved, allowing the study of synaptic transmission. However, the development of the cells within an organotypic slice can differ from that which would occurin vivo so some caution is required.
Finally, cellular processes and ion channel dynamics can be studied in cul- tured cells, unlike in organotypic cultures, the cells do not form a network and so synaptic transmission can not be studied using this preparation. However, cultured cells are well suited to the study of ion channels and other proteins, such as con- nexins, as mutant lines can be produced rapidly. Ion channel properties can also be studied in relative isolation by reconstituting them in artificial membranes.