Electrophysiological investigations define receptive field and other functional properties. These are carried out by intra- or extracellular recordings, usually from the soma but also from axon processes. Axonal conduction velocity is another important parameter. This is analyzed by measuring the latencies of spikes evoked by antidromic activation of axons by electrical stimulation. Temporal relationships between the firing times of two neurons can also be assessed by cross-correlation techniques, although these are more often used for inferring whether two neurons are connected.

A particularly elegant approach is intracellular or intraaxonal microelectrode recording combined with intracellular microinjections of tracers such as HRP or biocytin. In this way, physiological characteristics can frequently be correlated with morphological specializations. For example, thalamocortical connections to primary visual cortex are subdivided into several functionally distinct categories concerned with form or motion vision. Axons conveying "information" related to form (from the parvocellular layers of the lateral geniculate nucleus) have properties consistent with achieving fine spatial resolution. Among other specialized features, their terminal arbors are smaller than those of axons concerned with motion vision (from the magnocellular layers).

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