Proceedings of The Physiological Society

Trinity College Dublin (2003) J Physiol 551P, C52

Communications

Simultaneous optical and electrical recording from respiratory-rhythmic neurones in situ using micro-optoelectrodes

Peter M.J Bradley*, Sergey Kasparov*, David Murphy† and Julian F.R Paton*

* Department of Physiology, University of Bristol BS8 1TD and †URCN, University of Bristol, Bristol Royal Infirmary, Marlborough Street, Bristol, UK


Detection of intracellular calcium transients in mammalian neurones is restricted to in vitro slice preparations or dissociated cells. In the present study we attempted to record calcium transients from neurones within an intact brainstem. We have studied respiratory-rhythmic neurons using a calcium-sensitive dye in conjunction with a micro-optoelectrode. This approach was designed to allow simultaneous recording of extracellular electrical activity and changes in fluorescence corresponding to transient changes in intracellular calcium ion composition.

Lengths of optical fibre (outer diameter 125 µm, core diameter 62.5 µm) were flame-pulled to a taper; the tips were then broken back to achieve core diameters ranging from 5 to 10 µm. This end of the fibre was placed into a saline (2 M NaCl) filled glass microelectrode (0.5 MV) along with a silver wire, and sealed with dental wax. The other end of the fibre led to a photomultiplier tube for signal detection. The calcium-sensitive dye Fluo-4-AM was infused into the hypoglossal motor nucleus (5-15 µl, 50-200 µM at 0.2 µl min-1), which is inspiratory modulated, of the working heart-brainstem preparation (Paton, 1996). This in situ, unanaesthetized, arterially perfused, decerebrate rat preparation was established under halothane anaesthesia which was withdrawn when decerebration produced insentience. (Animals were killed humanely.) Phrenic nerve discharge was monitored continuously as an output of central respiratory drive. Excitatory 488 nm laser light was guided into a 600 µm optical fibre and positioned on the surface of the medulla for illumination of the dye-loaded region. The micro-optoelectrode was then tracked down through the area using a stepper motor.

Transient changes in fluorescence in phase with phrenic nerve discharge and correlated to mass extracellular electrical activity from inspiratory-modulated hypoglossal motoneurones were observed (n = 10). These remained stable for hours. No transient fluorescent changes were detected either above or below the hypoglossal motor nucleus where respiratory activity was not detected electrically. During elevations in central respiratory drive following stimulation of peripheral chemoreceptors (arterial injection of sodium cyanide, 50 µl, 0.03 %), the frequency of these transients increased; there was also a rise in baseline fluorescence (n = 44). Overdosing with sodium pentobarbitone (600 mg kg-1) abolished both the respiratory-related optical and electrical signals (n = 6).

This technique is being applied to investigate the relative roles of transmembrane versus intracellularly released calcium transients during different functional output states of the respiratory network.

P.M.J.B. is funded by a UOB Scholarship. Royal Society and British Heart Foundation funded research.

Where applicable, experiments conform with Society ethical requirements