Comparison of three intracellular markers for combined electrophysiological, morphological and immunohistochemical analyses

Hypothalamic paraventricular and supraoptic neurons were recorded intracellularly in coronal slices and injected with Lucifer yellow, ethidium bromide or biocytin. Electrical properties, morphological staining and neurophysin immunohistochemistry were compared among the 3 markers. Lucifer yellow electrodes had a high resistance and frequently blocked during experiments. Neurons recorded with Lucifer yellow electrodes had low input resistances and low-amplitude, broad spikes. Lucifer yellow labeling in whole mount was highly fluorescent, revealing distal dendrites and axons. Of cells injected with Lucifer yellow, 64% were recovered but were faint after immunohistochemical processing. Recordings with ethidium bromide electrodes were similar to controls, although electrode blockage sometimes occurred. Only somata and proximal dendrites of ethidium bromide-filled neurons were visible in whole-mount. Forty percent of cells injected with ethidium bromide were recovered after immunohistochemical processing; these were invariably faint. Recordings with biocytin-filled electrodes were similar to control recordings. Biocytin-filled, HRP-labeled cells showed distal dendrites and often dendritic spines and axons in 50–75-μm sections. Seventy percent of biocytin-injected cells labeled with fluorescent markers were recovered and remained strongly labeled after immunohistochemical processing. Biocytin had the best electrical and staining properties for combined electrophysiological and anatomical studies.