Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, C14
A novel viral tool to study ATP-mediated signalling in the brain
M. Figueiredo1, V. Kasimov2, N. Marina2, A. V. Gourine2, A. G. Teschemacher1, S. Kasparov1
1. School of Physiology-Pharmacology, University of Bristol, Bristol, United Kingdom. 2. Neuroscience, Physiology & Pharmacology, University College London, London, United Kingdom.
For a long time astrocytes were seen as cells that only provide structural and metabolic support for neurones but this view is now changing rapidly. ATP has a pivotal role in the astrocyte-to-neurone communication and it is considered to be a major “gliotransmitter”1. Due to the lack of selective antagonists, studying ATP-mediated signalling in vivo and especially in chronic experiments is complicated. In order to overcome these obstacles, we have developed a viral vector encoding the Transmembrane Prostatic Acid Phosphatase (TMPAP)2. This lentiviral vector employs an elongation factor 1-alpha (EF1α) promoter to drive the expression of TMPAP, an enzyme that functions as an ecto-5’-nucleotidase. In order to visualise expression, TMPAP is fused to EGFP. The expression of TMPAP-EGFP was tested in primary astrocytes using a SP1 Leica confocal microscope. It was confirmed that the construct is correctly targeted to the plasma membrane. Characterization of the novel vector, LVV-SW-EF1a-TMPAP-EGFP, was performed in primary cultures of rat astrocytes. As previously demonstrated when mechanically stimulated, astrocytes in cultures generate Ca2+ waves which propagate via spread of ATP. Cultures of astrocytes were prepared from the cerebral cortices, cerebellum and brainstem of 2-days-old (p2) Wistar rats and transduced with either LVV-SW-EF1a-TMPAP-EGFP or LVV-EF1a-EGFP which expressed EGFP as a control. Astrocytes were loaded with Rhod-2-AM and intracellular Ca2+ [Ca2+]i was monitored using a SP1 Leica confocal microscope at 34oC. To trigger a Ca2+ wave, astrocytes were mechanically stimulated with a patch pipette and changes in [Ca2+]i in the surrounding cells assessed. A line was drawn from the mechanically stimulated cell until each cell that responded and an average distance of Ca2+ wave spread was calculated. Mechanically stimulated TMPAP-EGFP expressing astrocytes showed a spread of Ca2+ wave of only 98 ± 6.6 µm (n=34) compared to EGFP expressing astrocytes where Ca2+ wave spread to 432 ± 40 µm (n=49), p<0.001, Unpaired t-Test. In a different experiment, astrocytes were loaded with Fura2-AM and mechanically stimulated as above. It was demonstrated that expression of TMPAP significantly shortened the duration of the stimulation-induced Ca2+ response at the site of stimulation. P2Y receptor antagonist MRS2179 (10 μM) also suppressed stimulation-induced Ca2+ waves, indicating that they are at least partially mediated by ATP. In summary, expression of TMPAP limits the spread of ATP-mediated signalling in cultured astrocytes, although it does not abolish it completely. LVV-SW-EF1a-TMPAP-EGFP is a novel tool to study purinergic signalling in the brain. It is currently used to investigate the role of ATP in cardio-respiratory control in vivo.
Where applicable, experiments conform with Society ethical requirements