Neuron
Article
Genomic Anatomy of the Hippocampus
Carol L. Thompson,1 Sayan D. Pathak,1 Andreas Jeromin,1 Lydia L. Ng,1 Cameron R. MacPherson,2
Marty T. Mortrud,1 Allison Cusick,1 Zackery L. Riley,1 Susan M. Sunkin,1 Amy Bernard,1 Ralph B. Puchalski,1
Fred H. Gage,3 Allan R. Jones,1 Vladimir B. Bajic,2 Michael J. Hawrylycz,1 and Ed S. Lein1,*
1Allen Institute for Brain Science, Seattle, WA 98103, USA
2South African National Bioinformatics Institute, University of the Western Cape, Bellville 7535, Cape Town, South Africa
3Salk Institute for Biological Studies, La Jolla, CA 92037, USA
*Correspondence: edl@alleninstitute.org
DOI 10.1016/j.neuron.2008.12.008
SUMMARY
Availability of genome-scale in situ hybridization
data allows systematic analysis of genetic neuroana-
tomical architecture. Within the hippocampus, elec-
trophysiology and lesion and imaging studies
demonstrate functional heterogeneity along the sep-
totemporal axis, although precise underlying
circuitry and molecular substrates remain uncharac-
terized. Application of unbiased statistical compo-
nent analyses to genome-scale hippocampal gene
expression data revealed robust septotemporal
molecular heterogeneity, leading to the identification
of a large cohort of genes with robust regionalized
hippocampal expression. Manual mapping of hetero-
geneous CA3 pyramidal neuron expression patterns
demonstrates an unexpectedly complex molecular
parcellation into a relatively coherent set of nine
expression domains in the septal/temporal and prox-
imal/distal axes with reciprocal, nonoverlapping
boundaries. Unique combinatorial profiles of adhe-
sion molecules within these domains suggest corre-
sponding differential connectivity, which is demon-
strated for CA3 projections to the lateral septum
using retrograde labeling. This complex, discrete
molecular architecture provides a novel paradigm
for predicting functional differentiation across the
full septotemporal extent of the hippocampus.
INTRODUCTION
The role of the hippocampus in learning and memory is well es-
tablished, with increasing evidence for a role in anxiety-related
behaviors as well (Bannerman et al., 2004). These functions are
differentially distributed along the septotemporal axis of the
hippocampus. Septal (dorsal) hippocampal lesions result in
selective impairment in spatial memory tasks but not anxiety-
related measures, whereas temporal (ventral) lesions result in
the converse impairments (Bannerman et al., 2002; Moser and
Moser, 1998b). Functional differentiation along the long axis of
the hippocampus is evolutionarily conserved and has been
described in rats (Bannerman et al., 2002; Jung et al., 1994;
Moser and Moser, 1998a; Vann et al., 2000), monkeys (Colombo
1010 Neuron 60, 1010–1021, December 26, 2008 ª2008 Elsevier Inc.
et al., 1998), and humans (Small et al., 2001) using various
approaches, including selective lesions and behavioral analysis,
c-fos activation, electrophysiology, and functional MRI.
Copious anatomical evidence exists for differential afferent,
efferent, and intrahippocampal connectivity along the septotem-
poral axis of the hippocampus that could underlie functional
differences. The entorhinal cortex, providing the major input to
the dentate gyrus (DG) via the perforant path, can be divided
into bands projecting to different septotemporal levels of the
DG. Lateral and intermediate bands through the medial and
lateral entorhinal cortex, which convey most of the information
from sensory cortical areas via the perirhinal and postrhinal
cortices, preferentially target the septal half and third quarter of
the DG, respectively. A medial band through the entorhinal
cortex, in contrast, targets the most temporal quarter of the
DG (Burwell and Amaral, 1998; Dolorfo and Amaral, 1998; Ruth
et al., 1988; van Groen et al., 2003). Amygdalar hippocampal
afferents selectively target temporal portions of CA3, CA1, and
subiculum (Petrovich et al., 2001). Differential afferent projec-
tions are also reflected by neurochemical localization, with
greater cholinergic innervation of septal hippocampus (Amaral
and Kurz, 1985; Milner et al., 1983) and greater temporal inner-
vation from fibers containing dopamine (Verney et al., 1985),
noradrenaline, and serotonin (Gage and Thompson, 1980; Haring
and Davis, 1985; Kohler et al., 1981) and various neuropeptides
(Caffe et al., 1987; Gall et al., 1981; Kohler et al., 1987; Mantyh
et al., 1984; Pazos et al., 1985; van Leeuwen et al., 1985).
In contrast to the simple lamellar model of intrahippocampal
connectivity (Anderson et al., 1971), projections from the DG to
CA3 to CA1 exhibit complex proximal/distal and septotemporal
topography (Amaral and Witter, 1989; Ishizuka et al., 1990).
Furthermore, recurrent associational projections in the hilus
and CA3 project extensively along the septotemporal axis (Ishi-
zuka et al., 1990). Hippocampal efferents project topographically
onto the lateral septum, with septotemporal levels of CA3 and
CA1 targeting distinct portions of the septum that in turn recipro-
cally innervate functionally discrete hypothalamic regions (Risold
and Swanson, 1997). In addition, there is reciprocal connectivity
between the temporal hippocampus and the amygdala (Petro-
vich et al., 2001), and temporal CA1 and subiculum selectively
project to prefrontal cortex (Verwer et al., 1997).
Septal and temporal hippocampus also differ in cellular and
circuit properties. Septal hippocampus has more place cells
than temporal hippocampus, and temporal place cells have
lower spatial selectivity (Jung et al., 1994). Temporal CA1 shows
greatly reduced long-term potentiation (LTP) induction relative to
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Genomic Anatomy of the Hippocampus
septal CA1 (Papatheodoropoulos and Kostopoulos, 2000),
whereas temporal CA1 selectively exhibits long-term depression
(LTD) with low-frequency burst stimulation (LFBS) (Izaki et al.,
2000). In models of epilepsy, temporal hippocampus exhibits
more rapid kindling (Racine et al., 1977), and slices through tem-
poral hippocampus exhibit greater epileptiform bursting in high
potassium (Bragdon et al., 1986). In contrast, septal CA1 shows
selective vulnerability to ischemic insults (Ashton et al., 1989).
Differential functional connectivity and intrinsic cellular
function are likely to be reflected by specific patterns of gene
