Laser-capture microdissection and transcriptional profiling of the dorsomedial nucleus of the hypothalamus
Identifying neuronal molecular markers with restricted patterns of expression is a crucial step in dissecting the numerous pathways and functions of the brain. While the dorsomedial nucleus of the hypothalamus (DMH) has been implicated in a host of physiological processes, current functional studies have been limited by the lack of molecular markers specific for DMH. Identification of such markers would facilitate the development of mouse models with DMH-specific genetic manipulations. Here we used a combination of laser-capture microdissection (LCM) and gene expression profiling to identify genes that are highly expressed within the DMH relative to adjacent hypothalamic regions. Six of the most highly expressed of these genes, Gpr50, 4930511J11Rik, Pcsk5, Grp, Sulf1, and Rorβ, were further characterized by real-time polymerase chain reaction (PCR) analysis and in situ hybridization histochemistry. The genes identified in this article will provide the basis for future gene-targeted approaches for studying DMH function. J. Comp. Neurol. 520:3617-3632, 2012. © 2012 Wiley Periodicals, Inc.
Laser-Capture Microdissection and Transcriptional
Proﬁling of the Dorsomedial Nucleus of the
Syann Lee, Angie L. Bookout, Charlotte E. Lee, Laurent Gautron, Matthew J. Harper, Carol F. Elias,
Bradford B. Lowell, and Joel K. Elmquist
Department of Internal Medicine and Department of Pharmacology, Division of Hypothalamic Research, University of Texas
Southwestern Medical Center, Dallas, Texas 75390-9077
Identifying neuronal molecular markers with restricted
patterns of expression is a crucial step in dissecting
the numerous pathways and functions of the brain.
While the dorsomedial nucleus of the hypothalamus
(DMH) has been implicated in a host of physiological
processes, current functional studies have been limited
by the lack of molecular markers specific for DMH.
Identification of such markers would facilitate the devel-
opment of mouse models with DMH-specific genetic
manipulations. Here we used a combination of laser-
capture microdissection (LCM) and gene expression
profiling to identify genes that are highly expressed
within the DMH relative to adjacent hypothalamic
regions. Six of the most highly expressed of these
genes, Gpr50, 4930511J11Rik, Pcsk5, Grp, Sulf1, and
Rorb, were further characterized by real-time polymer-
ase chain reaction (PCR) analysis and in situ hybridiza-
tion histochemistry. The genes identified in this article
will provide the basis for future gene-targeted
approaches for studying DMH function. J. Comp. Neu-
rol. 520:3617–3632, 2012.
2012 Wiley Periodicals, Inc.
INDEXING TERMS: DMH; LCM; hypothalamus
The dorsomedial nucleus of the hypothalamus (DMH)
has been implicated in a wide range of physiological proc-
esses, ranging from reproduction, thermogenesis, stress
response, pancreatic nerve activity, and plasma glucose
levels (Elmquist et al., 1998b; Thompson and Swanson,
1998; Zhang et al., 2011). Recently, there has been
renewed interest in the potential role of the DMH in regu-
lating food intake and circadian rhythms (Chou et al.,
2003; Gooley et al., 2006). The DMH is positioned
between key circadian and metabolic centers, and thus is
ideally situated to integrate signals from these two sys-
tems. It receives projections from circadian nuclei, includ-
ing the superchiasmatic nucleus (SCN) and subparaven-
tricular zone (SPZ) (Watts, 1991; Kalsbeek et al., 1996;
Huang et al., 2011). In turn, the DMH sends abundant
projections to the paraventricular hypothalamic nucleus
(PVH), preoptic area, arcuate nucleus (ARC), and lateral
hypothalamus, important centers for energy balance,
sleep, and wakefulness (Elmquist et al., 1998a; Chou
et al., 2003; Gautron et al., 2010).
Functional and molecular studies also support the role
of DMH in metabolism. DMH expresses a number of neu-
ropeptides known to be important in energy homeostasis,
including leptin receptor (LepR), neuropeptide Y, (Npy),
orexin, cholecystokinin (Cck), several opioids, melanocor-
tin 4 receptor (Mc4r), cocaine and amphetamine-regu-
lated transcript (Cart), melanin concentrating hormone
(Mch), and agouti-related peptide (Agrp) (Bellinger and
Bernardis, 2002). Importantly, DMH neurons also
respond to changes in metabolic stimuli. For instance,
mice fed a high-fat diet (HFD) show increases in Npy
expression and c-fos immunoreactivity within the DMH
Grant sponsor: National Institutes of Health; Grant numbers: P01
DK088761, PL1 DK081182, and UL1 RR024923; Grant numbers:
R01DK53301 and RL1DK081185 (to J.K.E.).
