Exp Brain Res (1992) 90:297-301 nResearch 9 Springer-Verlag1992 Immunohistochemistry and neurochemistry of the habenulo-interpeduncular connection after partial developmental depletion of habenular cholinergic neurons in the rat A. Fasolo 1, M. Virgili 2, G.C. Panzica 3, and A. Contestabile z 1Department of Animal Biology,Universityof Torino, Via AccademiaAlbertina 17, 1-10123 Torino, Italy 2 Department of Biology,Universityof Bologna, 1-40126 Bologna, Italy 3 Department of Human Anatomyand Physiology, Universityof Torino, 1-10126 Torino, Italy Received July 31, 1991/Accepted March 9, 1992 Summary. The habenulo-interpeduncular system of the rat constitutes an interesting model to address quantita- tively problems related to synaptogenesis and to the interactions between neuronal populations after selective alteration of these elements during development. In the present study this has been achieved by experimentally reducing, through gestational treatment with methyl- azoxymethanol acetate (MAM), the population of cholin- ergic neurons of the medial habenula which projects to the interpeduncular nucleus. Immunohistochemical analysis gave evidence that the topographical localization of the cholinergic and the substance P-containing populations in the medial habenula was not altered by MAM treatment. Furthermore, the topographical distribution of choliner- gic fibers and terminals in the interpeduncular nucleus, which reflects the habenulo-interpeduncular projection as well as cholinergic projections coming from different sources, was substantially preserved. The same was also true concerning the terminal distribution of substance P in the interpeduncular nucleus. Quantitative radioassays demonstrated a sizable decrease of overall ChAT activity in both the habenulae and the interpeduncular nucleus. By comparison of 1 month-old and 3 month-old animals it appeared that this effect was partially reversed with age in the interpeduncular nucleus. Key words: Methylazoxymethanol acetate - Microence- phaly Choline acetyltransferase Substance P Rat Introduction The habenulo-interpeduncular connection through the fasciculus retroflexus has been extensively investigated with reference to its anatomy and neurochemistry (Her- kenham and Nauta 1979; Contestabile and Flumerfelt Correspondence to: A. Fasolo 1981; Contestabile and Fonnum 1983; Groenewegen et al. 1986; Contestabile et al. 1987; Kawaja et al. 1990). The habenulae are the source of a massive and topographically ordered projection to different subnuclei of the inter- peduncular nucleus. This projection originates mainly from cholinergic and substance P-containing cell groups differentially localized in the medial habenula; their ter- minal fields have a complementary distribution in the interpeduncular nucleus. An interesting feature of the habenulo-interpeduncular system is its peculiar synaptic plasticity which can be revealed both during normal development and as a consequence of different types of lesions (Lenn 1978; Murray et al. 1979; Lenn and With- more 1989). This neural system may, therefore, constitute an useful model to study the effects of developmental alterations of the quantitative relationships between two interconnected nuclei as well as the balance between different neurotransmitter systems. This approach is made feasible by the use of agents which interfere with normal brain development by killing dividing cells (Johnston and Coyle 1982). This approach has proven particularly useful when applied to the habenulo-interpeduncular cholinergic system (Virgili'et al. 1988) in which the generation times of the projection neurons (medial habenula) and of the receiving neurons (interpeduncular nucleus) do not over- lap temporally. Neurogenesis in the interpeduncular nu- cleus is, indeed, completed by the end of the 15th gesta- tional day while the birth date of medial habenular neurons essentially occurs between gestational days 16 and 18 (Lenn and Bayer 1986). By injecting pregnant mothers with methylazoxymethanol acetate (MAM), at the beginning of gestational day 17, we found in young- adult rats a significant decrease of the cholinergic levels in both nuclei, as assessed by the radiochemical assay for choline acetyltransferase (CHAT) (Virgili et al. 1988). How- ever, the mere measurement of the enzymatic activity does not provide any information concerning the preservation or the disruption of the topographical patterns of choliner- gic neurons in MAM-treated animals. We therefore de- cided to further investigate this experimental model by means of immunohistochemical analysis of ChAT and