expression. There is extensive evidence for gene expression
specifically delineating classically defined hippocampal
subfields CA1, CA2, CA3, and the DG (Datson et al., 2004;
Lein et al., 2004, Lein et al., 2005). Recently, several genes
have been identified with differential septotemporal expression
in CA1 (Leonardo et al., 2006), CA3, and DG (Lein et al., 2006).
The recent availability of genome-scale in situ hybridization
(ISH) data (Lein et al., 2006) allows a ‘‘genomic anatomy’’
approach to understanding the functional architecture of the
hippocampus based on correlated expression patterns across
many genes. The present study uses a combination of statistical
component analysis techniques and more traditional manual
anatomical boundary mapping to assess the molecular and
cellular architecture of the hippocampus, identify boundaries
and domains within hippocampal subfields, and generate
a high-resolution, molecularly defined anatomical map that can
be related to differential functional properties along the septo-
temporal axis of the hippocampus.
RESULTS
Computational Analysis of Hippocampal
Gene Expression
The Allen Brain Atlas (ABA) provides a powerful data set for anal-
ysis of statistical relationships between different spatially sepa-
rated neuronal populations. These data consist of cellular reso-
lution ISH data for >20,000 unique transcripts sampled across
the entire adult (P56) C57BL/6J mouse brain, algorithmically
quantified and mapped to a common anatomical coordinate
framework (Lein et al., 2006). This framework allows the applica-
tion of clustering and component-based methods to understand
molecular anatomical subdivisions of brain architecture. In the
present context, dimensions of expression level and 3D spatial
coordinate can be analyzed to identify principal spatial domains
displaying similar gene expression profiles. Applied to the hippo-
campus, the resulting relational or ‘‘genomic anatomy’’ should
correlate with conventional anatomical delineations (e.g., DG,
CA1, CA3) based on previous work (Lein et al., 2004, 2006),
but may also suggest more complex anatomical parcellation
reflecting differential ontogeny and function of specific portions
of the hippocampus. Various matrix decomposition methods
exist for identifying principal features of multidimensional data
sets, including principal components analysis (PCA) (Hastie
et al., 2001), independent component analysis (ICA) (Bell and
Sejnowski, 1997), and nonnegative matrix factorization (NMF)
(Lee and Seung, 1999). NMF in particular has been successfully
applied to identify complex spatial and topological features and
is particularly appropriate for image-based gene expression data
Neuron 60, 1010–1021, December 26, 2008 ª2008 Elsevier Inc. 1011
since the dimensions of space and expression level are corre-
lated and nonnegative.
NMF-based methods were applied to data from the ABA
(www.brain-map.org). As described (Lein et al., 2006; Ng et al.,
2007), images of uniformly (100–200 mm) spaced sections for
each gene were algorithmically quantified and aligned in 3D to
a common anatomical framework derived from a Nissl-based
reference atlas (Dong, 2008). This framework consists both of
gross anatomical delineations (e.g., hippocampus) and an
underlying coordinate grid of (200 mm)3 3D voxels spanning the
entire brain. These data were restricted to a matrix of voxels
(n = 1290) spanning the hippocampus over a set of genes, where
the matrix elements represent expression level for each gene at
every voxel location. The original NMF method (Lee and Seung,
1999) does not explicitly account for local spatial relationships,
and variants that do so have recently been described to enhance
detection of local topological structure in facial feature detection
(Zhang et al., 2008). Since neighboring (200 mm)3 voxels tend to
exhibit highly correlated gene expression profiles in the ABA data
set, we took a similar approach by modifying the NMF method
(mNMF) to incorporate neighborhood constraints via Markov
Random Fields (Besag, 1986) (see Experimental Procedures).
mNMF decomposition was performed using 2686 genes from
the ABA showing hippocampal expression and for which coronal
plane data was available, as these data register more accurately
to the coronal reference model used (Table S1 available online).
Since the ABA was generated iteratively (Lein et al., 2006), with
screening in the sagittal plane followed by coronal replicates
for genes displaying regionalized expression, this coronal gene
set is highly selective for heterogeneous, nonubiquitous expres-
sion patterns. The number of decomposition modes must be set
a priori and was varied from 2 to 20 (Document S1). The data
reduction afforded by mNMF allows readily interpretable results
by plotting the resulting modes, each consisting of a set of
(mostly) spatially contiguous voxels partitioning the hippo-
campal volume, back on to sections of the anatomical reference
model (Figure 1). The initial modes correspond to canonical
neuroanatomical subdivisions consisting of discrete neuronal
subtypes, providing an intuitive validation of the approach to
obtain meaningful partitioning of hippocampal anatomy based
on gene expression. For example, the initial two modes corre-
spond to small granule cells of the DG and pyramidal neurons
of Ammon’s horn (Figure 1, second row). Decomposition into
three modes further divided the pyramidal cell layer into canon-
ical CA1 and CA3 fields (Figure 1, third row). Surprisingly, four
modes differentiated a discrete temporal domain spanning all
three subfields at the temporal pole (Figure 1, fourth row), indi-
cating that temporal hippocampus displays a molecular profile
distinct from the septal hippocampus. Increasing numbers of
modes further divides the hippocampus, although these data
are less interpretable and the statistical fit of the data as
measured by intermode contrast declines steeply beyond four
modes (Document S1). A similar result partitioning the hippo-
campus into the septal DG, septal CA1, septal CA3, and the
temporal hippocampus was also obtained using a recursive
correlation-based clustering method (Document S2).
Another approach that empirically matched individual gene
expression patterns well was to apply mNMF to the voxel sets
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Genomic Anatomy of the Hippocampus

