Genomic–Anatomic Evidence for Distinct Functional Domains in Hippocampal Field CA1

Genomic–anatomic evidence for distinct functional domains in hippocampal field CA1

Hong-Wei Donga,1, Larry W. Swansonb, Lin Chenc, Michael S. Fanselowd, and Arthur W. Togaa

aLaboratory of Neuro Imaging and Department of Neurology, School of Medicine, University of California, Los Angeles, CA 90095-7334; bDepartment of Biological Sciences, University of Southern California, Los Angeles, CA 90089; cDepartment of Genomic Science, University of Washington, Seattle, WA 98195; and dDepartment of Psychology and Brain Institute, University of California, Los Angeles, CA 90095-1563

Edited by Edward G. Jones, University of California, Davis, CA, and approved May 8, 2009 (received for review December 15, 2008) Functional heterogeneity has been investigated for decades in the two of the most complex cerebral functions—cognition and hippocampal region of the mammalian cerebral cortex, and evi- emotion. dence for vaguely defined ‘‘dorsal’’ and ‘‘ventral’’ regions is emerging. Direct evidence that hippocampal field CA1 displays clear Results regional, laminar, and pyramidal neuron differentiation is pre- More than 4,000 genes are expressed in the hippocampal for- sented here, based on a systematic high-resolution analysis of a mation, based on data on the ABA web site. We first screened publicly accessible, genome-wide expression digital library (Allen expression patterns of Ͼ2,000 genes in the hippocampal region, Brain Atlas) [Lein et al. (2007) Genome-wide atlas of gene expres- based on expression level, expression density, and gene cluster- sion in the adult mouse brain. Nature 445:168–176]. First, genetic ing. From this, we carefully analyzed expression patterns of 48 markers reveal distinct spatial expression domains and subdo- genes in the category field CA1 pyramidal layer and 400 ‘‘return’’ mains along the longitudinal (dorsal/septal/posterior to ventral/ genes when we interrogated points in dorsal, intermediate, temporal/anterior) axis of field CA1. Second, genetic markers ventral, and ventral tip parts of field CA1 (Fig. 1 A–C). Although divide field CA1 pyramidal neurons into multiple subtypes with these genes display very heterogeneous expression patterns, we characteristic laminar distributions. And third, subcortical brain found that many display consistent regional specificities in field regions receiving axonal projections from molecularly distinct CA1. Using these genes as molecular spatial markers, we have spatial domains of field CA1 display distinct global gene expression divided field CA1 into three distinct domains along the dorso- patterns, suggesting that field CA1 spatial domains may be genet- ventral or longitudinal axis (Fig. 1D and Fig. S1): dorsal (CA1d), ically wired independently to form distinct functional networks intermediate (CA1i), and ventral (CA1v) domains. They are related to cognition and emotion. Insights emerging from this visualized simultaneously in transverse mouse brain levels between 2.7 and 2.9 mm caudal to bregma where the maximal genomic–anatomic approach provide a starting point for a de- dorsoventral extent of field CA1 is displayed (Fig. 1 D–F and Fig. tailed analysis of differential hippocampal structure–function S1B). At these levels CA1d, CA1i, and CA1v occupy approxi- organization. mately the dorsal, intermediate, and ventral thirds of field CA1, respectively. The CA1d–CA1i border here is parallel to the genetics ͉ genomics ͉ hippocampus ͉ learning and memory ͉ ventral edge of the dentate gyrus lateral blade, whereas the neuroanatomy CA1i–CA1v border is approximately level with the dorsal edge of the rhinal fissure (Fig. 1D and Fig. S1B). Rostral and dorsal he basic outlines of hippocampal architecture were established to these transverse levels CA1d extends to the rostral (septal) tip Tby the pioneering work of Ramoń y Cajal (1) and his student of field CA1 (Fig. 1E and Fig. S1A), whereas caudally domain Lorente de No´(2), who named field CA3 (with large pyramidal CA1d is gradually replaced by pyramidal neurons of the dorsal neurons and mossy fibers), field CA2 (with large pyramids but no subiculum (Fig. S1 C and D), which is distinguished from domain mossy fibers), and field CA1 (with small pyramids). The hippocam- CA1d by lack of a stratum oriens. In contrast, domain CA1v pus and other interconnected medial temporal lobe cortical areas extends rostrally and ventrally for only a short distance (Fig. 1E). have been known for some time to be critical for learning and Dorsolaterally domain CA1v is progressively displaced by the memory (3), and more recently the fact has become clear that other ventral subiculum, until it merges with domain CA1i at the functions are subserved as well (4–6). Overall, the evidence sug- caudal end of field CA1 (Fig. 1E and Fig. S1 C and D). gests that the ‘‘dorsal’’ (septal or in humans posterior) hippocampus The extent of domain CA1d is uniquely defined by strong is involved in navigation and related spatial memory, whereas in expression of Wfs1 (Figs. 1 D–F and 2 and Fig. S1 A–D) and a contrast the ‘‘ventral’’ (temporal or in humans anterior) hippocam- number of other gene markers including Nov, Kcnh7, Ndst4, and pus influences stress responses and motivated or emotional behav- 2610017I09Rik, whereas domain CA1v is defined by strong and iors. Experimental neuroanatomical work has established in rats unique expression of many other gene markers, including Dcn, and primates that different transverse levels along the dorsoventral Grp, Htr2c, Col5a1, and Gpc3 (Figs. 1 D–F,2,and3andFig. S1 or longitudinal axis establish extrinsic axonal connections in a highly A–D). (The full names of these genes and image series numbers differentiated and topographic way (7–11). Nevertheless, the exact of their corresponding full sets of gene expression images in the spatial arrangement of the dorsal and ventral hippocampus has not, ABA are listed in Table S1.) The middle third of field CA1 until now, been clearly established. Here, we directly parcel field (CA1i) is characterized most obviously by the absence or much CA1 into multiple, spatially distinct molecular domains and subdomains using robust gene markers selected from a comprehensive Author contributions: H.-W.D., L.W.S., M.S.F., and A.W.T. designed research; H.-W.D. and digital gene expression library (Allen Brain Atlas [ABA], www. L.C. performed research; H.-W.D. analyzed data; and H.-W.D., L.W.S., L.C., M.S.F., and brain-map.org) (12) combined with hippocampal cytoarchitectonic A.W.T. wrote the paper. analysis. The evidence indicates that field CA1 is not a homogenous The authors declare no conflict of interest. cortical area but instead displays clear regional and laminar spec- This article is a PNAS Direct Submission. ificity. These basic structural insights immediately suggest genetic, 1To whom correspondence should be addressed. E-mail: [email protected]. physiological, and behavioral experiments to clarify exactly how This article contains supporting information online at www.pnas.org/cgi/content/full/ these molecular expression domains are differentially involved in 0812608106/DCSupplemental.