*CORRESPONDENCE TO: Joel K. Elmquist, DVM, PhD, Division of
Hypothalamic Research, Departments of Internal Medicine and
Pharmacology, University of Texas Southwestern Medical Center, 5323
Harry Hines Blvd., Dallas, TX 75390-9051.
2012 Wiley Periodicals, Inc.
Received December 23, 2011; Revised March 10, 2012; Accepted March
Published online April 2, 2012 in Wiley Online Library (wileyonlinelibrary.
The Journal of Comparative Neurology | Research in Systems Neuroscience 520:3617–3632 (2012) 3617
(Guan et al., 1998; Lin and Huang, 1999). Furthermore,
intravenous injections of the adipocyte-derived hormone
leptin, a key metabolic hormone, strongly activate neu-
rons within the DMH (Elmquist et al., 1998b; Elias et al.,
The role of DMH in circadian rhythmicity, however, is
not as clear. While the circadian genes, period homolog 1
(Per1) and period homolog 2 (Per2), have been shown to
oscillate in the compact DMH in a food entrainable man-
ner (Mieda et al., 2006), DMH ablation experiments have
produced variable effects on food intake (Bellinger and
Bernardis, 2002), food anticipatory behavior, and other
circadian rhythms (Chou et al., 2003; Gooley et al., 2006;
Landry et al., 2006; Landry et al., 2007; Moriya et al.,
2009). While these discrepancies are most likely due to
variations in lesioning methods and experimental designs,
neuron-specific, genetic manipulations are ultimately
needed to resolve these issues.
The genetic and chemical makeup of DMH is poorly
understood. While other hypothalamic nuclei can be
defined by molecular markers, such as Agrp in the arcu-
ate (ARC), or steroidogenic factor 1 (Sf-1) in the ventro-
medial hypothalamus (VMH), no such marker genes have
been identified in the DMH. Identifying DMH-specific
genes would be invaluable for studying cell lineages, cell
biology, and the creation of a set of molecular tools for
genetic DMH deletion studies.
With recent advances in whole genome sequencing
and high-throughput gene expression assays, it is now
possible to conduct large-scale efforts to identify and
map the expression of all genes within the rodent brain
(Gray et al., 2004; Lein et al., 2007). While these
approaches have produced comprehensive expression
databases, their high-throughput natures have necessi-
tated the use of broad neuroanatomical classifications,
and have preferentially selected for highly expressed
genes. However, genes that are expressed at low le vels,
or in small, discrete regions of the brain, such as the
DMH, are often overlooked.
To address these limitations, we set out to use a neuro-
anatomically guided approach to identify genes that are
highly expressed in the ventral DMH relative to other
hypothalamic nuclei. Laser-capture microdissection
(LCM) was used to isolate the DMH and three adjacent
nuclei: the PVH, ARC, and the dorsal medial portion of the
ventromedial hypothalamus (dmVMH). Gene expression
arrays were used to create preliminary expression pro-
files of each nucleus, and to identify genes that were
expressed preferentially in the DMH relative to the other
nuclei. RNA in situ hybridization histochemistry and real-
time quantitative polymerase chain reaction (qPCR) were
performed on the putative DMH genes to confirm their
expression patterns within the hypothalamus, and
throughout the entire brain. Using this approach, we have
compiled the first compr ehensive description of genes
from the adult DMH.
MATERIALS AND METHODS
Animals and tissue collection
Animal protocols were approved by the Beth Israel
Deaconess Medical Center and the University of Texas
Southwestern Medical Center Animal Care and Use Com-
mittees. Adult male C57Bl/6 mice were housed with ad
libitum access to both food and water in a light (12/12-
hour on/off, 7
AM to 7 PM) and temperature (21.5–
For the microarray expression analysis, C57Bl/6 male
mice (6–7 weeks old, n ¼ 2, Taconic, Farms, German-
town, NY) were fed standard chow ad libitum and sacri-
ficed via intraperitoneal injections of chloral hydrate
PM. Brains were rapidly isolated, imbedded
in OCT compound (Tissue-Tek, Sakura Finetek, Torrance,
CA), frozen on dry ice, and stored at 80
For diet-indu ced obesity studies, 6-week-old C57Bl/6
males (Jackson laboratories, Bar Harbor, ME) were fed ei-
ther a standard maintenance chow (n ¼ 24; Harlan Teklad
TD.7912, Madison, WI) or an HFD with 42% of calories
derived from fat (n ¼ 12; Harlan Teklad TD.88137). Mice
were maintain ed on their respective diets for 12–13
weeks. Half of the mice on standard chow (n ¼ 12) were
fasted for 24 hours prior to being sacrificed. Six-week-old
ob/ob males (n ¼ 24, Jackson laboratories) were fed
standard maintenance chow for 12–13 weeks. Fed mice
were injected with either 100 lg of recombinant mouse
leptin (n ¼ 12; A.F. Parlow, National Hormone and Pep-
tide Program) or saline (n ¼ 12) and sacrificed 45
minutes later. All mice were sacrificed between 7–9
and brains were isolated as described above.
