Galanin and cholecystokinin in cultured magnocellular neurons isolated from adult rat supraoptic nuclei: a correlative light and scanning electron microscopical study ADELAIDA SANCHEZ 1 , MARIO BILINSKI 2à , VALERIA GONZALEZ NICOLINI 2 , MARCELO J. VILLAR 2 and JUAN H. TRAMEZZANI 2 1 Facultad de Ciencias Veterinarias, Ca Âtedra de Histologõ Âa y Embriologõ Âa, Universidad de Buenos Aires, and 2 Instituto de Neurobiologõ Âa, Serrano 669, Buenos Aires, Argentina Received 3 January 1997 and in revised form 15 April 1997 Summary Cultured magnocellular neurons, isolated from adult rat supraoptic nuclei, were characterized by immunocytochemistry, using the avidin±biotin±peroxidase complex and antisera to vasopressin, oxytocin, galanin and cholecystokinin. Light microscope examination of the immunostained cultures revealed the presence of vasopressin- and oxytocin-like immunoreactivity, as well as neurons containing either galanin- or cholecystokinin-like immunoreactivity. In contrast, no signi®cant galanin- or cholecystokinin-like immunoreactivity could be observed in freshly dispersed cells. Correlative scanning electron microscopical observations in the secondary electron imaging mode revealed that the stained neurons appeared signi®cantly brighter than the unstained structures. Complementary observations with toad brain sections (preoptic area), immunostained for galanin, led to the same result. Considering previous results, it is suggested that the presence of galanin- and cholecystokinin-like immunoreactivity in the cultured neurons and its virtual absence in freshly dispersed cells is indicating a participation of these peptides in the regenerative processes taking place during culture. It is further concluded that the avidin±biotin±peroxidase method is suitable for correlative light and scanning electron microscopical studies of smooth surfaces and cultured cells. Introduction The magnocellular hypothalamic neural system con- sists of peptidergic neurons located in the paraven- tricular and supraoptic nuclei. Separate sub- populations of magnocellular neurons are the site of synthesis of either vasopressin (VP) or oxytocin (OXY), which are transported to and released from the neurohypophysis (see Brownstein et al., 1980; Castel et al., 1984). Evidence has been obtained for the coexistence of the mentioned hormones with other peptides in these cells. VPergic magnocellular neurons were shown to exhibit immunoreactivity for galanin (Melander et al., 1986; Ro È kaeus et al., 1988; Gaymann & Martin, 1989; Sko®tsch et al., 1989; Meister et al., 1990), dynorphin (Watson et al., 1982; Whitnall et al., 1983), Leu-enkephalin (Martin & Voigt, 1981; Martin et al., 1983) and angiotensin II (Kilcoyne et al., 1980). On the other hand, immunor- eactivity for cholecystokinin (Beinfeld et al., 1980; Vanderhaeghen et al., 1981; Martin et al., 1983; Tsuruo et al., 1988; Meister et al., 1990), corticotro- pin-releasing factor (Burlet et al., 1983; Sawchenko et al., 1984), thyrotropin-releasing factor (Tsuruo et al., 1988), Met-enkephalin (Martin & Voigt, 1981; Martin et al., 1983; Vanderhaeghen et al., 1983; Adachi et al., 1985; Gaymann & Martin, 1987) and renin (Fuxe et al., 1982) could be detected in OXYergic magnocel- lular neurons. Finally, nitric oxide synthase-like immunoreactivity has been shown in VPergic and OXYergic neurons (see Villar et al., 1994). Very little is known about the functions of the coexisting peptides in these neurons, but some results suggest a possible role for galanin (GAL) and cholecystokinin (CCK) in regenerative pro- Histochemical Journal 29, 631±638 (1997) 0018±2214 # 1997 Chapman & Hall à To whom correspondence should be sent.