Fluorescent indicators have become useful tools for the study of intracellular function. However, conventional imaging techniques are usually limited to in vitro preparations. We have developed a fibre optic probe or ‘optrode’ to record real-time changes in fluorescence corresponding to intracellular processes within the intact central nervous system.

The optrode consists of two 125 µm optical fibres placed side by side along with a tungsten microelectrode for simultaneous extracellular recording. 488 nm laser light was launched down one fibre, whilst the other fibre collected the resulting green fluorescence. This was long-pass filtered to remove residual blue light and focused onto the face of a photo-multiplier tube for detection.

Three experiments have been performed. First, cellular activity was recorded from brainstem slices (n = 4) loaded with 15 µM Oregon Green-BAPTA-1AM (OG-I), a cell-permeant calcium-sensitive dye. Increases in fluorescence F (ΔF/F = 250 %, S.E.M. = 34.4 %) were observed from the ventrolateral medulla during perfusion with a high potassium medium (30 mM) to depolarise cells. Second, we extended this study to examine calcium transients in respiratory modulated hypoglossal motoneurones in the in situ working heart-brainstem preparation of rat (WHBP; Paton, 1996). Following local microinjection of OG-I (20 µl at 0.4 µl min^-1), recordings show transient changes in fluorescence that correlate with mass extracellular activity and phrenic nerve discharge (n = 2). Third, the ability to monitor changes in gene expression in the brains of freely moving animals should be useful in linking neuronal gene function to physiological parameters and behaviour. The hippocampi of rats were microinjected with a replication-deficient adenovirus expressing enhanced GFP under control of the tetracycline system (tet-off; see Harding et al. 1998). Using this system eGFP expression can be switched off and on by administration and withdrawal of doxycycline to drinking water, respectively. In anaesthetised in vivo rats (53.33 mg kg^-1 ketamine + 0.33 mg kg^-1 medetomidine), eGFP expression was detected in the hippocampi of animals without dox treatment vs. undetected in dox treated (n = 2). Animals were humanely killed with an overdose of pentobarbitone.

These preliminary data support the use and further refinement of a single optical fibre probe for the detection of fluorescence from discrete regions of intact brain.

P.M.J.B. is supported by a University of Bristol scholarship. The British Heart Foundation funded research.