11794–11799 ͉ PNAS ͉ July 14, 2009 ͉ vol. 106 ͉ no. 28 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0812608106 Downloaded by guest on September 23, 2021

CA1d A B C

C i1A

CA1v

D1 Fig. 1. (A–C) Overall gene expression ‘‘heat maps’’ spd CA1d for ‘‘seed voxel’’ points in dorsal, intermediate, ventral D domains of field CA1. (D) One transverse level of the CTX sps ARA (15) displaying the maximal dorsoventral scope of field CA1 and its corresponding Nissl-stained histolog- CA1d 212 μm ical images. Distributions of five marker genes, Wfs1 CA1d DGlb (blue), Zbtb20 (green), Dcn (purple), Htr2c (red), and Grp (yellow) are plotted on this level to reveal three

hf spd CA1d molecular domains of ﬁeld CA1 (CA1d, CA1i, and

CA1i CA1v). (D1, D2, D3) High-resolution Nissl images that sps contain CA1d, CA1i, and CA1v, respectively (see text

CA1i for details). (E) A three-dimensional model of Am- CA1i D2 rf mon’s horn (in the context of the whole mouse brain). rf CA3 PERI D3 vd The overall shape of CA1 and its molecular domains vid revealed by CA1 gene markers Wfs1 (blue), Dcn (pur- CA1v-spd sps CA1v viv CA1v ple), and Htr2c (red) occupy the outside surface of the ENTl C-shaped cylinder of Ammon’s horn, with the dorsal vvm end (CA1d) extending much more rostral than the spm ventral end (CA1v). These two domains merge at the

1310 μm caudalmost end of CA1. (F) Three-dimensional expression patterns of 4 representative genes, Wfs1 (blue), Wfs1 Zbtb20 Dcn Htr2c Grp Grp (yellow), Dcn (purple), and Htr2c (red), in one transverse plane of field CA1. These genes show dis- E F tinct regional specificities and clearly define the spatial extent of CA1d (Wfs1), CA1v (Grp, Htr2c, and Dcn), and CA1i (revealed here by lack of these gene markers). CA1d CA1d CA1d Three-dimensional images of CA1 were generated in DG CA3 BrainExplore (http://www.brain-map.org), one three-

PERI CA1i CA1i dimensional model of the ARA (15, 40). See Fig. S1 for CA3

ENTl CA1v more detailed mapping of these genes in 4 represen- rf tative levels of the ARA. CTX, cerebral cortex; DGlb, dentate gyrus, lateral blade; ENTl, lateral entorhinal CA1v CA1v area; PERI, perirhinal cortical area. (Scale bars: D, 1,310 ␮m; D1, D2, D3, 212 ␮m to match the same magniﬁ- cations of the same images on ABA web site.)

