A NOVEL SINGLE-CELL STAINING PROCEDURE PERFORMED IN-VIVO UNDER ELECTROPHYSIOLOGICAL CONTROL - MORPHOFUNCTIONAL FEATURES OF JUXTACELLULARLY LABELED THALAMIC CELLS AND OTHER CENTRAL NEURONS WITH BIOCYTIN OR NEUROBIOTIN

We describe a novel and very effective single-cell labeling method wit h unique advantages for revealing the axonal and dendritic fields of a ny extracellularly recorded neuron. This procedure involves the use of fine glass micro-pipettes (tip diameter: approximate to 1 mu m), whic h contain biocytin or Neurobiotin dissolved in a salt solution, for th e simultaneous juxtacellular recording and tracer iontophoresis. Once a neuron is well-isolated and identified, low intensity (< 10 nA) posi tive-current pulses are injected by way of the micro-electrode such as to modulate its firing. Juxtacellular tracer iontophoresis may last a s long as the cell electrophysiologically remains in good health, whil e determining some of its physiological properties. Control experiment s, including the selective killing of previously injected cells, provi de convincing evidence that it is the stained unit that was recorded a nd 'tickled' by the juxtamembranous iontophoretic pulses, Electrophysi ological and histochemical data further show that neuronal filling cou ld occur during an electrically induced, transient, physical micro-dam age of a somatic or dendritic membrane patch. This simple, single-cell staining method has been used to label several types of rat brain neu rons, including projection neurons and interneurons. Its success rate (> 86%) far exceeds that obtained by direct intracellular injections o f tracers as shown by the labeling of a large sample of 100 individual cells (from 115 attempts) in the thalamic reticular (Rt) nucleus of 3 3 rats. We thereby demonstrate that Rt cells project to restricted reg ions of a single thalamic nucleus, including anterior thalamic nuclei, and that the thalamus and Rt complex have reciprocal connections. The juxtacellular procedure thus represents an ideal directed single-cell labeling tool for determination of functional properties, for subsequ ent identification, for delineation of overall neuronal architecture a nd for tracing neuronal pathways, provided care is taken to avoid the possible drawbacks and pitfalls that are illustrated and discussed in the present paper.