Freshly isolated astrocyte (FIA) preparations: a useful single cell system for studying astrocyte properties.

Astrocytes are cell constituents of the mammalian CNS whose intricate relationships with neurons, blood vessels and meninges in situ are well documented. These relationships and their complex morphologies imply numerous functions. Over the past quarter century or so, however, the main experimental basis for determining which roles are likely have been derived from studies on primary astrocyte cultures, usually prepared from neonatal rodent brains. We list a number of examples where these cultures have shown quantitative and qualitative differences from the properties exhibited by astrocytes in situ. The absence of an adequate reliable database makes proposals of likely hypotheses of astrocyte function difficult to formulate. In this article we describe representative studies from our laboratory showing that freshly isolated astrocytes (FIAs), can be used to determine the properties of astrocytes that seem more in concordance with the properties exhibited in situ. Although the cells are most easily isolated from < or =15 day old rat hippocampi they can be isolated from up to 30 day old rats. The examples we describe are that several different types of K(+) currents can be determined by patch clamp electrophysiology, of all the mGluRs only mGluR3 and 5 were detected by single cell RT-PCR, and that single cell Ca(2+) imaging shows that the mGluR5 receptor is functional. It was found that the frequency of cells expressing mGluR5 declines with the age of the animal with the mGluR5b type splice variant replacing the mGluR5a type, as occurs in the intact brain. It is concluded that FIAs can be used to determine the individual characteristics of astrocytes and their properties without the problems of indirect effects inherent in a heterogeneous system such as the slice, and without the problem of cultures unpredictably reflecting the in situ state. The FIAs obviously cannot be used to study interactions of astrocytes with the other CNS components but we propose that they will provide a good database on which hypotheses regarding such interactions can be tested in slices. FIAs can also be isolated from brain slices or intact brain after various pharmacological or electrophysiological perturbations to determine the changes in astrocyte properties that correlate with the perturbations.