Ultrastructural detection of neuronally transported choleragenoid by postembedding immunocytochemistry in freeze-substituted Lowicryl HM20™ embedded tissue

Choleragenoid (cholera toxin B-fragment; CTB) is an anterograde, retrograde and transganglionic neuronal tracer. We describe a method for detecting CTB-labeled neuronal cell bodies, neurites and boutons at the ultrastructural level, using postembedding immunogold techniques on freeze-substituted Lowicryl HM20? embedded nervous tissue. Primary afferents and motoneurons were labeled by injection of CTB in the dorsal ramus of the C2 spinal nerve of the rat. Following fixation with paraformaldehyde (4%) and glutaraldehyde (0.25%), tissue sections from the spinal cord C2 segment were freeze-substituted and embedded in Lowicryl HM20? and subsequently processed with postembedding immunocytochemistry for CTB and glutamate. Immunogold particles indicating CTB immunoreactivity were found over primary afferents and motoneurons. In primary afferents in the central cervical nucleus (CCN) and motor nuclei, immunogold labeling was seen in boutons over vesicle-containing axoplasm and to a lesser extent over axoplasm devoid of vesicles, but not over mitochondria or axolemma. In motoneurons, immunogold particles were seen over the Golgi apparatus in the soma and over lysosomes in both soma and dendrites. Quantification of glutamate-like immunoreactivity in 20 CTB-labeled and 20 CTB-negative boutons in the neuropil was found similar, indicating that CTB does not interfere with the immunocytochemical detection of neuronal epitopes such as the transmitter substance glutamate.