Proceedings of The Physiological Society

University College Dublin (2009) Proc Physiol Soc 15, C104

Oral Communications

Chemosensitive astrocytes in the rostral ventrolateral medulla

N. Marina1, V. Kasimov1, M. Figuereido2, S. Kasparov2, A. Gourine1

1. Neuroscience, Physiology and Pharmacology, University College London, London, United Kingdom. 2. Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom.

ATP is released from the chemosensitive regions located on the ventral surface of the medulla oblongata in response to changes in arterial PCO2 and pH, which results in the activation of the respiratory network (1, 2). In vitro studies using brainstem slices have shown that increases in PCO2 or [H+] results in the generation of intracellular calcium waves ([Ca2+]i) in ventral surface astrocytes and this effect can be mimicked by the application of ATP. In the present study, we used an in vivo preparation to detect astrocytic calcium responses in ventral surface astrocytes induced by respiratory acidosis. Methods: Six male Sprague Dawley rats were used in accordance with the UK Animals (Scientific Procedures) Act 1986 and associated guidelines. Animals were anaesthetised with a mixture of ketamine (60 mg kg-1 I.M.) and medetomidine (250 µg kg-1 I.M.) and received bilateral microinjections into the rostroventrolateral medulla (2µl each side) of an adenoviral vector encoding a Ca2+ sensitive protein Case12 (3) under control of an enhanced GFAP promoter. Anaesthesia was reversed with atipemazole (250 mg kg-1 I.M.) and post-operative care was taken. Seven days after injections, animals were anaesthetised with propofol (30 mg kg-1 h-1, I.V.), paralysed with gallamine triethiodide (10 mg kg-1, I.V.; then 1-2 mg kg-1 h-1, I.V.) and artificially ventilated. The adequacy of anaesthesia during neuromuscular blockade was assessed by monitoring heart rate and blood pressure stability. The ventral surface of the medulla was exposed and continuously perfused with HBSS (pH 7.4) and phrenic nerve activity was recorded. GFP expression in the ventral surface of the medulla was visualized using a fluorescence microscope. Case12 fluorescence was used as an indicator of [Ca2+]i specifically in astroglia. At the end of recordings, animals were intracardially perfused with 4% paraformaldehyde and the brainstems were subsequently processed for immunohistochemical detection of Phox2B and GFAP. Results: Lowering ventral surface pH from 7.4 to 7.2 increased phrenic nerve activity and resulted in profound elevations in [Ca2+]i in surface astrocytes. Immunohistochemical analysis of the injected areas revealed strong GFP expression in GFAP-expressing astrocytes located in the marginal area of the ventral medullary surface. These astrocytes were often seen surrounding blood vessels and in close contact with chemosensitive Phox2B-immunoreactive neurons of the retrotrapezoid nucleus (RTN). Conclusion: Medullary astrocytes surrounding ventral penetrating arteries could play an important role in integrating chemosensory information by sensing physiological changes in arterial PCO2 and pH. We suggest that these astrocytes shape the chemosensory responses of the RTN neurons and the central respiratory network as a whole.

Where applicable, experiments conform with Society ethical requirements