Anterograde and retrograde filling of central neuronal systems with horseradish peroxidase under in vitro conditions

Abstract

In this study, a simple technique for neuronal tracing with horseradish peroxidase (HRP) in vitro is described. It can be applied to excised small brains of poikilothermic vertebrates, or to trimmed blocks or slices of brain tissue from homoiothermic vertebrates. The filling technique relies on the diffusion of HRP into transected axons of supravital neurons, and gives a Golgi-like staining of the filled neuronal profiles. The incubation is carried out at 4°C, which minimizes unspecific background staining caused by pinocytotic uptake of HRP. The technique was tested for visualizing (i) intrapineal neurons projecting to the brain in the rainbow trout, (ii) pinealofugal projections to the brain in the rainbow trout and the three-spined stickleback, (iii) retinal ganglion cells in the rainbow trout, and (iv) central pinealopetal projections in the golden hamster. The strength of this method lies in the possibility to perform very accurate applications of HRP to excised brains or brain slices under a stereo microscope, the simple processing of tissue, and the Golgi-like filling of the neuronal profiles. This technique may provide an important complement to currently used HRP-tracing techniques.