Ultrastructural metabolic activity following quick-freezing and freeze-substitution in tetrahydrofuran in the superior cervical ganglion.

A method of quick-freezing and freeze-substitution has been developed for localizing diffusible substances such as 2-deoxyglucose-6-phosphate (2-DG-6-P) ultrastructurally in neural tissue. Quick-freezing under pressure provides well preserved tissue down to 30-35 microns from the surface. This allows blocks of neural tissue to be quick-frozen and analysed for diffusible substances in areas removed from the freezing face. Freeze-substitution in tetrahydrofuran following quick-freezing was found to dissolve and remove 2-deoxyglucose (2-DG) but not 2-DG-6-P. Consequently, this technique extends the ability to analyse localization of glucose utilization to postsynaptic as well as presynaptic sites. We have applied the technique to isolated superior cervical ganglion while provoking selective increases in energy metabolism. Exposure to an elevated extracellular potassium (12 mM) concentration produced a pattern of metabolic activity with enhanced neuropil labelling (neuronal and glial processes). With antidromic stimulation of the external carotid nerves, deoxyglucose uptake in neuronal and glial soma in the caudal portion of the ganglion was enhanced more than neuropil labelling. This caudal region corresponds to the region of origin of the cell bodies of the external carotid nerve. Results from this technique suggest that the contribution of glia to overall rate of energy metabolism may be significant and that this is a promising method for correlating the relationship between functional activity and cellular electrical activity.