For circadian studies, 10-week-old C57Bl/6 males (n
¼ 6; Jackson Laboratories) were fed maintenance chow
(TD.2916 Teklad Global Diet, Harlan Teklad) and sacri-
ficed at lights on (7
AM) or lights off (7 PM). Animals sacri-
ficed during the dark phase were decapitated in complete
darkness to reduce light-induced gene expression
changes. Brains were isolated as described above.
Brains were cryosectioned at a thickness of 14–30 lm
and thaw-mounted onto either silane-coated glass slides
(Labscientific, Livingston, NJ) or silane-coated PEN mem-
brane glass slides (Applied Biosystems, Foster City, CA)
and stored at 80
C. Slides were lightly fixed in 75%
ethanol immediately prior to thionin staining. Slides were
then dehydrated in a graded ethanol series followed by 5
minutes in xylenes. The Arcturus Autopix (Applied
Lee et al.
3618 The Journal of Comparative Neurology | Research in Systems Neuroscience
Biosystems) and Arcturus Veritas Microdissection System
(Applied Biosystems) were used to isolate the superchias-
matic nucleus (SCN) (0.34 mm to 0.92 mm from
Bregma), retrochiasmatic nucleus (RCN) (0.94 mm to
1.06 mm from Bregma), PVH (0.70 mm to 1.22 mm
from Bregma), dmVMH (1.34 mm to 1.70 mm from
Bregma), vlVMH (1.34 mm to 1.70 mm from Bregma),
ARC (1.46 mm to 1.70 mm from Bregma), and ventral
DMH (1.94 mm from Bregma), as defined by Paxinos
and Franklin (2001). RNA was extracted using a PicoPure
RNA Isolation Kit (Applied Biosystems) with an on-column
DNAseI treatment to remove genomic contamination
(Qiagen, Valencia, CA) and stored at 80
C. RNA was
visualized on the Experion Automated Electrophoresis
system (Bio-Rad, Hercules, CA). The anatomic specificity
of the LCM dissections was confirmed by qPCR for
the expression of known marker genes in each nucleus
Amplification and microarray hybridization
One ng of total RNA underwent two rounds of linear
amplification using the Arcturus RiboAmp HS RNA Ampli-
fication Kit (Molecular Devices, Palo Alt o, CA). RNA tran-
script was labeled with the ENZO BioArray High Yield RNA
Transcript Labeling Kit (Enzo Biochem, New York, NY).
The resulting labeled cRNA was cleaned up via the Affy-
metrix GeneChip Cleanup Module (Affymetrix, Santa
Clara, CA). Samples were quantified using the Agilent
2100 Bioanalyzer (Agilent, Santa Clara, CA). Samples
were hybridized to the GeneCh ip 430 2.0 Array
Microarray data were analyzed using the GeneSpring
GX 7.3 Gene Expression Analysis software (Agilent).
Briefly, linear signals were generated using the GCRMA
summarization algorithm (Wu et al., 2004). To normalize
the values, measurements less than 0.01 were set to
0.01. Each chip was then normalized to the 50th percen-
tile of all measurements in that sample, and each gene
was then normalized to its median value across all sam-
ples. Logarithms of the expression ratios were used as
the basis for statistical analysis and the Cross-Gene Error
Model (CGEM) was applied because of the small sample
numbers. Genes were filtered based on the control
strength as calculated by CGEM. Genes were considered
‘‘DMH-enriched’’ if they were upregulated at least 2-fold
in the DMH compared to the PVH, ARC, and VMH. Twelve
genes showing the highest hybridization signals in the
DMH relative to the other nuclei were selected for further
analysis by RNA in situ hybridization and qPCR expression
Generation of in situ hybridization
histochemistry (ISHH) probes
Probes for RNA in situ hybridization were derived from
PCR fragments amplified with iTaq DNA polymerase (Bio-
Rad) from cDNA generated with the SuperScript III First-
Strand Synthesis System for RT-PCR (Invitrogen, Carls-
bad, CA) from total mouse hypothalamic RNA (BD Bio-
sciences, Palo Alto, CA). The PCR products were cloned
with the TOPO TA Cloning Kit for Sequencing (Invitrogen).
