Initial report on combined in vivo single cell recording and intracellular staining

This paper reports preliminary data obtained from the combination of extracellular single unit recording, microiontophoretic testing and intracellular staining of single neurons in the diencephalon of the anesthetized rat. Sixty neurons were recorded extracellularly and iontophoretically tested with glass multibarrelled micropipettes. Thirty-four of these neurons were identified by antidromic invasion from median eminence stimulation. Seventeen of these antidromically identified neurons were subsequently impaled with the micropipette and intracellularly stained with the fluorescent dye lucifer yellow-CH. The average diameter of the antidromically identified neuronal cell bodies was 7.4 microns. The iontophoretic response profiles of these stained neurons were similar to the profiles of non-stained antidromically identified neurons. Four of the remaining 26 neurons were synaptically activated from median eminence stimulation and were successfully marked with lucifer yellow. Average soma diameters of these neurons was 12.8 microns. Twenty-two neurons were not antidromically or orthodromically identified from median eminence stimulation. Three of these neurons were intracellularly stained with lucifer yellow and their soma diameters averaged 6.9 microns. Approximately 50% of all staining attempts, subsequent to extracellular recording and iontophoretic testing, were successful. The combination of these techniques is therefore a feasible approach to the in vivo study of the physiologic, pharmacologic and morphologic properties of single neurons.