weaker expression of the above markers (Fig. 1 D–F and Fig. S1 and then CA1vd (Fig. 2). Many other genes, including Zdhhc7 A–D). However, many genes have expression patterns that help and Pole4, are expressed progressively more strongly in CA1vid, to distinguish domain CA1i more directly. For example, Zbtb20 CA1vd, and even CA1i—but not in CA1viv and CA1vv. and Gpt2 are expressed in both domains CA1d and CA1i but not The size and shape of field CA1 pyramidal neurons and in CA1v, with Zbtb20 expressed more strongly in CA1i than differences with field CA3 pyramids are firmly established (1, 2), CA1d (Fig. S1 B–D) and the reverse occurring for Gpt2.Of and boundaries between these two hippocampal cortical areas fundamental importance, although the expression of many genes are clearly visualized with molecular markers (13, 14). However, in field CA1 may not be restricted to CA1d, CA1i, or CA1v, the these pyramidal neuron populations have long been considered expression does consistently respect one of the boundaries relatively uniform within an area. The expression pattern het- between them. Because genes expressed in domain CA1i are erogeneity described above next led us to reexamine carefully more commonly also expressed in CA1d but not CA1v, CA1i is field CA1 cytoarchitecture based on high-resolution Nissl- possibly simply a subdomain of CA1d. stained images presented in the Allen Reference Atlas (ARA) The concept of subdomains is an important one, especially in (15). We found that field CA1 cytoarchitecture is clearly heter- domain CA1v where four are obvious (Figs. 1D and 2 and Fig. ogeneous both regionally and with respect to pyramidal neuron S1B): dorsolateral (CA1vd), intermediodorsal (CA1vid), inter- layers (Figs. 1D and 3 and Fig. S1 A–D). Following Swanson in medioventral (CA1viv), and ventromedial tip (CA1vv). The tiny rats (16), we first divided the pyramidal layer into superficial and CA1vv is very distinctive because of weak Grp expression (Figs. deep sublayers. The cytoarchitecture of domain CA1d is char- 1D and 2 and Fig. S1 B–D) and much stronger, localized Htr2c acterized by a remarkably darkly stained, densely packed super- and Col5a1 expression (Figs. 1 D–F and 2 and Fig. S1 B–D). In ficial pyramidal layer (CA1d-sps) and a thin deep layer with addition, a number of other genes such as Gpr101 (Fig. 2) and many fewer pyramids (CA1d-spd; Fig. 1D1 and Fig. S1 A–D), as Dlk1are expressed almost exclusively in CA1vv. Genes expressed originally described by Lorente de No´(2). The CA1d-spd is in the other three subdomains display more or less overlapping considerably thinner rostrally, with mostly only one row of or gradient-type patterns. For example, Tc1568100 and pyramids (Fig. S1 A and B), but progressively thickens caudally Loc432748 are weakly expressed in subdomain CA1vv, are into 3 or 4 rows of loosely arranged pyramids (Fig. S1 C and D).

heavily expressed in CA1viv, and become progressively more Nevertheless, expression patterns for most genes (such as Nov NEUROSCIENCE restricted to a single sublayer of pyramidal neurons in CA1vid and Ndst4) are very consistent from rostral to caudal in trans-

Dong et al. PNAS ͉ July 14, 2009 ͉ vol. 106 ͉ no. 28 ͉ 11795 Downloaded by guest on September 23, 2021 Wfs1 Zbtb20 Grp Col5a1 Tc1568100 Gpr101

CA1d CA1d CA1d CA1d CA2 A CA3 606 μm

CA1d CA1d CA1d CA1d CA1d CA1d

CA1i CA1i C CA1i CA1i A1 CA1i

B i rf rf a rf rf rf a rf b b c v CA1v c CA1v CA1 d CA1v CA1v d d CA1v 842 μm

Fig. 2. Digital images of six representative genes expressed specifically in three molecular domains of field CA1: CA1d (Wfs1), CA1i (Zbtb20), CA1v (Grp), and the CA1v subdomains, CA1vd, CA1vid, and CA1viv (Col5a1 and Tc1568100) and CA1vv (Gpr101 and Col5a1) at two different rostrocaudal levels (A, B) of field CA1. CA1v subdomains are labeled: a (CA1vd), b (CA1vid), c (CA1viv), and d (CA1vv). Images in (C) are gene expression heat maps of corresponding images in (B) (for definition of heat map, see ABA web site, http://www.brain-map.org) that display relative intensities of gene expression signals. All images were downloaded from the ABA web site. (Gene names and the image series numbers of their original digital images are listed in Table S1.) Overall expression patterns of Wfs1, Zbtb20, and Grp in the hippocampus are schematically mapped in more detail in Fig. S1. All images in the same panels have the same magnifications. Scale bars are displayed in the lower right corner of Gpr101 images.

