Brain Struct Funct (2011) 216:301–317 307 Fig. 4 Calbindin-immunoreactive (CaBP-ir) and green-ﬂuorescent protein expressing pyramidal cells in a the septal CA1 and b the distal mid-septotemporal CA1 of thy1-GFP (M line) transgenic mice. c CaBP-ir CA1 pyramidal cells in septal CA1 of Wistar rat. Some of the voids left by unstained deep cells are marked with an asterisk. d Zinc-containing pyramidal cells in mid-septotemporal, mid-prox- imodistal CA1 of Wistar-Kyoto rat. e CaBP-ir CA1 pyramidal cells in temporal one-half of th fox CA1. Lightly CaBP-ir superﬁcial cells are separated from a tier of strongly CaBP-ir, large deep pyramids by target areas—calbindin and zinc. They are discussed in some detail below. Efforts have been made to rationally deﬁne functional domains along the hippocampal septo-temporal axis. A number of genes have been identiﬁed that may deﬁne such domains (Fanselow and Dong 2010; Dong et al. 2009; Thompson et al. 2008), and some of them are expressed preferentially in either superﬁcial or deep pyramidal cells of both CA1 (e.g. Nov and Nr3c2 in superﬁcial and Ndst4 and Astn2 in deep septal pyramidal cells) and CA3 (potassium channel subunit, Kcnq5, in superﬁcial and procollagen gene Col6a1 and suppressor of tumorigenicity 18, St18, in deep mid-septotemporal pyramidal cells). Their laminar distri- butions appear to coincide with those of calbindin and zinc. Calbindin. The distribution of calbindin immunoreactive (CaBP-ir) pyramidal cells in CA1 of the rat hippocampus probably represents the earliest neurochemical evidence for a radial subdivision of the layer. Throughout most of the transverse extent of the layer, CaBP-ir pyramidal cells are located in the cell-dense superﬁcial part, while deeper pyramidal cells do not contain this protein (Baimbridge and virtually unstained cells. f Cells in the septal three-quarters of the elephant shrew CA1 pyramidal cell layer (delimited by open arrows) are completely unstained. The ﬁlled arrow marks the boundary between stratum radiatum and stratum lacunosum-moleculare. g CaBP-ir deep CA1 pyramidal cells in the temporal CA1 of the elephant shrew. h CaBP-ir deep CA3 pyramidal cells in naked mole rats, mf: CaBP-ir mossy ﬁbers. Scalebars a, c and d 20 lm; b, g and h 50 lm; e and f 100 lm Miller 1982; Rami et al. 1987b; Sloviter 1989; Celio 1990). In the septal hippocampus, CaBP-ir pyramidal cells form a superﬁcial mono- or bilayer above unstained deeper cells (Fig. 4c; also compare with Fig. 2l, m). Only at the prox- imal limit of CA1, where CaBP-ir becomes weak superﬁ- cially, CaBP-ir appears in some of the deepest pyramidal cells. The deepest cells are also stained distally, in the vicinity of the subiculum. Baimbridge et al. (1991) have shown that there was virtually no overlap in the labeling of CaBP-negative and CaBP-ir pyramidal cells when animals were injected with 3H-thymidine on E16 and E18, respectively. Notably, calbindin expression is apparently controlled by Zbtb20—being down-regulated in cells in which the expression of Zbtb20 has been blocked and appearing in deep pyramidal cells as the expression of Zbtb20 is induced in these cells (Nielsen et al. 2010). Despite their change in location, pyramidal cells in CA1 of reeler mice retain their identities with regard to calbindin expression, which is strongest in the deep sublayer but weak or absent in superﬁcial cells (Fig. 3d). 123