The probe positions and reference GenBank sequences
are as follows: the Gpr50 probe includes positions 1218–
1737 of GenBank accession number NM_010340. The
4930511J11Rik probe corresponds to nucleotide posi-
tions 784–1354 of GenBank accession number
BC027071. The Pcsk5 probe span s nucleoti des 2314–
2743 of GenBank accession number BC013068. The Grp
probe contains nucleotides 17–456 of GenBank acces-
sion number NM_175012. The Sulf1 probe is designed
from positions 3932–4547 of GenBank accession number
NM_172294. The Rorb probe spans 877–1381 of Gen-
Bank accession number NM_146095.1. Antisense and
S-labeled probes were generated with MAXI-
script In Vitro Transcription Kits (Ambion, Austin, TX).
ISHH of coronal sections through the entire mouse
brain were performed as previously described (Marcus
et al., 2001; Kishi et al., 2003; Zigman et al., 2006).
Briefly, 6–8-week-old male C57Bl/6 mice (n ¼ 3, Taconic
Farms) were intracardially perfused with 10% formalin
PM and brains were sectioned, then
mounted onto SuperFrost slides (Fisher Scientific, Pitts-
burgh, PA). Sections were hybridized with either sense or
S-labeled probes. ISHH signal was visualized
first on autoradiographic film, then on slides dipped in
Kodak NTB Autoradiography Emulsion (Carestream
Health, Rochester, NY) and expos ed for 1–3 weeks,
developed in Kodak Dektol Black & White Paper Devel-
oper (Eastman Kodak, Rochester, NY), and counter-
stained with thionin. The hybridization signal was esti-
mated subjectively. Control procedures included
hybridization with sense probes and tissue pretreatment
with RNase A (200 lg/ml). No specific hybridization was
observed following either procedure.
Nine-week-old C57Bl/6 males (n ¼ 3, Taconic Farms)
were fasted overnight and then intraperitoneally injected
with 100 lg of recombinant mouse leptin (A.F. Parlow,
National Hormone and Peptide Program) or phosphate-
buffered saline (PBS) and perfused with 10% formalin 45
Dmh-enriched genes identified by LCM
The Journal of Comparative Neurology | Research in Systems Neuroscience 3619
minutes later. Free-floating brain sections were proc-
essed sequentially by ISHH for the DMH-enriched genes
and IHC for phosphorylated STAT3 (p-STAT3) as previ-
ously described (Liu et al., 2003; Zigman et al., 2006).
Briefly, after ISHH tissues were incubated overnight at
C in primary rabbit p-STAT3 antiserum (Cell Signaling
Technology, Danvers, MA). Next, tissues were incubated
with a biotinylated donkey antirabbit secondary (1:1,000;
Jackson ImmunoResearch Laboratories, West Grove PA)
for 1 hour at room temperature, followed by a 1-hour
incubation in avidin-biotin complex from the Vectastain
Elite ABC Kit (Vector Laboratories, Burlingame, CA,
1:500). The sections were then incubated in a solution of
0.04% diaminobenzidine tetrahydrochloride (DAB; Sigma-
Aldrich, St. Lou is, MO). Sections were mounted onto
SuperFrost slides, visualized with x-ray film, followed by
photographic emulsion, as described above.
The p-STAT3 antisera used in this study is commer-
cially available and has been tested and reported for use
in IHC (Scott et al., 2009). The key features are summar-
ized in Table 1. The antiserum recognizes a single band
with the predicted molecular weight on western blots of
cell extracts from SK-N-MC neuroblastoma cells (manu-
facturer’s datasheet). The antibody specificity was tested
by enzyme-linked immunosorbent assay (ELISA), as well
as by reabsorption of the antiserum with a synthetic
phospho-STAT3 peptide (Scott et al., 2009). Furthermore,
in these experiments p-STAT3 immunoreactivity was only
detected in the brains of animals treated with leptin, and
not in saline controls (data not shown).
Production of photomicrographs
Images were obtained with a Carl Zeiss Axioskop 2
microscope and a Zeiss Stemi 2000-C dissecting micro-
scope using both brightfield and darkfield optics. Images
were captured using a Zeiss digital camera and the Axio-
vision 3.1 software. Adobe PhotoShop CS2 (San Jose, CA)
was used to combine the images into plates, make minor
adjustments to contrast and brightness, and remove any
obvious dust from the darkfield images.
