Intercellular communication of type 1 astrocytes via gap junctions

Type 1 astrocytes in situ and in vitro form a syncytium via gap junction complexes so have a communicating cytosol (Finkbeiner-S 1992). Type 1 astrocytes in

vitro and in situ respond to a variety of stimuli, e.g. neurotransmitters, by an increase in

[Ca^^ji. The rise in [Ca^^]i appears to propagate from one cell to another within the syncytium forming Ca^^ waves travelling at 10-20tims'^ (Cornell-Bell-A. H. et al 1990: Dani-JW & Smith-S.J., 1992). Interestingly this form of signalling requires extracellular Ca^\ Mechanical stimulation also causes a rise in [Ca^^ji which propagates between astrocytes, but this form of intercellular signalling was not dependent upon external [Ca^^] levels. Thus this form of communication is thought to involve second messenger systems such as InsPs (Charles-A.C. e ta l 1993; Charles-A.C. e ta l 1991).

Several models have been proposed to describe how this cellular based Ca^^ signalling occurs. As the intercellular signalling is fairly rapid there is some doubt whether diffusion of the mediator would be fast enough. If internal [Ca^^] is raised Ca^^ ions can diffuse through gap junctions into neighbouring cells (Dunlap-K. et al 1987). Cellular [Ca^^ji are tightly controlled by buffering and removal from the cytosol. In addition the process of Ca^^ diffusion is slow (Allbritton-N.L. et al 1992). The communicating agents could be other diffrisible second messenger molecules such as InsPs (Bennett-M. V. L. e ta l 1991).

Carter et al have shown in porcine endothelial cells that gap junctions consisting of connexin 43 and 37 are permeable to caged InsPg, which has similar size and valency as InsPs molecules (valency 5', Mr 635 Da , Carter-T.D. e ta l 1996).

Rat cerebellar glial cells at 5DIV (Chapter 3) have connexin 43 (Dermietzel-R. et al 1991, Massa-P.T. & Mugnaini-E. 1985, Mugnaini-E. 1986). The possibility that caged InsPs can pass through astrocytic gap junctions and induce a [Ca^^]i rise in the adjacent glial cells in vitro after photolysis was investigated. Paired type 1 astrocytes were loaded with 500pM furaptra and 40 or 80 [lM caged InsPs as described above. The fluorescence from the patched cell was not recorded as it was shielded by the rectangular diaphragm from the PMT. The furaptra fluorescence signal took longer to equilibrate in paired cells than single cells, average loading time for single cells 419+63 n=10 cells, compared to 640+146, n=10.

Figure 4.3.1 An investigation into astrocytic intercellular communication.

Photograph A shows a pair of astrocytes 8 DIV The cell on the lefti was whole cell

clamped at resting membrane potential . The pipette contained 500pM furaptra and 40/80pM caged InsPs.

Photograph B depicts the same frame as photograph A, when illuminated with light of

wavelength 420nm. The picture shows that the cell on therighthas been infiltrated with the furaptra, via the cell on the right.

Graph C illustrates the time taken to load the second astrocyte through the first. The

fluorescent signal from the un-patched cell took over 20 minutes to equilibrate. The graph shows the fluorescence measured as photon counts plotted as a function of time in seconds

Panel Z) is of a fluorescent trace of an InsPs induced [Ca^^i change as a result of photolysis

of caged InsPs loaded through a single adjacent cell initial InsPs concentration was 80pM. The response shows a 20|iM increase in [Ca^^Ji as a result of photolysis of caged InsPs in the un-patched .cell.

(A) PATCHED PAIRED ASTROCYTES

iN vm o

(B) PAIRED ASTROCYTE LOADED \MTH500|iM FURAPTRA

(Q LOADING TIME OF INDICATOR

(D)[Ca li RISE ΠUN-PATCHED CELL

650000 500000 350000 300000 250000 200000 150000 0 100 200 300 400 500 600 700 800 900100011001330 lO nM IC a TIME (8)

Figure 4.3 .1 A shows a bright field photograph of a patched pair of type 1 cultured astrocytes. Figure 4.3.l.B shows the same pair of cells filled with SOOpM of furaptra excited with light of 420nm and recorded at >490nm. The photographs were taken after the completion of the experiment and show clearly that the Ca^^ indicator furaptra diffused through into the second cell. Figure 4.3.1 C shows the loading profile for the cell pair used in figure D. Loading in paired cells took 1.5 times as long as the indicator loading time in single astrocytes. Figure 4.3. l .D shows a typical InsPs evoked [Ca^^]i rise. This type of result was seen in 7 out of the 10 pairs o f type 1 astrocytes tested.

The diffusion of free [Ca^^]i is slow due to buffering (Allbritton-N.L. et al 1992),

so at this time scale the [Ca^^Ji rise is likely to be a result o f InsPs released activity in the un-patched, coupled cell. However a better test would be to measure the delay times of the response in the coupled cell and compare it with single cells. This was difficult in these experiments as the cells were grown on glass coverslips, so the initial rising phase was obscured by the phosphorescence artefact. These experiments were difficult to perform on astrocytes cultured on quartz with poly-L-lysine, as they did not readily form healthy monolayers (see section 4.6.1).

In conclusion these experiments show that furaptra and caged InsPs can diffuse fi-om one type 1 astrocyte via gap junction complexes, as photolysis elicits a [Ca^^ji rise in the adjacent cell. Thus it is possible that the endogenous InsPs molecules could pass between cells and so act as a diffusible second messenger involved in [Ca^^Ji waves of interastrocytic communication.