Figure 1. NMF Decomposition of Hippocampal Gene Expression

(A) (Row 1) Series of Nissl-stained sections spanning the rostrocaudal extent of the hippocampus, with major fields annotated. (Rows 2–4) Results of NMF
classification of hippocampal voxels into two through four modes, respectively, color coded and plotted onto the same Nissl sections in row 1. Two modes differ-
entiate DG (blue) from the CA fields (red). Three modes correlate closely to CA1 (red), CA3 (green), and the DG (light blue). Addition of a fourth mode delineates
a zone at the temporal pole of the hippocampus (yellow, arrows) spanning all subfields. (Rows 4–6) NMF decomposition of CA1, CA3, and DG derived from three-
mode decomposition of the entire hippocampus in row 3. The results of a two-mode decomposition are shown, dividing each subfield into rostral/septal (red) and
caudal/temporal (green) division (arrows).
(B and C) Heterogeneous septotemporal expression of individual gene patterns match NMF boundaries. Kctd4 is expressed in caudal ([B], right panels) but not
rostral (left panel) CA1 and avoids the temporal portion of CA3 (arrowhead). Trhr is expressed in temporal DG (arrows) and temporal CA3 (arrowheads). DG, CA1,
CA2, CA3: major hippocampal subfields; vSub: ventral subiculum.

contained in modes identified from the whole hippocampus
decomposition (iterative mNMF). Decomposition of CA1, CA3,
and dentate gyrus voxel sets from the initial three-mode partition
further subdivided each region along the septotemporal axis of
the hippocampus (Document S1). The first two modes for each
region are shown in Figure 1A, along with individual genes
matching these modes (Figures 1B and 1C). CA1 is divided
into rostral and caudal domains more or less in the coronal plane
(Figure 1A), a pattern seen for the gene Kctd4 (Figure 1B). The
DG is divided into dorsal/septal and ventral/temporal domains

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Genomic Anatomy of the Hippocampus
(Figure 1A), mirrored by expression of thyrotropin-releasing
hormone receptor (Trhr, Figure 1C). CA3 is divided into septal
and temporal domains (Figure 1A), matching septal expression
of Kctd4 (Figure 1B) and temporal expression of Trhr
(Figure 1C). This boundary is very robust and recapitulated by
many individual gene expression patterns, as described further
below. These individual gene examples demonstrate that
regardless of higher-order cross-subfield correlations, heteroge-
neous expression within each hippocampal subfield frequently
occurs independently (e.g., regionalized expression of Kctd4 in
CA1 and CA3 but uniform expression in the DG), and at least
for the hippocampus an iterative NMF strategy is an effective
means of predicting novel genetic partitions based on
large-scale gene expression data. Decomposition of individual
subfields into additional modes produces further septotemporal
divisions, potentially indicating a significantly more complex par-
titioning along this axis (Document S1).
Septotemporal Heterogeneity in CA3
Manual Mapping of Gene Expression Boundaries
To gain a more detailed understanding of molecular partitioning
along the septotemporal axis of the hippocampus, cellular reso-
lution expression patterns of 6000 genes including the set
analyzed by mNMF were manually analyzed. CA3 demonstrated
a particularly high degree of heterogeneity, and over 300 genes
were identified with robust regionalized expression patterns
(Table S2). Many different individual gene expression patterns
are observed in CA3, with differential expression along the sep-
totemporal and proximal/distal (between the DG and CA2) axes,
and less frequently between the inner and outer pyramidal cell
populations in the radial dimension (Lein et al., 2006). Genes
with particularly discrete boundaries allowed a detailed mapping
of expression domains across the full extent of CA3 on to a refer-
ence Nissl series, facilitated by the uniform, densely sampled
plane of section-matched expression data available for each
gene in the ABA. Approached in this way, gene expression
boundaries delineating nine discrete subdivisions within the
CA3 pyramidal cell layer were identified, with numerous genes
recapitulating each of these boundaries. The majority of the
differentially expressed genes in CA3 obey these boundaries
(Table S2), and any individual gene expression pattern could
therefore be described as comprising some subset of these
expression ‘‘domains.’’ It should be noted that great variability
exists from gene to gene with respect to enrichment versus binary
restriction to a particular region of CA3. Furthermore, many genes
display some gradient-like characteristics, but tend either to
taper off at one of these boundaries or appear to some degree
as step gradients, showing signal drop-off at these boundaries.
Figure 2 illustrates the boundaries and domains identified in
CA3. To describe these complex anatomical data, individual
genes demonstrating each observed boundary are shown, with
approximate locations plotted on a plane-matched series of
Nissl-stained sections spanning the entire rostrocaudal extent
of the hippocampus. CA3 in this Nissl series is defined here as
the portion of the pyramidal cell layer between the DG and the
CA2 subfield (defined on the basis of robust and consistent
boundaries delineated by many genes: Figure S1) (Lein et al.,
2005). The regions or domains of CA3 between boundaries are
Neuron 60, 1010–1021, December 26, 2008 ª2008 Elsevier Inc. 1013
numbered 1–9 according to their proximal/distal and septotem-
poral locations in CA3. Regions 1–3 cover roughly the septal third
of CA3 (from proximal to distal), regions 4–6 span approximately
the mid-septotemporal half (from proximal to distal), and regions
7–9 cover the temporal pole of CA3. Genes were selected to
demonstrate these boundaries where possible as series of exten-
sions from the septal pole of CA3, with each gene adding one
additional domain in the septal-to-temporal axis to the extent of
the previous gene. For example, Ttn1 is expressed only in the
septal, proximal-most (to the DG) portion of CA3 (region 1;
Figure 2). Fmo1 is expressed in regions 1 and 2, extending slightly
more temporally and distally. Prkcd is expressed in regions 1–3,
and so on up to Dkk3, which is expressed in regions 1–8,
excluding only the temporal-most tip of CA3 comprising region
9. Differentiation between small temporal regions 7–9 is shown
in Figure S2. Certain boundaries are observed more frequently
than others. For example, the boundaries between regions 4/7
and 6/7 are particularly robust and frequently observed, and
these correspond well to the temporal CA3 division identified
by NMF in Figure 1A. To demonstrate the reproducibility of these
boundaries across multiple genes and specimens, 24 additional
genes displaying these boundaries are shown in Figure S3.
The complexity of the subdivisions delineated by gene expres-
sion is best appreciated by mapping subdomains onto a 3D
model of CA3 (Figure 3). Viewed in 3D, the septal two-thirds of
CA3 is clearly divided into a series of diagonal bands oriented
septal-distal (toward CA2) to temporal-proximal (toward the
DG; Figure 3B). This banding is highly reminiscent of the organi-
zation of recurrent associational projections (Ishizuka et al.,
1990), as discussed below. The temporal pole, on the other
hand, consists of a series of domains wrapped around the prox-
imal and distal edges.
Combinatorial Molecular Profiles of CA3 Domains
Many more complex patterns are observed as well, consisting
of subsets of the domains described above. Each subdivision
of CA3 defined above displays a distinct molecular profile
defined by combinatorial patterns of gene expression, exempli-
fied by genes involved in differential cell adhesion, ionic conduc-
tance, and transcriptional regulation. Genes displaying robust
regional expression in each of these functional ontological cate-
gories are plotted as a series of heat maps representing densi-
tometric quantification of ISH signal, with subdivisions of CA3
organized linearly from septal to temporal as effectively a ‘‘flat
map’’ of CA3 (Figure 4). For example, many cell adhesion genes
exhibit complex, partially overlapping expression patterns,
predominantly starting at either the septal or temporal pole of
CA3 and ending at different boundaries in between. Individual
domains are not necessarily defined by single genes; rather,
each domain is defined by the combinatorial expression of sets
of adhesion molecules, such that each CA3 subdivision
expresses a unique complement of adhesion molecules. Similar
patterns for ion channels suggest heterogeneity in septotemporal
physiological properties as well. Many specific expression
patterns are recapitulated across multiple genes (Table S2). A
series of genes selectively expressed in regions 7–9 are shown
in Figure S4. Interestingly, the CA3 NMF decomposition
described above gives a similar pattern of septotemporal parti-
tioning when the CA3 volume is decomposed into more modes
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Genomic Anatomy of the Hippocampus

