Brain Research, 447 (1988) 369-375 Elsevier BRE 22869 369 A new combination of WGA-HRP anterograde tracing and GABA immunocytochemistry applied to afferents of the cat inferior olive at the ultrastructural level C.I. de Zeeuw, J.C. Holstege, F. Calkoen, T.J.H. Ruigrok and J. Voogd Department of Anatomy HB13, Medical Faculty, Erasmus University. Ronerdam (The Netherlands) (Accepted 28 January 1988) Key words: Anterograde tracing: Wheatgerm-agglutinated horseradish peroxidase (WGA-HRP): ;,-Aminobutyric acid (GABA) immunocytochemistry: Inferior olive: Electron microscopy: Cat In order to identify cerebellar terminals in the cat inferior olive which contain 7-aminobutyric acid (GABA), a technique was deve- loped combining anterograde transport of wheatgerm agglutinine-conjugated horseradish pcroxidase (WGA-HRP) with gold-immu- noeytochemistry. With this technique both the HRP reaction pro:~uct and the immunogold labelling can be visualized in a single ultra- thin section. Our results suggest that most, if not all of the WGA-HRP-labelled cerebellar terminals in the rostral medial accessory olive (MAO) and the rostral principal olive (PO) are GABAergic. In an additional experiment the GABAergic innervation of the ros- tral MAO was studied in combination with WGA-HRP anterograde tracing from the rostral mesencephalon. In this case the WGA-HRP-labelled terminals were never found to be GABA-positive. There exists a reciprocal connection between the inferior olive and the cerebellar nuclei, which is topo- graphically organized 15"17. The cerebellar innerva- tion of the inferior olive is largely GABAergic as demonstrated lightmicroscopically in the rat by means of retrograde transport of horseradish perox- idase (HRP) combined with glutamatedecarboxylase (GAD) immunohistochemistry9. We studied the ce- rebellar GABAergic innervation of the cat inferior olive at the ultrastructural level to answer the ques- tion whether all cerebe!lo-o!ivary terminals are GABAergic. For this purpose a technique was deve- loped combining postembedding ),-aminobutyric acid (GABA)-immunogold staining with anterograde transport of WGA-HRP. For HRP histochemistry tetramethylbenzidine (TMB) was uscd as a chromo- gen. The incubation in TMB was followed by a stabi- lization procedure with diaminobenzidine-cobalt (DAB-Co) t'. In order to determine whether the stabi- lization procedure could induce false positive label- ling with GABA, the same technique was applied to another, presumably non-GABAergic system. In this additional experiment WGA-HRP was injected in the nucleus of Darkschewitsch which is known to project to the medial accessory olive (MAO) I°. In two cats 0.5 Id WGA-HRP (7% in saline) was injected bilaterally in the interposed and lateral nu- clei of the cerebellum under Nembutal anaesthesia (Fig. 1A). In another cat a similar injection was made in the rostral part of the nucleus of Darkschewitsch and its surrounding area (Fig. 1B). After a survival time of 3 days the cats were deeply anaesthetized with Nembutal and perfused transcardially with 100 ml 0.9% saline in 0.1 M phosphate buffer (PB) (pH 7.3) under artificial respiration, followed by 2 liter 5% glutaraldehyde in 0.1 M PB (pH 7.3). The brain- stem was removed and a block containing the inferior GlOve was dissected ~nd left in the fixattve tor one hour. Subsequently the inferior olive was cut trans- versely into slices of 80 Ftm on a vibratome. The vi- bratome sections were incubated with TMB in 0.05 M acetate buffer (AB) (pH 4.8), rinsed twice in 0.05 Correspondence: C.I. de Zeeuw, Department of Anatomy HBI3, Medical Faculty, Erasmus University. P.O. Box 1738.31)(11) DR Rotterdam, The Netherlands. 0006-8993/88/$l)3.50© 1988 Elsevier Science Publishers B.V. (Biomedical Division)