verse sections of field CA1d, although a few genes (including Lct) and CA1v-spd but weakly in CA1v-spm (Figs. 2 and 3 and Fig. show much stronger expression in rostral levels of CA1d than in S1B); Dcn is expressed strongly in CA1v-sps but weakly in more caudal levels, and a few others (including Kit) present the CA1v-spd and CA1v-spm (Fig. 3 and Fig. S1 B–D), whereas reverse gradient. Wfs1 and Nr4a1 are expressed in both CA1d- Htr2c and Gpc3 are strongly expressed in CA1vv, but only in the sps and CA1d-spd sublayers, Nov and Nr3c2 tend to express more deep layer of CA1viv, CA1vid, and CA1vd (Figs. 1 D–F and 3 and specifically in CA1d-sps, and Ndst4 and Astn2 are expressed Fig. S1 B–D). Second, Tc1568100 and Loc432748 are expressed preferentially in CA1d-spd (Fig. 3). Some neurons in the stratum strongly in the CA1viv-sps, but only the CA1vid-spm and CA1vd- oriens also express Astn2. Compared with domain CA1d, the spm (Fig. 2), whereas Prss23 is expressed specifically in the deep superficial pyramidal layer of CA1i (CA1i-sps) is much less layer of CA1viv, CA1vid, and CA1vd and in CA1i (Fig. 3). And dense and more lightly stained, whereas the deep layer (CA1i- third, Prss12 is expressed specifically only in the striatum oriens spd) is relatively thicker with 3 or 4 rows of pyramids (Fig. 1D2 of domain CA1v (Fig. 3). Interestingly, some genes display and Fig. S1B). laminar differences in different domains. For example, Calb1 is CA1v displays even more complex cytoarchitecture. In addi- expressed selectively in the superficial layer of CA1d and CA1viv tion to superficial (CA1v-sps) and deep (CA1v-spd) pyramidal but is expressed more strongly in the deep layer of CA1i, CA1vd, layers, a middle sublayer (CA1v-spm) containing smaller pyra- and CA1vid (Fig. 3). mids appears to be sandwiched between the other layers in Next, we examined global spatial gene expression pattern subdomains CA1vd and CA1vid (Fig. 1D3 and Fig. S1B). In the correlations between these field CA1 molecular domains and progressively more ventral CA1viv, the middle pyramidal sub- other brain regions (Fig. 4; all correlation coefficients provided layer disappears, and pyramidal neuron morphology becomes in Table S2). We found that domain CA1d exhibits a high level less distinguishable between the superficial and the deep layers of intrastructural correlation between different transverse (Fig. 1 D and D3 and Fig. S1B). On reaching subdomain planes of CA1d, but lower correlations with domain CA1v, CA1vv—the ventral tip of field CA1—pyramids align in an confirming their molecular specificities across the dorsoventral apparently uniform, evenly stained, thick layer of approximately rather than the rostrocaudal axis. As expected from earlier 7–9 neuronal rows (Fig. 1D and Fig. S1B). This cytoarchitectonic results (above), domain CA1i expression patterns are correlated sublamination pattern in CA1v is substantiated by gene expres- more with those of CA1d than with CA1v. Most interestingly, sion patterns. First, Grp is expressed strongly in both CA1v-sps probabilistic coexpression correlations of domains CA1d and

Fig. 3. Gene markers showing regional and laminar Wfs1 Nov Nr3c2 Ndst4 Calb1 CA1d-spd CA1d-sps specificities of field CA1 pyramidal neurons. For high- CA1d-spd resolution Nissl-stained cytoarchitecture and delineation CA1d-sps CA1d-sps CA1i-spd CA1d-sps 105 μm of pyramidal neuron sublayers, see Fig. 1 and Fig. S1. CA1v subdomains are labeled: a (CA1vd), b (CA1vid), c Prss12 b Grp Dcn a Gpc3 rf (CA1viv), and d (CA1vv). All digital images were down- a c loaded from the ABA gene expression library. (Gene b b pd spm 1v-sps CA1v 1v CA c names and the image series numbers of their original d CA so sps c CA1v-s digital images are listed in Table S1.) Images in the same d CA1v-spd 421 μm d 1011 μm rows have the same magnifications.

11796 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0812608106 Dong et al. Downloaded by guest on September 23, 2021 A B C CA1d

gerB

CA1i 1i m

- = a CA1v 2

CA1d a b cc CA1i B

MB CA1v .2- = a

CA1d a’ CA1d b’ c’

CTX

CEA amgerB 3V TH MEA RE LA 656.1- = LA HY

c” r a” b” e ACA

LS = amg CP LS

.0 BST 0

ac ac ac 92 MPO MPO Fig. 4. Global gene coexpression ‘‘energy’’ maps result from setting seed voxel points in domain CA1d (A), CA1i (B), and CA1v (C)at three representative transverse levels of the [0.5, 1] mouse brain at approximately bregma Ϫ2.739 (a, b, c), Ϫ1.656 (aЈ,bЈ,cЈ), and 0.029 (aЉ,bЉ,cЉ). D E The heat map energy deﬁnition is shown at the LSc LSc bottom. The coexpression correlation coefﬁ- cients of these CA1 molecular domains with LSr other brain structures are listed in Table S2. For a detailed description of AGEA spatial gene expression correlation map, see Ng et al. (24). (D and E) Correlated gene expression patterns LSv LSv of domain CA1d marker gene Matn2 and CA1v Matn2 421µm Gpc3 421µm marker gene Gpc3 in the lateral septal nucleus. ac ac (Scale bars: D and E, 421 ␮m.)

