A method for specific transmitter identification of retrogradely labeled neurons: immunofluorescence combined with fluorescence tracing.

In the present article a method is described which allows the delineation of the projections of a single neuron as well as the identification of one or more of its chemical components. The technique is a combination of retrograde tracing and fluorescent dyes based on the work of Kuypers and collaborators and indirect immunofluorescence histochemistry as originally described by Coons and collaborators. The crucial parameters including the selection of the dyes, the injection technique and tissue processing as well as the appropriate immunohistochemical fluorescent markers and filter combinations are discussed. The method of choice involves the use of the retrogradely transported dyes Fast Blue, True Blue or Propidium Iodide, and in addition, for double labeling experiments, Diamidino Yellow or Primuline. They are combined with FITC (Propidium Iodide) or TRITC (Fast Blue, True Blue, Diamidino Yellow, Primuline) as immunofluorescence markers.

Attempts to combine 2-deoxyglucose autoradiography and tyrosine hydroxylase immunohistochemistry.

The possibilities were analysed to combine the 2-deoxyglucose technique and indirect immunofluorescence histochemistry using tyrosine hydroxylase antiserum, with the aim to study functional activity in immunohistochemically characterized single neurons. Since the product measured with the 2-deoxyglucose method is water soluble and since immunohistochemistry requires that sections repeatedly run through aqueous media, the 2-deoxyglucose method was carried out before fixation and immunohistochemistry. The routine rapid thaw-mounting at + 60 degrees C of sections for 2-deoxyglucose autoradiography was found not to be compatible with immunohistochemistry. Instead a new mounting technique based on "gluing" the sections on to the object slide with a mixture of a standard mounting medium (Permount) and xylene was used to avoid diffusion at this stage. Two procedures were outlined, both starting with unfixed brains cut on a cryostat. In Method I autoradiographic sheet film was used. After autoradiographic exposure, the section was immersion-fixed in formalin, processed for immunohistochemistry, analysed and photographed in a fluorescence microscope and the results compared with the autoradiographic distribution patterns on the film. However, only the low resolution of the routine 2-deoxyglucose technique was obtained, which did not allow analysis of activity in single cells. In Method II, liquid emulsion applied by the loop technique was used. After exposure, autoradiographic developing and fixation, dehydration, mounting, analysis and photography of autoradiographs in the light microscope, the cover-slip was removed, the sections rehydrated and processed for indirect immunofluorescence histochemistry. With this procedure single autoradiographically labeled cells were observed, some of which contained tyrosine hydroxylase. Thus, with Method II it may in the future be possible to monitor functional activity in single immunohistochemically identified neuronal cell bodies. In order to obtain a useful and reliable method for this purpose, however, further extensive work with regard to, for example, quantification will be required.

Improved resolution of the 2-deoxy-D-glucose technique.

It was attempted to improve the resolution of the 2-deoxyglucose method. Two principle changes in the procedure were introduced: the gluing of the sections on to the object slide at--20 degrees C and the application of the emulsion with the loop technique. With this approach autoradiographs with grain accumulations over single cell bodies could be observed in many brain regions in addition to a diffuse activity over neuropil.

The pontocerebellar system in the rat: an HRP study. II. Hemispheral components.

The projection of basilar pontine neurons to the cerebellar hemispheres was studied to pigmented rats by means of the retrograde transport of horseradish peroxidase. Injections of horseradish peroxidase were restricted to the lateral aspects of the lobulus simplex (11 cases), crus I (26 cases), crus II (23 cases), and paramedian lobule (18 cases). The main focus of labeled neurons following lobulus simplex injections of horseradish peroxidase was located in the ventral pons, at rostral levels. Interestingly, the majority of labeled cells were distributed ipsilateral to the injection site. After crus I injections, however, labeled neurons were most evident contralaterally , although labeled ipsilateral cells were conspicuous rostrally. The majority of labeled cells were characteristically distributed along the medial, ventral, and lateral perimeters of the pontine gray. This pattern of labeling contrasts with that in cases of crus II injections, in which the main focus of labeled somata occupied more central regions of medial and ventral portions of the pons. Similarly, the pattern of labeling following injections into the paramedian lobule largely avoided the medial and lateral perimeters of the pontine gray, while numerous labeled somata occupied the central region of the pons. In addition to the pontine regions described above, labeled cells were observed in various cases in the dorsal peduncular region, the lateral and dorsolateral areas, and the nuclear reticularis tegmenti pontis (NRTP) where three separate zones of labeling could be discerned in various cases. Several general organizational features were derived from these studies. Although specific quantitation procedures were not applied, the number of ipsilaterally labeled neurons was impressive in some cases, as was the mirror-image location of certain ipsi- and contralateral cell clusters. It was also noted that certain, similarly located clusters of labeled pontine neurons were present in cases in which injections were made into different cerebellar lobules, at least raising the possibility that some pontine neurons might give rise to divergent projections of multiple cerebellar locations, Moreover, it was evident that the location of certain clusters of labeled neurons was congruent with terminal zones of various pontine afferent systems, particularly those of the sensorimotor cortex. Combining the latter finding with the preceeding notion regarding pontocerebellar divergence suggests a mechanism by which sensorimotor information might be transmitted to several different cerebellar locations.