Figure 2. Mapping Gene Expression Boundaries in CA3

Nine divisions of the CA3 pyramidal cell layer can be identified on the basis of gene expression boundaries, displayed on a panel of eight coronal planes of section
through the entire hippocampus moving from rostral (top) to caudal (bottom). Exemplar genes for each boundary are presented mainly as a series of extensions
from the septal pole, with the first gene (Ttn) expressed solely in the most septal region of CA3 proximal to the DG and each gene moving to the right expressed in

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Figure 3. Three-Dimensional Modeling of CA3 Molecular Anatomy

Color-coded 3D models of major hippocampal subfields CA1 (red), CA2 (dark blue), CA3 (green), and DG (yellow) (A) and gene expression-based subdivisions of
CA3 (B). Four different orientations demonstrate the organization of CA3 subdivisions, which can be seen to divide septal CA3 into a series of diagonal bands
oriented septal-distal (toward CA2) to temporal-proximal (toward DG). CA2 (dark blue) is included in both models as a fiducial reference, and color coding of
CA3 divisions matches that in Figure 2. 3D orientation bars: lateral, red; ventral, green; rostral, blue. Scale bar, 1 mm.

than described in Figure 1. A direct comparison of the CA3 model
from Figure 2 to the NMF decomposition is shown in Figure S5.
While these partitions were not easily matched to individual
gene patterns, the additive parts-based nature of the NMF
approach appears to predict spatially coherent domains that are
reflected by the combinatorial overlap of expression patterns.
Reciprocal, Nonoverlapping CA3 Gene
Expression Domains
CA3 gene expression boundaries are reciprocal, such that indi-
vidual genes delineate each boundary from both sides. For
example, Fmo1 and Mas1 reciprocally delineate the boundary
between regions 2 and 3 (Figures 5A and 5B), Itga7 and Plagl1
the boundary between regions 3 and 5 (Figures 3C and 3D),
Ptgs2 and Coch the boundary between regions 6 and 7 (Figures
5E and 5F), and Loxl1 and Coch the boundary between regions 4
and 7 (Figures 5G and 5H). Interestingly, the expression bound-
aries for these genes are stable across late postnatal develop-
ment, indicating that this partitioning is established fairly early
in development (Figure S6). To determine how discrete these
gene expression boundaries are at a cellular level, double fluores-
cent ISH was used to label mRNAs for these same pairs of highly
restricted genes reciprocally abutting each border on the same
tissue sections. In each case expression proved to be mutually
exclusive, with no detectable coexpression in cells at the bound-
aries. In some cases the boundaries were extremely sharp,
as between regions 6 and 7 (Figures 5K and 5O) and 4 and 7
(Figures 5G and 5H). In other cases, cells labeled for each gene
commingle at the boundary (e.g., Plagl1 and Itga7; Figures 5C
and 5D), but no cells were observed that expressed both genes.
An additional level of complexity is presented by a single-cell-
thick band of neurons along the border of the pyramidal cell layer
and stratum oriens that strongly expresses a small set of genes
including the procollagen gene Col6a1 (Figures 6A and 6I) and
suppressor of tumorigenicity 18 (St18; Figures 6E and 6I). These
appear to be pyramidal neurons, since most genes are coordi-
nately expressed throughout the pyramidal cell layer including
this outer layer, and because GABAergic cell markers (and
markers for nonneuronal cell populations) do not delineate a
similar population along this boundary (data not shown). Some
genes also appeared to selectively lack expression along the
inner portion of the pyramidal cell layer, such as the potassium
channel subunit Kcnq5. Colabeling for Kcnq5 with these two
outer layer markers demonstrates, as above, that these genes
label nonoverlapping populations of pyramidal cells (Figures
6A–6H). Further heterogeneity exists within this outer population,
as St18 colabels with Col6a1 entirely in septal CA3, but in a
decreasing subset moving temporally along the septotemporal
axis (Figures 6I–6O). In general, the areal extent of labeled cells
in this band adjacent to stratum oriens mirrors areal boundaries
described above for the full radial thickness of CA3, and this
expression is also annotated in Table S2 when possible.
Hippocampal Connectivity and Gene
Expression Boundaries
It is well established that there is heterogeneous afferent,
efferent, and intrinsic hippocampal connectivity that varies along
the transverse and septotemporal axes, and tracing studies have
indicated that relatively discrete borders are associated with
many of these projections (Dolorfo and Amaral, 1998; Ishizuka
et al., 1990; Risold and Swanson, 1997; van Groen et al., 2003;
Verwer et al., 1997). Many of the molecular patterns we observe
correlate with trends reported by tracing studies (Figure 7). For
example, anterograde and retrograde labeling studies in rat
subdivide the DG into three rough domains comprising the
septal half and subsequent two more temporal quarters based
on connections from discrete bands in the entorhinal cortex

