APPROACH TO TRACING NEURON NETWORKS IN THE CEREBRAL-CORTEX AND BASAL GANGLIA - COMBINATION OF GOLGI STAINING, RETROGRADE TRANSPORT OF HORSERADISH-PEROXIDASE AND ANTEROGRADE DEGENERATION OF SYNAPTIC BOUTONS IN THE SAME MATERIAL

A procedure is described that makes it possible to study, in the light microscope and in the electron microscope, neurons that have been labelled by the retrograde transport of horseradish peroxidase in brain tissue that is also subsequently stained by the Golgi method. The procedure involves the use of o-tolidine (3,3′-dimethylbenzidine) as a substrate for horseradish peroxidase; the reaction end product from this substrate can be formed in blocks of tissue of the size needed for the Golgi method and can be recognized both in the light and in the electron microscope. In order to detect the reaction end product within Golgi-stained neurons in the light microscope, the Golgi stain is replaced by gold ('gold-toning'). It then becomes possible to identify the type of efferent neuron (in an area we call B), on the basis of its characteristic shape and dendritic morphology, that projects to the area (C) where horseradish peroxidase was injected. Furthermore, the afferent input to such a characterized efferent neuron can be studied if a lesion had previously been placed in a third area (A) that projects to area B; degenerating terminal boutons making contact with any part of the Golgi-stained, horseradish peroxidase labelled neuron can then be identified in the electron microscope. In this way, the existence of a chain of neurons within a network can be established starting with a neuron in area A, through a monosynaptic link to a neuron (in area B) that sends a projection to area C. In addition, if the region that is stained by the Golgi method contains Golgi-stained local circuit interneurons, it is possible to study the input from these interneurons to an efferent neuron that has been labelled retrogradely with horseradish peroxidase. The procedure is illustrated by three specific applications that have provided the following new information concerning connections in the brain of the rat. 1. (i) Some of the neurons that project from the striatum to the substantia nigra are medium sized and have densely spinous dendrites. 2. (ii) One type of striatonigral neuron terminates (symmetrical synapses) on smooth dendrites of nigrothalamic neurons in the substantia nigra pars reticulata. 3. (iii) A specific type of local circuit interneuron (the 'chandelier cell' or 'axo-axonic' cell) in the visual cortex terminates (symmetrical synapses) on the axon initial segments of pyramidal cells that project to the contralateral visual cortex. Some possible functional implications of these findings are discussed. It is suggested that further applications of the procedure might provide direct information about many aspects of synaptic connectivity in neuron networks in parts of the central nervous system that are too complex to study by conventional morphological methods. © 1979.