The habenular complexes represent phylogenetically constant structures in the diencephalon of all vertebrates. Available evidence suggests that this area is engaged in a variety of important biological functions, such as reproductive behaviors, central pain processing, nutrition, sleep‐wake cycles, stress responses, and learning. Based on Nissl‐stained sections, one medial nucleus and two lateral nuclei (divisions) have been widely accepted in the rat. Cytochemical, hodologic, and functional studies suggest a considerably more complex subnuclear structure. To improve our knowledge of the precise structural composition of the habenular complexes, we have systematically investigated their fine ultrastructure in the rat. Based on the detailed analysis of complete series of large, semithin sections supplemented with electron photomicrographs of selected fields, clear criteria for the delineation of five distinct subnuclei of the medial and ten subnuclei of the lateral habenular complexes were elaborated for the first time. All 15 subnuclei were reconstructed, and their dimensions were determined. A medial and lateral stria medullaris were described. Different roots of the fasciculus retroflexus were differentiated within the medial and lateral habenular complexes. The topographical relationships with respect to the adjacent habenular areas as well as to the neighboring thalamic nuclei were identified and demonstrated. The new understanding of the subnuclear organization of the habenular complexes certainly will facilitate further functional investigations. Whether the newly identified subnuclei finally will be recognized as functionally distinct awaits ongoing immunocytochemical, hodologic, and functional studies. J. Comp. Neurol. 407:130–150, 1999. © 1999 Wiley‐Liss, Inc.

Haptenylation of primary antibodies is a useful technique for multiple purposes. It is a technically straightforward procedure, as many haptens are available as N-hydroxysuccinimide esters or isothiocyanates. Unfortunately, the hapten group may become covalently attached to or close to the combining site of antibodies, lectins, or other ligand-binding proteins during the process of haptenylation. Thus, the interaction of the corresponding protein with its ligand may become severely hampered. To overcome this restriction, we developed a novel procedure for the haptenylation of polyclonal antibodies that combines purification and haptenylation. Haptenylation during adsorption to the affinity matrix combines two advantages: the antigen binding site is protected and the labeling procedure becomes most convenient, as overlabeled proteins and unreacted haptens are easily removed by simple washing. Haptenylation during adsorption to the affinity matrix is a two-phase reaction, which requires different conditions to the conventional procedure. To obtain such optimal conditions, stabilities and reactivities of N-hydroxysuccinimide esters and isothiocyanate groups were investigated with a newly developed assay. Based on this information, antibodies against two recently described calcium-binding proteins, NCS-1 and NVP-3, were biotinylated or digoxigenylated. The haptenylated antibodies were successfully applied for biochemical determination and simultaneous immunoenzymatic double labeling of the two proteins.