the previous regions as well as one additional region, generally slightly more temporal and distal to the DG. The regions or expression domains circumscribed by
these boundaries are numbered 1–9 based on their relative septal to temporal location. For each gene, the boundary of its expression is shown by a black bar
across CA3, and color-coded arrowheads and numbers delineate the boundaries and domains between boundaries as they are added moving from left to right.
These boundaries are compiled onto a plane-matched Nissl series in the left-most column. The most temporal tip (region 9) is not shown by gene expression but is
delineated with black arrows in the Nissl series and for Dkk (regions 1–8). CA2 and the boundary between CA1 and ventral subiculum are delineated by blue bars in
all sections. Scale bars: rows 1 and 2, 421 mm; rows 3–8, 842 mm.

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Figure 4. Combinatorial Profiles of Cell Adhesion/Axon Guidance Molecules, Ion Channels, and Transcription Factors in CA3 Subdivisions
Heat map representation of densitometric quantification of gene expression in each functional category across nine subdivisions of CA3 from septal to temporal,
demonstrating clustering of enriched genes along septal to temporal subdivisions. Data are normalized to [0,1] corresponding to minimum and maximum
measurements for that gene, with the corresponding continuous color scale ranging from black (0) to bright copper (1). Assignment of genes to functional cate-
gories based on AmiGO (Ashburner et al., 2000), DAVID (Dennis et al., 2003), MGI, or PANTHER (Mi et al., 2005) annotation.

(Burwell and Amaral, 1998). Likewise, we identified genes that
differentially label septal and temporal DG, dividing the DG into
three domains through their overlap (Figures 7 and S7). The
temporal portion of CA1 specifically projects to frontal cortex
(Verwer et al., 1997), a pattern observed in regionalized CA1
gene expression (Figures 7 and S7). Within CA3, gene expression
boundaries divide the septal 2/3 into a series of diagonal bands
oriented distal/septal to proximal/temporal, similar to the orienta-
tion of autoassociational collaterals of pyramidal cells within CA3
(Ishizuka et al., 1990) (Figure 7). Amygdalar inputs target very
specific small regions in temporal CA3 (Petrovich et al., 2001),
similar to the domain structure of gene expression in this region.
A topographically organized projection of CA3 to the lateral
septum has also been described, with different septotemporal
levels of CA3 projecting to distinct portions of the septum (Risold
and Swanson, 1997), and previous studies have argued that this
topography is established via opposing gradients of axon guid-
ance molecules and their receptors in the hippocampus and

Figure 5. Gene Expression Defines Recip-

rocal Boundaries in CA3
(A–H) ISH data for a set of genes exemplifying the
reciprocal, nonoverlapping nature of CA3 gene
expression boundaries. (A) Fmo1, (B) Mas1, (C)
Itga7, (D) Plagl1, (E) Ptgs2, (F and G) Coch, (H)
Loxl1. White arrows in (A)–(H) indicate the location
of CA2, and black arrows and corresponding
numbers indicate the specific boundary being
illustrated in each panel.
(I–P) Double fluorescent ISH for pairs of genes
defining reciprocal boundaries in CA3 at low
magnification (I–L) or high magnification (M–P).
Sections are counterstained with DAPI (blue).
(I and M) Mas1 (green) and Fmo1 (red), (J and
NJ) Plagl1 (green) and Itga7 (red), (K and O) Coch
(green) and Ptgs2 (red), (L and P) Coch (green)
and Loxl1 (red). Scale bars: (A and B) 500 mm,
(C–H) 500 mm, (I–L) 1 mm, (M–P) 250 mm.

