RESTING MICROGLIAL CELLS IN-VITRO - ANALYSIS OF MORPHOLOGY AND ADHESION MOLECULE EXPRESSION IN ORGANOTYPIC HIPPOCAMPAL SLICE CULTURES

Neurons in organotypic hippocampal slice cultures (OHSCs) are known to preserve morphological and physiological features of the in vivo situ ation; however, little is known about the properties of microglial cel ls under these in vitro conditions. In this study, we addressed the qu estion whether microglial cells in OHSCs are initially activated follo wing explantation but return to a resting state during in vitro cultiv ation. Thus, we analyzed a) microglial cell morphology, b) microglial cell distribution, and c) expression of integrin adhesion molecules as putative markers of microglial activation. Hippocampal slices fixed i mmediately following explantation showed only resting microglial cells , mainly located in the paraventricular regions. After 3 days in vitro (div) OHSC surfaces were covered by activated microglia, whereas inte rmediate layers contained fewer microglial cells, giving the slices a sandwich-like appearance with the intact hippocampal formation being s urrounded by glial tissue. After 3 div, microglial cells in intermedia te layers of OHSCs showed activated morphology with ovaloid cytoplasm and no or merely few cytoplasmic processes; after 6 div, however, an i ncreasing degree of ramification could be observed. After 9 div, micro glia in intermediate layers had almost regained the morphological appe arance of resting cells with filigrane cytoplasmic processes extended in all directions. The integrin adhesion molecules LFA-1 (alpha and be ta chains) and VLA-4 were expressed on most microglial cells with acti vated morphology, as verified by co-localization with double immunoflu orescence labeling for LFA-1 or VLA-4 and Griffonia simplicifolia isol ectin B-4 (GFS-B-4). In contrast, only low levels of integrin adhesion molecule expression were also found on reactive astrocytes along slic e surfaces. However, LFA-1 or VLA-4 were never found on ramified micro glial cells, and double immunofluorescence labeling of LFA-1 or VLA-4 with ramified GFS-B-4(+) microglia never occurred. We conclude that a) originally resting microglial cells activated in an early phase of in vitro culture but regain a resting status after at least 6 div; and b ) integrin adhesion molecules LFA1 and VLA-4 are potential markers of microglial activation, as they were found on activated but never on re sting microglial cells. This enables further investigations on immunol ogical and electrophysiological features of resting and activated micr oglial cells under in vitro conditions. (C) 1996 Wiley-Liss, Inc.