CA1v with other brain regions are very distinctive. For example, hypothalamic structures mediating the expression of different although both domains exhibit very high correlation values with classes of innate motivated behavior. To explore potential the cortical mantle as a whole, domain CA1v shows higher genetic mechanisms underlying this topographic wiring pattern, correlation values than CA1d with cortical amygdalar areas and we compared coexpression patterns of CA1 domain-specific the basolateral amygdalar complex, both of which share direct genes with those of the lateral septal nucleus. We found that a bidirectional axonal connections with CA1v (10, 11, 17, 18). In number of CA1 domain-specific genes are also expressed in the terms of subcortical regions, CA1v displays strikingly higher lateral septal nucleus and that their coexpression correlations coexpression correlations than CA1d with neuron populations respect the topologically ordered dorsal-to-dorsal and ventral- associated with autonomic, neuroendocrine, and affective be- to-ventral organization of their axonal projection connectivity. haviors, including the central and medial amygdalar nuclei, For example, genes that show strong expression in domain CA1d, intermediate and ventral parts of the lateral septal nucleus, bed such as Wfs1 and Matn2, are also strongly expressed in the caudal nuclei of the stria terminalis, and hypothalamus—all of which end of the caudal and rostral lateral septal nucleus (LSc, LSr), share massive direct and indirect neuronal connectivity with which are topologically dorsal (Fig. 4D). In contrast, Dlk1 and CA1v (9, 10, 17–19). These obvious correlations strongly suggest Gpr101—the CA1vv (and ventral subicular) marker genes—also that CA1d and CA1v are genetically wired independently with show strong expression patterns in the ventral lateral septal different functional specificities. nucleus (LSv). Genes that show significant expression in more Finally, Risold and Swanson (9, 16) proposed the existence of dorsal subdomains of CA1v are most likely also to coexpress in a series of structure–function domains along the longitudinal ventral levels of the LSr. Thus, Loc432748, a specific marker axis of field CA1 and adjacent subiculum that send topograph- gene for CA1viv, presents specific signal only in a specific zone

ically organized projections to the three major parts of the lateral of the LSr. Gpc3 and Htr2c, which are strongly expressed in NEUROSCIENCE septal nucleus that in turn share bidirectional connections with CA1vv and the rest of domain CA1v, are strongly expressed in