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septum (Zhang et al., 1996). To test directly whether molecular
boundaries observed in CA3 correlate with efferent connectivity,
we combined retrograde labeling from CA3 axon terminals in the
septum with ISH for a marker gene delineating the boundaries
described above. Injections of Alexa Fluor 555-conjugated
cholera toxin into the lateral septum back-labeled CA3 pyramidal
cells. Three cases are shown in Figure 8, demonstrating that cells
retrogradely labeled from the caudal lateral septum consistently
avoid the portion of CA3 proximal to the dentate gyrus (Figures
8C, 8H, and 8M), corresponding closely to the domain 2/3
boundary described above. Colabeling these sections for
a marker of this boundary, Fmo1 (see Figures 2 and 5) showed
a close apposition of this gene expression boundary with the
extent of backlabelled neurons (Figures 8E, 8J, and 8O).
DISCUSSION
The availability of genome-scale ISH data provides great oppor-
tunities to understand the detailed cellular and functional archi-
Neuron 60, 1010–1021, December 26, 2008 ª2008 Elsevier Inc. 1017
Figure 6. Inner and Outer CA3 Pyramidal
Cells Display Nonoverlapping Gene Expres-
sion Profiles
Double fluorescent ISH for pairs of genes differen-
tiating inner (adjacent to stratum radiatum) from
outer pyramidal cells in CA3 at low (A, E, and I)
and high (B–D, F–H, and J–O) magnification.
Kcnq5 (green) is nonoverlapping with Col6a1
(red; [A–D]) or with St18 (red; [E–H]), shown as indi-
vidual gene labeling (B and C and F and G) or
colabeling (A and D and E and H). (I–O) St18 labels
a subpopulation of Col6a-expressing cells with
differential colabeling in septal (J and M), midsep-
totemporal (K and N), or temporal CA3 (L and O)
shown with (J–L) and without DAPI (M–O). Scale
bars: (A and E) 1 mm, (B–D and F–H) 50 mm, (I)
500 mm, (J–O) 50 mm.
tecture of the nervous system on the premise that molecular
phenotype reflects and/or underlies function. These data allow
a data-driven, unbiased ‘‘genomic anatomy’’ approach that takes
advantage of large-scale cellular resolution data to reveal organi-
zational principles that are difficult to glean based on single-gene
or targeted hypothesis-driven experimentation. Application of
statistical component analysis and detailed mapping of gene
expression to the hippocampus reveals a complex yet discrete
molecular architecture defined by highly combinatorial expression
patterns of many genes. These findings provide a molecular
substrate for previously described anatomical data demonstrating
complex in vivo hippocampal architecture, with a level of complex-
ity in stark contrast to a lamellar model of hippocampal circuitry
reflecting that portion of intrinsic circuitry preserved in an in vitro
transverse hippocampal slice. In particular, the current findings
provide strong evidence for molecular regionality that could underlie
functional differentiation along the septotemporal axis of the hippo-
campus. This approach should be generally informative applied to
other brain regions or other large-scale ISH data sets as well.
Figure 7. Similarities between Molecular
Domains and Extra- and Intrahippocampal
Connectivity
(Top panels) Schematic views of the topography of
projections from the entorhinal cortex to the DG,
forward and recurrent projections from CA3, and
from the amygdala onto the entire hippocampal
formation. (Bottom panels) 3D modeling of molec-
ularly defined subdivisions of DG, CA3, and CA1
delineated by gene expression. Temporal CA1 is
delimited by the gene Dio3 (red), while the DG is
divided into three regions based on the expression
of the genes Cyp7b1 (septal, red), Trhr (temporal,
green), and their overlap (middle, yellow). Entorhi-
nal-dentate (Burwell and Amaral, 1998 [Reprinted
with permission of Wiley-Liss, Inc., a subsidiary
of John Wiley & Sons, Inc]), CA3 intrahippocampal
(Deadwyler and Hampson, 1999 [Reprinted with
permission of Wiley-Liss, Inc., a subsidiary of
John Wiley & Sons, Inc]), originally modified from
(Ishizuka et al., 1990 [Reprinted with permission
of Wiley-Liss, Inc., a subsidiary of John Wiley &
Sons, Inc]), and amygdalohippocampal (Petrovich
et al., 2001 [Reprinted with permission of Elsevier])
schematics reprinted with permission.

