Proc. R. Soc. Lond. B. 199 , 463-476 (1977) Printed in Great Britain The resolution of neuronal assemblies after cobalt injection into neuropil B y N. J. S traus FELDt and K. H ausen J f j EuropeanMolecular Biology Laboratory, Heidelberg, Federal Republic of Germany J Max Planck Institut fur biologische Kybernetik, Tubingen, Federal Republic of Germany (Communicated by Sir John Kendrew, F.R.S. - Received 17 August 1977) [Plates 1 and 2] Cobalt ions are incorporated by many intemeurons of the insect central nervous system after injection of cobalt chloride or cobalt acetate into the neuropil. Entire nerve cells are subsequently revealed by sulphide precipitation which is followed by silver intensification of whole brains and ganglia. The number of neurons resolved depends upon the optimal timing of an injection phase, followed by a period of diffusion. These, and the site of injection, can be adjusted so as to demonstrate arrangements within and between complete populations of morphological classes of interneurons. This novel use of cobalt reveals cytoarchitectural features of neuropil that are refractory to other histological procedures and shows that in columnar visual neuropil the nerve cell arrangements are limited to a few basic modes of assembly. I ntroduction Much of our knowledge about the structure of the central nervous system is derived from classical histological techniques. These either resolve single neurons that are unpredictably impregnated by the Golgi method, or any of its modifications, or resolve patterns of neurofibrillae after treatment by a reduced silver technique (see Ramon-Moliner 1967; Boycott, Gray & Guillery 1961). Upon these methods all modern procedures for structural investigation find a frame of reference. These include diverse techniques such as, for example, staining for degeneration (see Heimer 1970), tracing pathways with radioactive amino acids (LaVail & LaVail 1972) or with horseradish peroxidase (Cowan et al. 1972), and resolving single neurons by electrophoresis of Procion dyes or cobalt into axons or cell bodies (Stretton & Kravitz 1968; Pitman, Tweedle & Cohen 1972). Introduction of dye or cobalt into severed connectives, fasciculi or peripheral nerve bundles con- stitutes one method for resolving groups of neuronal components rather than entire nerve cells (see, for example, Llinas 1973; Gregory 1974; Altman & Tyrer 1974; Sz^kely & Galiyas 1975). Apart from rare exceptions these methods consistently fail to demonstrate l6 [ 463 ] Vol. 199. B.