Dong et al. PNAS ͉ July 14, 2009 ͉ vol. 106 ͉ no. 28 ͉ 11797 Downloaded by guest on September 23, 2021 the LSv and LSr but not in LSc (Fig. 4E). Man1a and Zdhhc7, The model of three major CA1 molecular domains presented which are expressed throughout field CA1 (except in CA1vv), here shows interesting correlations with the 9 even more striking express strongly in the LSc and LSr (but not LSv). By comparison molecular regions recently reported along the longitudinal axis of the topology and topography of these gene expression cor- of mouse field CA3 (25). The observations of Thompson et al., relations with the hippocampal functional domains defined by together with our own in fields CA1 and CA3, suggest that field Risold and Swanson in rat (9, 16), CA1vv defined here appears CA3—and thus Ammon’s horn as a whole—is also divided into to correspond well to their Domain 5—the ventral tip of field three major molecular domains: (i) a dorsal domain (CA3d) CA1 and subiculum, which sends projections specifically to the including their regions 1–3 that is accompanied by domain LSv (9)—one of the primary sources of cerebral inputs to the CA1d; (ii) an intermediate domain (CA3i) including their re- hypothalamic neuroendocrine zone and region controlling feed- gions 4 and 5 that is accompanied by domain CA1i; and (iii)a ing behavior. The progressively more dorsally located CA1viv, ventral domain (CA3v) including their regions 6–7 that is CA1vid, and CA1vd may correspond to their Domains 2–4. accompanied by domain CA1v. The domains CA3d and CA3i These CA1v subdomains send topographic projections to (regions 1–5) form approximately the dorsal half of field CA3, different zones of the LSr, which in turn project to the whereas the domain CA3v (regions 6 and 7) forms approximately hypothalamic medial column that controls two basic classes of the ventral half. social behavior—reproduction and defense (9). Finally, CA1d Ramo´n y Cajal first noted structural differences along the and CA1i together may correspond to their Domain 1, which hippocampal longitudinal axis, related to what he called the presumably innervates LSc and LSr levels that in turn project to the superior and inferior perforant paths (1) and what would later theta-rhythm-controlling supramammillary nucleus (8). The dens- be referred to loosely as the dorsal and ventral hippocampus, est outputs of dorsal CA1 have been shown to be to the dorsal respectively (9, 27). Lorente de No´(2) vaguely divided the subiculum (11)—the major source of the postcommissural fornix ‘‘Ammonic system’’ into three longitudinal segments based on pathway innervating the medial and lateral mammillary nuclei and now discredited connectional data and also divided field CA3 anterior thalamic complex. As a matter of fact, this dorsal CA1 to and field CA1 into three parallel, longitudinally running zones subiculum projection is the major hippocampal component that (a, b, and c for both). Lorente de No´’s (2) subfields CA3a, CA3b, processes spatial memory (20–22). and CA3c appear to correspond well with three molecular regions identified by Thompson et al. (25): region 3 (Mas1 Discussion expression) with CA3a, region 2 (Fmo1 expression) with CA3b, Many gene markers have been used as reproducible landmarks and region 1 (Ttn1 expression) with CA3c. In field CA1, at least for defining nervous system divisions at various stages of devel- some genes (such as Lct and Kit) were found here to show opment (23) and in the adult (15). This is an extension of the differential expression patterns across the topological medial to lateral (transverse) axis, but correspondences with Lorente de revolutionary chemical neuroanatomy approach that began in No’s subfields CA1a, CA1b, and CA1c are not yet clear. the 1950s with cholinesterase histochemistry, followed by histo- Extensive work since the early 1970s established that intra- fluorescence and then immunohistochemistry (see ref. 15). The hippocampal circuitry, typified by the classical trisynaptic circuit resulting chemoarchitectonic data greatly refined classical cyto- from entorhinal area to dentate gyrus to field CA3 to field CA1, architectonics based on Nissl staining. The present study, com- runs transverse to the longitudinal axis and that specific extrinsic bined with two recent reports (24, 25), supply proof-of-principle inputs and outputs are associated with specific regions along the examples of how the genomic–neuroanatomic approach can longitudinal axis of the hippocampal formation (8–11, 17–19, further refine architectonic maps and identify neuronal pheno- 28–33). The dorsal half of field CA1 has a high concentration of types amenable to selective experimental genetic manipulation. place neurons (e.g., ref. 34) and sends most of its extrinsic Although raw data about these expression patterns (digitized cortical projections, either directly or indirectly via dorsal sub- images) are freely available on the ABA web site and powerful iculum, to the cortical retrosplenial area (11, 35), which is online informatics tools enable users conveniently to identify involved in cognitive processing of visual sensory information at regionally specific gene expression patterns, synthesis and inter- least in part for spatial and contextual memory. The dorsal pretation of this information relative to existing structural, subiculum receives its strongest input from dorsal CA1 and sends functional, and behavioral knowledge is still required. Specifi- massive projections to the mammillary and anterior thalamic cally, we provide here a testable model to guide and stimulate nuclei, two regions containing abundant navigation neurons further analysis of hippocampal structure–function in health and (36). These two diencephalic regions in turn project back to the disease. dorsal hippocampus and retrosplenial area (35). This and other Our genetic–anatomic analysis and the current axonal con- evidence (see refs. 4–6, 20, and 21) suggest that the dorsal half nectivity literature (7–11, 17–19, 26) suggest that domains CA1d of field CA1 (domains CA1d and CA1i) is critically involved in and CA1i lie in approximately the dorsal half of the hippocam- cognitive spatial memory processing, at least in rodents. pus, and they display strong global gene expression correlations The most distinguishing feature of connectivity associated primarily with other parts of cerebral cortex and subcortical with the ventral half of field CA1 (domain CA1v and its 4 regions innervated by dorsal hippocampus and related to theta- subdomains) is dense bidirectional connections with amygdalar rhythm modulation and navigation. In contrast, domain CA1v regions implicated in emotional behaviors (10, 11, 17, 18). lies in approximately the ventral half of the hippocampus, and Ventral but not dorsal field CA1 also projects directly (19) to the this domain shares strong gene coexpression patterns and pro- hypothalamic periventricular region and medial zone, which jections with cerebral cortex and subcortical regions mediating integrate neuroendocrine, autonomic, and somatic motor re- neuroendocrine, autonomic, and goal-oriented or emotional sponses associated with three basic classes of motivated behav- behavioral responses. The natural boundary between CA1i and iors common to all animals: ingestive (feeding and drinking), CA1v in mice coincides with the rhinal fissure’s dorsal border, a reproductive (sexual and parental), and defensive (fight or boundary shown by certain CA1 domain-specific genes and by flight) (26). These direct descending projections from ventral not other cortical marker genes, including Col5a1, which is strongly dorsal CA1 are reinforced by multiple indirect pathways involv- expressed in isocortical layers 6a and 6b (Fig. 2). The evidence ing relays in the ventral subiculum, amygdalar region, lateral suggests that the dorsal and ventral halves of field CA1 are septal nucleus, and bed nuclei of the stria terminalis (9–11, genetically wired independently to support cognitive and emo- 17–19, 26, 33, 37), all of which display gene coexpression patterns tional responses, respectively (4–6). correlated with those of CA1v as shown here.