Figure 8. CA3 Efferent Projections to the Lateral Septum Mirror Molecular Boundaries
Combined fluorescent ISH with retrograde labeling of CA3 pyramidal cells following injection of Alexa Fluor 488 conjugated-CTB into dorsal caudal lateral septum.
Injection sites for three separate cases (16, 17, 18) are shown in panels (B), (G), and (L), with corresponding atlas plates in panels (A), (F), and (K) (Dong, 2008). CTB
(red) labeling in CA3 (C, H, and M) closely apposes the expression domain of Fmo1 mRNA (green; [D, I, and N]). The merged image is shown at higher magnification
in (E), (J), and (O). Arrows denote boundary between Fmo1 and CTB labeling. Scale bars: 500 mm (B–D, G–I, and L–N) and 200 mm (E, J, and O).
Hippocampal Boundaries, Domains,
and Cellular Identities
A number of organizational principles have emerged from this
broad analysis of hippocampal gene expression. Perhaps the
most striking finding is the degree of molecular diversity within
excitatory neuronal populations, generally considered fairly
homogeneous. Gene expression patterns define contiguous
groups of neurons in each hippocampal subfield, generally
dividing the septal or dorsal hippocampus from the temporal or
ventral hippocampus. In all subfields, the sharpest gene expres-
sion boundaries involve the most temporal quarter of the hippo-
campus, roughly the region defined as a principle mode by the
NMF analysis. Gene expression in each hippocampal subfield
appears to be regulated independently. CA3 molecular architec-
ture is characterized by a series of reciprocal, nonoverlapping
boundaries extending along the septal/temporal and proximal/
distal axes, and graded expression in CA3 frequently appears
as a step gradient, in contrast to more continuous gradients
observed in DG and CA1. Although these regions were not
analyzed in the same detail as CA3, gene expression ‘‘bound-
aries’’ identified in these regions appear less discrete, and for
CA1 in particular, are not reciprocal in the sense that we could
not identify genes that abut each boundary from both sides.
Several functional categories are highly represented among
differentially expressed genes in CA3, suggestive of the func-
tional relevance for this restricted expression. By far the most
abundant broad category, including more than half of all differen-
tially expressed genes, involves differential cell adhesion, axon
guidance, and cell-cell communication (including neuropeptides
and neurotransmitter receptors). This high representation
suggests that a great deal of septotemporal heterogeneity
relates to the establishment and maintenance of differential
1018 Neuron 60, 1010–1021, December 26, 2008 ª2008 Elsevier Inc.
Neuron
Genomic Anatomy of the Hippocampus
functional connectivity. Previous studies have postulated links
between differential cell adhesion molecule expression and
axon guidance in the process of establishing topographic spec-
ificity in hippocampal projections. For example, the ephrin
receptor Epha5 is expressed in a medial to lateral gradient in
the developing hippocampus, with its corresponding ligands ex-
pressed in a reciprocal fashion in hippocampal target tissues
such as the lateral septum (Zhang et al., 1996). While axon guid-
ance and establishment of topographic specificity are generally
considered developmental phenomena, there is increasing
evidence that cell adhesion molecules are involved in synaptic
plasticity and maintenance of neural networks in adult animals
(de Wit and Verhaagen, 2003; Pinkstaff et al., 1999). Further-
more, it is worth noting that neurogenesis persists in the adult
DG, and it may be necessary to maintain cues for axonal path-
finding for newly generated granule cells to establish appropriate
connectivity within CA3 (Zhao et al., 2006). For example, several
homophilic cell adhesion molecules, including Pcdh21, Robo2,
Cdh9, and Cdh8, are expressed both in septal DG and in septal
CA3. The majority of the remaining differentially expressed
genes relate in some way to intrinsic cellular functions, most
notably ion channels and transcription factors.
The systematic compilation of differentially expressed genes
provides a window into the molecular specification of cellular
identity. Clearly the unique molecular identity of a particular
cell is defined by the combinatorial overlap of many expressed
genes. In CA3, we have shown nine discrete regions along the
septotemporal axis that can be defined by gene expression.
Many of these regions are not represented uniquely by expres-
sion of any single gene, but rather can be defined by the overlap
of many gene expression domains, providing each CA3 subdivi-
sion with a unique complement of expressed genes. Notably,
Neuron
Genomic Anatomy of the Hippocampus
members of many different cell adhesion (cadherins, collagens,
IgG superfamily) and axon guidance (ephrins, slits, robos, sem-
aphorins) families exhibit combinatorial expression in a cascade
of partially overlapping domains. While it is common to discuss
the transcription factor code specifying the fate of a cell during
development, it would appear that unique adhesion molecule
and ion channel profiles may serve to determine functional
connectivity and intrinsic electrophysiological profiles of discrete
hippocampal neuronal populations as well.
Functional Correlates of Molecular Boundaries
To make sense of the complicated molecular anatomy of the
hippocampus, it is necessary to synthesize knowledge of func-
tional connectivity, molecular boundaries, and molecular func-
tion (e.g., Figures 4 and 7). A variety of tracing studies have indi-
cated that there are discontinuous features associated with
(topographically organized) intrinsic, afferent and efferent hippo-
campal projections, frequently with relatively discrete borders
(Dolorfo and Amaral, 1998; Ishizuka et al., 1990; Risold and
Swanson, 1997; van Groen et al., 2003; Verwer et al., 1997). In
the current study, we similarly found a consistent, relatively
discrete boundary of efferent CA3 projections to the caudal
lateral septum by retrograde labeling and established that this
boundary correlates with one of the molecular boundaries
(between regions 2 and 3). Many of the other molecular patterns
observed are consistent with trends observed by tracing studies
(Figure 7), which, along with the overrepresentation of cell adhe-
sion genes, suggests that a great deal of regionalized gene
expression reflects underlying hippocampal circuitry.
Differential physiological properties have also been reported
across septal and temporal hippocampus, likely the result of
differential ion channel expression (Figure 4). For example,
septal versus temporal CA1 pyramidal neurons exhibit differen-
tial induction of LTP and LTD (Izaki et al., 2000; Papatheodoro-
poulos and Kostopoulos, 2000), while epileptiform bursting can
be readily induced in CA3 of temporal but not septal hippo-
campal slices (Bragdon et al., 1986). Interestingly, this latter
work indicated that the greatest bursting could be induced in
the second-most temporal slice, suggesting a regionalized
expression of ion channels underlying these responses. The
hippocampus also receives differential innervation across its
septotemporal extent from various neurotransmitter systems,
with stronger temporal innervation by fibers containing dopamine
(Verney et al., 1985), serotonin (Gage and Thompson, 1980), NPY
(Kohler et al., 1987), and TRH (Pazos et al., 1985). There is a cor-
responding localized expression of cognate receptors in
temporal CA3 of the dopamine receptor Drd2, serotonin receptor
Htr4, NPY receptor Npy1r, and TRH receptor Trhr (see Table S2).
The comprehensive molecular description of ion channels and
receptors should be a powerful predictor for differential intrinsic
membrane properties and ligand-induced excitation among
discrete hippocampal neuronal populations and guide future
experimentation to understand their functional relevance.
Comprehensive molecular mapping based on analysis of
genome-wide ISH data provides a complex molecular framework
for considering the functional architecture of the hippocampus,
and also the means for functional analysis through targeted
genetic manipulation. This approach has been used to generate
Neuron 60, 1010–1021, December 26, 2008 ª2008 Elsevier Inc. 1019
CA1- and CA3-specific NMDA receptor knockouts, demonstrating
differential hippocampal subregion function in spatial learning
tasks (Nakazawa et al., 2002; Tsien et al., 1996). Similar transgenic
approaches to recapitulate discrete septotemporal hippocampal
subdivisions, coupled with an array of developing technologies
for selective cell ablation (Luquet et al., 2005), neuronal silencing
(Lerchner et al., 2007; Tan et al., 2006), and transneuronal labeling
(Wickersham et al., 2007), hold great promise for elucidating the
fine functional architecture of the hippocampus.
EXPERIMENTAL PROCEDURES
High-Throughput Nonisotopic In Situ Hybridization
All procedures were approved by the Allen Institute Institutional Animal Care
and Use Committee. P56 male C57BL/6J mice (Jackson Labs West, Sacra-
mento, CA) were used for most experiments, except for a small developmental
series using P14 and P28 animals. High-throughput data generation was per-
formed using a semiautomated nonisotopic digoxigenin-based colorimetric
ISH platform as described previously (Lein et al., 2006). Briefly, gene-specific
riboprobes were hybridized to 25 mm thick postfixed sections sampled uniformly
either across the entire brain or restricted to the hippocampus for high sampling
density series used for 3D reconstruction of gene expression patterns.
Nonnegative Matrix Factorization
ISH data were quantified and registered in 3D to a common anatomical refer-
ence framework provided by a Nissl-stained reference atlas as previously
described (Lein et al., 2006; Ng et al., 2007). Expression levels were calculated
for each gene for the set of (200 mm)3 voxels spanning the hippocampus. These
expression data were used to generate an N 3 V matrix A, representing N
genes in V 3D voxels. 2868 genes displaying expression above a minimum
threshold (Ng et al., 2007) and for which coronal ISH data were available
were used for this analysis across 1290 voxels representing the hippocampus
(see Table S1). To identify distinct subdivisions of the hippocampus based on
spatial gene expression patterns, the data were decomposed into defined
numbers of modes, each mode defined as some subset of the V voxels. We
extended the method of Non-Negative Factorization (NMF) (Lee and Seung,
1999) using Markov Random Fields (MRF) (Besag, 1986) to account for spatial
influence in the data. NMF is an iterative matrix factorization technique that
differs from principal components analysis in that the matrix factors are con-
strained to have positive and interpretable component entries. In the present
implementation (mNMF) the matrix factors are modified during each iteration
of the decomposition via a MRF model to incorporate gene expression from
a voxel’s spatial neighborhood. This additional use of MRF acts as a spatial
smoothing and yields more robust clusters. Details and the full decomposition
results for 2 to 20 modes are provided in Document S1.
Expression Boundary Mapping and Densitometric Quantification of
Colorimetric In Situ Hybridization Data
Gene expression boundaries were initially identified by mapping approximate
boundaries of 300 genes exhibiting regionalized CA3 expression onto a high-
density reference Nissl series. The vast majority of observed expression
patterns could be accounted for a set of expression boundaries dividing
CA3 into nine discrete subdomains. The boundaries exhibited by particularly
robust exemplars, such as those shown in Figure 2, were used for final
boundary delineations used to model these regions in 3D and to bound regions
for quantitative analysis of gene expression across subdomains. For the 103
genes in Figure 4, bitmap images of ISH data were quantified using Scion
Image (Scion Corporation, Frederick, MD) on 12 hippocampus-containing
coronal ISH sections per gene. For each gene a rectangular region of interest
was used to sample the mean density (average gray value) from each of the
nine subregions of CA3, with five measurements collected per CA3 subdomain
per gene where possible (several regions were only present on one to three
available brain sections). Transition areas between subdomains were avoided
when possible to improve the accuracy of the quantitation. It should be noted
that this method does not discriminate between expression in pyramidal cells
of the hippocampus and other cells such as interneurons, nonneuronal cells, and

