Brain Struct Funct (2011) 216:301–317 309 Fig. 5 Mid-septotemporal hippocampus of Wistar Kyoto rats, stained for zinc-containing neurons and ﬂuorescence of the retrograde tracer ﬂuorogold. a In proximal CA1, predominantly deep zinc-negative pyramids label after an injection into the ipsilateral lateral septum. b After a contralateral ﬂuorogold injection into the lateral septum, deep zinc-containing CA3 pyramids are not labeled, whereas c deep superﬁcial pyramidal cells. A number of retrograde tracing studies in rodents and primates have indeed shown a preference of labeled pyramidal cells to be located either deep or superﬁcially in the cell layer. These include the hippocampo-septal projection (Chronister and DeFrance 1979; see also below), projections from deep CA1 pyra- midal cells to the nucleus accumbens (McGeorge and Faull 1989), medial prefrontal (Barbas and Blatt 1995; Insausti and Mun˜ oz 2001; Roberts et al. 2007) and orbitofrontal cortices (Cavada et al. 2000), and projections from super- ﬁcial CA1 pyramidal cells to the medial temporal cortex (Yukie 2000; Insausti and Mun˜ oz 2001). In a study of hippocampo-septal connections (Sørensen et al. 1993) we found that projections to the septum originate from zinc- containing as well as zinc-negative pyramidal cells of much of CA1 while septal projections originate primarily from zinc-negative deep pyramidal cells in extreme prox- imal CA1 (Fig. 5a), a part of the layer that is likely to correspond to CA2. More strikingly, zinc-containing dee- ply situated cells in CA3 project almost exclusively to the ipsilateral septum while zinc-negative superﬁcial pyrami- dal cells have strong bilateral septal connections (Fig. 5b, c). In a study of the zinc-containing afferent circuitry of the striatum (Sørensen et al. 1995) we observed that CA1 pyramidal cells projecting to the ventral striatum/nucleus accumbens are found among the zinc-negative deep pyra- midal cells (Fig. 5d). Based on multiple injections of different tracers, Swanson et al. (1980) proposed that CA3 pyramidal cells form a population of neurons, which have virtually iden- tical efferent projections. Their sections were obtained from within the septal one-third of CA3, whereas zinc- containing cells are mainly located within the temporal half of CA3. Even at temporal levels, the estimates made at zinc-containing CA3 pyramids are labeled after an ipsilateral ﬂuorogold injection into the lateral septum. d Only deep zinc- negative pyramidal cells in distal CA1 are retrogradely labeled after an ipsilateral ﬂuorogold injection into the ventral striatum. Scalebars a–c 50 lm; d 25 lm septal levels (more than 50% multiple labeling, Swanson et al. 1980) provide sufﬁcient leeway from ‘‘virtual iden- tity’’ to accommodate different septal connections of zinc- containing and zinc-negative pyramidal cells. Afferent connections Deﬁning differences in the afferent connectivity of deep and superﬁcial pyramidal cells would require an identiﬁ- cation of individual cells postsynaptic to afferents that have been visualized either by tract tracing procedures or immunocytochemistry (e.g. Kajiwara et al. 2008). This task is not easily accomplished, and data on afferent connec- tions speciﬁc to deep or superﬁcial pyramidal cells are scarcer than on efferent connections. Early evidence con- cerns local inhibitory afferents as described by Lorente de No (1934), who states ‘‘… the second reason [to distin- guish both types of pyramids] is that the superﬁcial and deep pyramids have a different relation to the pyramidal, horizontal and polygonal basket cells. The superﬁcial pyramids are in contact with the end arborizations of pyramidal, horizontal and polygonal cells. The deep pyra- mids are chieﬂy in contact with the polygonal basket cells, and the deepest have almost no contact with the basket plexus.’’ Unfortunately, this observation is not being commented on further in the extensive literature on inhibitory hippocampal neurons and their targets within CA1 and CA3 (reviewed in Freund and Buzsa´ki 1996; Klausberger and Somogyi 2008). With regard to interneu- rons characterized by the presence of parvalbumin, the innervation of the two pyramidal cell layers in reeler mice does not show gross differences (Fig. 3e, Ishida et al. 1994). That inhibitory mechanisms of deep and superﬁcial pyramidal cells may differ is supported by the selective 123