ULTRASTRUCTURAL DETECTION OF NEURONALLY TRANSPORTED CHOLERAGENOID BY POSTEMBEDDING IMMUNOCYTOCHEMISTRY IN FREEZE-SUBSTITUTED LOWICRYL HM20(TM) EMBEDDED TISSUE

Choleragenoid (cholera toxin B-fragment; CTB) is an anterograde, retro grade and transganglionic neuronal tracer. We describe a method for de tecting CTB-labeled neuronal cell bodies, neurites and boutons at the ultrastructural level, using postembedding immunogold techniques on fr eeze-substituted Lowicryl HM20(TM) embedded nervous tissue. Primary af ferents and motoneurons were labeled by injection of CTB in the dorsal ramus of the C2 spinal nerve of the rat. Following fixation with para formaldehyde (4%) and glutaraldehyde (0.25%), tissue sections from the spinal cord C2 segment were freeze-substituted and embedded in Lowicr yl HM20(TM) and subsequently processed with postembedding immunocytoch emistry for CTB and glutamate. Immunogold particles indicating CTB imm unoreactivity were found over primary afferents and motoneurons. In pr imary afferents in the central cervical nucleus (CCN) and motor nuclei , immunogold labeling was seen in boutons over vesicle-containing axop lasm and to a lesser extent over axoplasm devoid of vesicles, but not over mitochondria or axolemma. In motoneurons, immunogold particles we re seen over the Golgi apparatus in the soma and over lysosomes in bot h soma and dendrites. Quantification of glutamate-like immunoreactivit y in 20 CTB-labeled and 20 CTB-negative boutons in the neuropil was fo und similar, indicating that CTB does not interfere with the immunocyt ochemical detection of neuronal epitopes such as the transmitter subst ance glutamate. (C) 1998 Elsevier Science B.V. All rights reserved.