the outer layer of CA3, and does not correct for changes in cell density across
different subdomains, tending to attenuate quantitative differences between
pyramidal cell expression between different subdomains and leading to gener-
ally lower values for relatively low density regions (e.g., region 1 in Figure 4). Due
to large variance in background levels between sections, a method for outlier
detection appropriate for small sample sizes, the median absolute deviation
statistic (MAD), was used prior to plotting results in Figure 4 (Wilcox, 2001).
3D Modeling of Hippocampal Gene Expression
Hippocampal expression patterns were visualized in 3D by either reconstruct-
ing the raw ISH data from aligned sections (DG) or by transposing expression
patterns onto a model of the hippocampus (CA1 and CA3) as described previ-
ously (Lein et al., 2005). Briefly, a reference model was created by aligning a set
of high-density Nissl images spanning the hippocampus in Adobe PhotoShop
(Adobe Systems, San Jose, CA), and digitally ‘‘dissecting’’ each structure of
interest (e.g., CA1, CA2, CA3, DG) from surrounding brain regions for individual
visualization. Observed gene expression patterns were transposed onto these
reference sets to generate independent aligned image series for each division
(except for DG patterns, for which raw ISH data were used on independent
templates). 3D modeling and surface rendering of each region and gene
expression-defined subdivision was performed using the 3D Constructor
plug-in of ImagePro Plus (v. 5.1; Media Cybernetics, Silver Spring, MD).
Fluorescent In Situ Hybridization
Double-label fluorescent ISH was performed using a variation of the colori-
metric protocol. Briefly, riboprobes were labeled with either digoxigenin-UTP
or dinitrophenyl-11-UTP (DNP; Perkin Elmer). A DNP-labeled probe and
a DIG-labeled probe were hybridized simultaneously. Tyramide signal amplifi-
cation was performed for each probe individually, using either anti-DIG-HRP
with tyramide-biotin, or anti-DNP-HRP with tyramide-DNP for amplification.
Visualization of signal was achieved using either streptavidin-Alexa Fluor
488 (Invitrogen/Molecular Probes) or anti-DNP-Alexa Fluor 555 (Invitrogen/
Molecular Probes), and automated fluorescence microscopy. See Document
S3 for complete methodological details.
Retrograde Labeling Combined with ISH
Stereotaxic injections of Alexa Fluor 555-conjugated cholera toxin B subunit
(Invitrogen) were made into the dorsal portion of the caudal lateral septum of
P56 mice using coordinates from Paxinos and Franklin (2004) (0.4 mm M/L;
0.15–0.3 mm A/P; 2.8 mm D/V) using a Picrospritzer and pulled glass pipette.
0.2–2.0 ml of 0.5% w/v labeled toxin was delivered under isoflurane anesthesia
using five pulses of 50–80 ms duration at 30 psi. Five to seven days postinjec-
tion to allow transport of the tracer, animals were sacrificed and brains pro-
cessed for fluorescent ISH using Alexa Fluor 488 detection to allow visualiza-
tion of retrogradely labeled cells and ISH signal on the same tissue sections.
SUPPLEMENTAL DATA
The Supplemental Data can be found with this article online at http://www.
neuron.org/supplemental/S0896-6273(08)01056-8.
ACKNOWLEDGMENTS
This work was sponsored by the Allen Institute for Brain Science. The authors
wish to thank the Allen Institute founders, P.G. Allen and J. Patton, for their
vision, encouragement, and support. The authors also wish to acknowledge
Theresa Zwingman and Maureen Howell for assistance with mining efforts to
identify regional hippocampal expression patterns; Simon Smith for assis-
tance with histological preparations; Jolene Kidney, Maureen Howell, Andrew
Boe, and Tracy Lemon for assistance with tract tracing and sectioning; John
Hohmann and John Morris for assistance with the developmental analysis of
hippocampal gene expression; Amanda Ebbert, Lon Luong, Kimberly Smith,
Melissa Reding, and Cathy Copeland for supporting production and scanning
of fluorescent ISH images; Chinh Dang for supporting database needs; and
Paul Wohnoutka for supporting data production.
Accepted: December 8, 2008
Published: December 24, 2008
1020 Neuron 60, 1010–1021, December 26, 2008 ª2008 Elsevier Inc.
Neuron
Genomic Anatomy of the Hippocampus
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