11798 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0812608106 Dong et al. Downloaded by guest on September 23, 2021 The observation that the dorsal half and ventral half of field Ramoń y Cajal (1) and Lorente de No´(2) observed deep and CA1 display distinct global gene expression correlations with superficial pyramids and noticed that they establish unique brain structures that they project to either directly or indirectly relationships with basket cells. However, the addition of multiple is striking. Although the mechanistic significance of this rela- genetic markers for differentiation of field CA1 pyramids opens tionship between CA1 molecular domains and their extrinsic many new possibilities for establishing analytical distinctions. circuitry is not yet clear, from the above the dorsal and ventral halves are obviously genetically wired independently for differ- Materials and Methods ent functional specializations. The former is selectively involved This study was based on a systematic, high-resolution analysis of Ͼ4,000 genes in cognitive aspects of the learning and memory associated with expressed in the hippocampal formation through a publicly accessible online navigation, exploration, and locomotion, whereas in contrast the gene expression digital library—the ABA. We first manually screened expression latter is part of the temporal lobe associated most directly with patterns of Ͼ2,000 genes under the Anatomic Search tool category ‘‘hippocam- motivational and emotional aspects of the classic Klu¨ver–Bucy pal region,’’ based on expression level, expression density, and gene clustering, Syndrome. In fact, at least two CA1v marker genes, Grp and and carefully analyzed expression patterns of 48 genes in the category ‘‘field CA1 Htr2c, have been implicated in psychiatric disorders (38, 39). pyramidal layer’’ under ‘‘fine structure search.’’ We then used the Gene Finder This and similar work (25) are part of a systematic examination tool of the Anatomic Gene Expression Atlas (AGEA) application (Fig. 1 A–C)to of relationships between gene expression patterns and neuronal filter candidate genes showing regional specificities. Detailed methodology of networks to clarify genetic contributions to brain structure and the AGEA has been described in a recent report (24). In brief, because all ABA function. However, the detection sensitivity and specificity for image-based, in situ hybridization data are spatially registered to the common many genes, especially those of low abundance, probably were anatomic framework of the ARA (15) with a standard coordinate system and compromised in the large-scale, high-throughput process of hierarchical ontology, the AGEA in principle enables users to view a comprehen- ABA data production (12). This would make the list of domain- sive list of genes expressed in any given three-dimensional voxel (‘‘seed voxel’’) of the mouse brain atlas using the Gene Finder application (24). With this strategy, specific markers assembled here rather incomplete, and many Ϸ genes critical for determining adult brain structure and connec- we carefully analyzed the expression of 400 ‘‘return’’ genes when we set seed voxel points in dorsal, intermediate, and ventral parts of field CA1. AGEA also tions likely are expressed during development not in the adult allows users to view global expression correlations associated with any seed voxel. (23). Even for the domain-specific markers listed here (Table With this tool, we have also analyzed gene expression correlations of different S1), information in Gene Ontology suggests many diverse (and domains of the CA1. unknown) functions whose possible role in neuronal development and adult structure–function remain to be investigated. ACKNOWLEDGMENTS. This work was supported in part by National Institutes Relative to isocortex, very little is known about laminar and of Health Grants MH083180 (to H.-W.D.), RR013642 (to A.W.T.), NS16686 (to regional differences in field CA1 pyramidal cell subtypes. Both L.W.S.), and MH62122 (to M.S.F.).

