Separation and culture of cells isolated from the developing rodent cerebellum

Currie, D. Neil (1981). Separation and culture of cells isolated from the developing rodent cerebellum. PhD thesis The Open University.



This thesis describes a project which had a simple central objective: to take an existing preparation of dissociated cells from the immature cerebellum and develop conditions in which these cells could survive and differentiate in monolayer culture. The aim was to develop a system in which brain development could be investigated, particularly at the biochemical level in a simplified environment.

The study of cellular differentiation in vitro has two advantages:

1. Aspects of a cell's development which are intrinsically programmed in the cell at that stage can be distinguished from aspects which are dependent on some extrinsic influence, for instance from another cell type.

2. Cultures have a more restricted cellular composition than the brain and therefore may go some way to overcoming the cellular heterogeneity of brain tissue which renders interpretation of most conventional biochemical measurements difficult.

To realise the first it is necessary to compare a cell's differentiation in vitro with its normal course of development in vivo. To realise the second, cultures must be of a simplified and defined composition in which biochemical measurements can be attributed to particular cell types. Both requirements imply the use of cultures which contain in quantified proportions a few cell types which can be specifically identified by reliable criteria, rather than in terms of such general categories as 'neurons'.

The use of a single area of the brain, the cerebellum, was an important first step in restricting the cellular composition of the cultures. The cerebellum contains large numbers of relatively few neuronal types which differ greatly in perikaryal size and therefore were amenable to a size-based separation of cell types by unit gravity sedimentation. Culture conditions were established for the survival and differentiation of the mixed cerebellar cell suspension and some of the separated cell fractions. This led to cultures containing a high proportion of granule neurons. However a fraction enriched in Purkinje cells did not survive well in culture. Finally, the composition of the cultures was further defined in a study involving cell type-specific immunological markers and the different uptake of the inhibitory neurotransmitter, GABA.

The description of this work is organised in seven more or less self-contained chapters, and can be informally divided into two parts. Chapter 1 is a general introduction to the problems of studying development, to the advantages of the cerebellum, in particular the detailed knowledge of some mechanisms of cerebellar development gained from work with mutants and X-irradiation and to the current knowledge, or lack of it, concerning the molecular basis of cellular interactions. Chapter 2 is a description of the cell isolation method used here, and of some improvements to the method effected during the course of this work. Chapter 3 completes the first part of the thesis and deals briefly with the history of brain cell separation techniques before detailing the separation of cerebellar cells by a unit gravity sedimentation technique. This was a joint project with James Cohen and Gary Dutton at The Open University and Graham Wilkin and Robert Balazs at MRC, Carshalton. Chapter 3 concentrates mainly on the part of the project which was the primary work of this author, that is the size analysis of cells using a Coulter counter and points out those parts of the work, in particular electron microscopy, which were performed by others.

The second part begins with Chapter 4, which is a review of nervous system culture work with the aim of establishing whether there are reasons why the contribution of cell cultures to our biochemical knowledge has been quite limited, although they have been in use for many years and would appear an ideal controlled system for biochemical studies. Chapter 5 is a description of the search for culture conditions in which one week postnatal cerebellar cells would survive and grow, and of their pattern of development in culture. Chapter 6 details the use of markers to define culture composition more closely; cerebellar cultures were found to be over 80% in a single neuronal type, the granule cell. Cultures of embryonic cerebellum were found to contain a distinct population of neurons which are absent from cultures of older animals, Chapter 7 briefly summarises some future directions for this work and suggests that defined cell culture systems in alliance with modern immunological techniques, may offer the best route to find the elusive cell surface recognition molecules which are generally believed to mediate the cellular interactions that control development.

Some of this work, together with related aspects which are primarily the work of others, has been published elsewhere. Copies of these papers are attached.

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