Patterns of neuronal excitation in complex populations can be mapped anatomically by activating ionotropic glutamate receptors in the presence of 1-amino-4-guanidobutane (AGB), a channel-permeant guanidinium analogue. Intracellular AGB signals were trapped with conventional glutaraldehyde fixation and were detected by probing registered serial thin sections with anti-AGB and anti-amino acid immunoglobulins, revealing both the accumulated AGB and the characteristic neurochemical signatures of individual cells. In isolated rabbit retina, both glutamate and the ionotropic glutamate receptor agonists alpha-amino-3-hydroxyl-5-methylisoxazole-4-propionic acid (AMPA), kainic acid (KA), and N-methyl-D-aspartic acid (NMDA) activated permeation of AGB into retinal neurons in dose-dependent and pharmacologically specific modes. Horizontal cells and bipolar cells were dominated by AMPA/KA receptor activation with little or no evidence of NMDA receptor involvement. Strong NMDA activation of AGB permeation was restricted to subsets of the amacrine and ganglion cell populations. Threshold agonist doses for the most responsive cell groups (AMPA, 300 nm; KA, 2 microM; NMDA, 63 microm; glutamate, 1 mM) were similar to values obtained from electrophysiological and neurotransmitter release measures. The threshold for activation of AGB permeation by exogenous glutamate was shifted to <200 microM in the presence of the glutamate transporter antagonist dihydrokainate, indicating substantial spatial buffering of extracellular glutamate levels in vitro. Agonist-activated permeation of AGB into neurons persisted under blockades of Na+ -dependent transporters, voltage-activated Ca2+ and Na+ channels, and ionotropic gamma-aminobutyric acid and glycine receptors. Cholinergic agonists evoked no permeation.