1. Ramoń y Cajal S (1901–1902) Estudios sobre la corteza cerebral humana. Trab Inst Cajal 22. Gigg J (2006) Constraints on hippocampal processing imposed by the connectivity Invest Biol 1:1–227. between CA1, subiculum and subicular targets. Behav Brain Res 174:265–271. 2. Lorente de No´RJ (1934) Studies on the structure of the cerebral cortex. II. Continuation 23. Puelles L, Martínez S, Martínez-de-la-Torre M, Rubenstein JLR (2004) Gene maps and of the study of the ammonic system. J Psychol Neurol 46:113–177. related histogenetic domains in the forebrain and midbrain. The Rat Nervous System, 3. Squire LR (1992) Memory and the hippocampus: A synthesis from findings with rats, ed Paxinos G (Elsevier, Amsterdam), pp 3–25. monkeys, and humans. Psychol Rev 99:195–231. 24. Ng L, et al. (2009) An anatomic gene expression atlas of the adult mouse brain. Nat 4. Moser MB, Moser EI (1998) Functional differentiation in the hippocampus. Hippocam- Neurosci 12:356–362. pus 8:608–619. 25. Thompson CL, et al. (2008) Genomic anatomy of the hippocampus. Neuron 60:1010– 5. Anagnostaras SG, Gale, GD, Fanselow MS (2002) The hippocampus and Pavlovian fear 1021. conditioning: Reply to Bast et al. Hippocampus 12:561–565. 26. Swanson LW (2000) Cerebral hemisphere regulation of motivated behavior. Brain Res 6. Kjelstrup KG, et al. (2002) Reduced fear expression after lesions of the ventral hip- 886:113–164. pocampus. Proc Natl Acad Sci USA 99:10825–10830. 27. Gloor P (1997) The Temporal Lobe and Limbic System (Oxford Univ Press, New York), 7. Swanson LW, Cowan WM (1975) Hippocampo-hypothalamic connections: Origin in pp 325–589. subicular cortex, not ammon’s horn. Science 189:303–304. 28. Witter MP (2000) Anatomical organization of the parahippocampal-hippocampal 8. Swanson LW, Cowan WM (1977) An autoradiographic study of the organization of the efferent connections of the hippocampal formation in the rat. J Comp Neurol 172:49–84. network. Ann N Y Acad Sci 911:1–24. 9. Risold PY, Swanson LW (1996) Structural evidence for functional domains in the rat 29. Witter MP, Van Hoesen GW, Amaral DG (1989) Topographical organization of the hippocampus. Science 272:1484–1486. entorhinal projection to the dentate gyrus of the monkey. J Neurosci 9:216–218. 10. Witter MP, Amaral DG (2004) Hippocampal formation. The Rat Nervous System,ed 30. Dolorfo CL, Amaral DG (1998) Entorhinal cortex of the rat: Organization of intrinsic Paxinos G (Elsevier, Amsterdam), pp 637–704. connections. J Comp Neurol 398:49–82. 11. Cenquizca LA, Swanson LW (2007) Spatial organization of direct hippocampal field 31. Van Groen T, Miettinen P, Kadish I (2003) The entorhinal cortex of the mouse: Organiza- CA1 axonal projections to the rest of the cerebral cortex. Brain Res Rev 56:1–26. tion of the projection to the hippocampal formation. Hippocampus 13:133–149. 12. Lein ES, et al. (2007) Genome-wide atlas of gene expression in the adult mouse brain. 32. Swanson LW, Wyss JM, Cowan WM (1978) An autoradiographic study of the organization Nature 445:168–176. of intrahippocampal association pathways in the rat. J Comp Neurol 181:681–715. 13. Lein ES, Callaway EM, Albright TD, Gage FH (2005) Redefining the boundaries of the 33. Swanson LW, Ko¨hler C, Bjo¨rklund A (1987) The limbic region. I: The septohippocam- hippocampal CA2 subfield in the mouse using gene expression and 3-dimensional pal system. Integrated Systems of the CNS, Part I. Handbook of Chemical Neuro- reconstruction. J Comp Neurol 485:1–18. anatomy, eds Ho¨kfelt T, Bjo¨rklund A, Swanson LW (Elsevier, Amsterdam), Vol 5, pp 14. Lein ES, Zhao X, Gage FH (2004) Defining a molecular atlas of the hippocampus using 125–277. DNA microarrays and high-throughput in situ hybridization. J Neurosci 24:3879–3889. 34. Jung MW, Wiener SI, McNaughton BL (1994) Comparison of spatial firing characteris- 15. Dong H-W (2007) The Allen Reference Atlas: A Digital Color Brain Atlas of the C57BL/6J tics of units in dorsal and ventral hippocampus of the rat. J Neurosci 14:7347–7356. Male Mouse (Wiley, Hoboken, NJ). 35. Risold PY, Thompson RH, Swanson LW (1997) The structural organization of connec- 16. Swanson LW (2004) Brain Maps: Structure of the Rat Brain (Elsevier, Amsterdam), 3rd Ed. tions between hypothalamus and cerebral cortex. Brain Res Brain Res Rev 24:197–254. 17. Pitkanen A, Pikkarainen M, Nurminen N, Ylinen A (2000) Reciprocal connections 36. Taube JS (2007) The head direction signal: Origins and sensory-motor integration. between the amygdala and the hippocampal formation, perirhinal cortex, and post- Annu Rev Neurosci 30:181–207. rhinal cortex in rat. Ann N Y Acad Sci 911:369–391. 37. Dong H-W, Petrovich GD, Swanson LW (2001) Topography of projections from amyg- 18. Petrovich GD, Canteras NS, Swanson LW (2001) Combinatorial amygdalar inputs to dala to bed nuclei of the stria terminalis. Brain Res Rev 38:192–246. hippocampal domains and hypothalamic behavior systems. Brain Res Rev 38:247–289. 19. Cenquizca LA, Swanson LW (2006) Analysis of direct hippocampal cortical field CA1 38. Roesler R, Henriques JA, Schwartsmann G (2006) Gastrin-releasing peptide receptor as axonal projections to diencephalon in the rat. J Comp Neurol 497:101–114. a molecular target for psychiatric and neurological disorders. CNS Neurol Disord Drug 20. Moser E, Moser MB, Andersen P (1993) Spatial learning impairment parallels the Targets 5:197–204. magnitude of dorsal hippocampal lesions, but is hardly present following ventral 39. Drago A, Serretti A (2008) Focus on HTR2C: A possible suggestion for genetic studies of lesions. J Neurosci 13:3916–3925. complex disorders. Am J Med Genet B Neuropsychiatr Genet, 10.1002/ajmg.b.30864, 21. Hunsaker MR, Fieldsted PM, Rosenberg JS, Kesner RP (2008) Dissociating the roles of in press. dorsal and ventral CA1 for the temporal processing of spatial locations, visual objects, 40. Lau, et al. (2008) Exploration and visualization of gene expression with neuroanatomy

and odors. Behav Neurosci 122:643–650. in the adult mouse brain. BMC Bioinformatics 18:9:153. NEUROSCIENCE

Dong et al. PNAS ͉ July 14, 2009 ͉ vol. 106 ͉ no. 28 ͉ 11799 Downloaded by guest on September 23, 2021