Quantitative PCR (qPCR)
All gene expression levels were measured with an
Applied Biosystems 7900HT Sequence Detection System
as previously described (Bookout et al., 2006a). Real-
time qPCR gene expression analysis was performed using
inventoried TaqMan Gene Expression Assays (Applied
Biosystems) and previously published gene assays (Fu
et al., 2005; Bookout et al., 2006b).
Anatomic specificity of the LCM-isolated nuclei was
confirmed by testing for the expression of the following
marker genes in each nuclei by qPCR, SCN: vasoactive in-
testinal peptide receptor 2 (Vipr2; ABI, Cat. no.
Mm00437316_m1), leptin receptor (Lepr; ABI, Cat. no.
Mm00440181_m1); RCN: Lepr; ARC: Neuropeptide Y
(Npy; ABI, Cat. no. Mm00445771_m1), proopiomelano-
cortin (Pomc; ABI, Cat. no. Mm00435874_m1), Lepr;
PVH: single-minded (Sim1; ABI, Cat. no.
Mm00441390_m1), arginine vasopressin (Avp; ABI, Cat.
no. Mm00437761_g1), Lepr; dmVMH and vlVMH: steroi-
dogenic factor 1 (Sf-1) (Fu et al., 2005; Bookout et al.,
2006b), estrogen receptor a (Esr1) (Fu et al., 2005),
estrogen receptor b (Esr2) (Fu et al., 2005), Lepr; DMH:
cocaine- and amphetamine-regulated transcript (Cart;
ABI, Cat. no. Mm00489086_m1), neurotensin (Nts; ABI,
Cat. no. Mm00481140_m1), Lepr. The expression of
marker genes were assayed in the individual nuclei sam-
ples and samples that did not show the expe cted pat-
terns of marker gene expression were excluded from fur-
Because of the low levels of starting material, 0.5 ng of
RNA from each isolated hypothalamic nuclei was converted
to cDNA using the High Capacity Reverse Transcription Kit
(Applied Biosystems) and subjected to 14 rounds of pream-
plification using the TaqMan Preamp mastermix (Applied
Biosystems) using a cocktail of the marker genes listed
above, as well as DMH-specific genes identified by microar-
ray analysis: Gpr50 (ABI, Cat. no. Mm00439147_m1),
4930511J11Rik (ABI, Cat. no. Mm00512483_m1), Pcsk5
(ABI, Cat. no. Mm01206138_m1), Grp (ABI, Cat. no.
Mm00612977_m1), Sulf1 (ABI, Cat. no.
Mm00552283_m1), Rorb (Fu et al., 2005). Primers for the
normalizer gene 18S were not included in the preamplifica-
tion assay mix. Prior to this study, the amplicons were
tested to confirm unbiased, uniform amplification. Preampli-
fied products were diluted 1/20 and PCR-amplified for 50
cycles with TaqMan Gene Expression Master Mix (Applied
Biosystems) with a final concentration of 900 mM of Taq-
Man Gene Expression Assays (Applied Biosystems). Analysis
of gene expression was performed using the TaqMan-based
Antigen Immunogen Manufacturer Dilution used
p-STAT3 Synthetic peptide,
from mouse ADPGSAAPyLKTKFIC
Cell Signaling Technologies (Danvers, MA)
cat. no. 9131L lot 9
Lee et al.
3620 The Journal of Comparative Neurology | Research in Systems Neuroscience
efficiency-corrected DCt assay. mRNAs with cycle times
30 cycles were considered below detection.
qPCR data were analyzed using ABI instrument soft-
ware SDS2.1. Baseline values of amplification plots were
set automatically and threshold values were kept con-
stant to obtain normalized cycle times and linear regres-
sion data. For each sample, normalized mRNA levels were
expressed as arbitrary units and were obtained by divid-
ing the averaged, efficiency-corrected values for each
gene by that for 18S RNA. The resulting values were mul-
tiplied by 10
for graphical representation and plotted 6
standard deviation from triplicate sample wells. Fold
changes were calculated by dividing normalized signal
intensities for fasted and HFD conditions by those
obtained under standard chow. Fold changes of less than
0.8 or greater than 1.2 were considered physiologically
LCM and microarray analysis
The DMH can be divided into two anatomically and
functionally distinct parts, the compact DMH, and the lep-
tin receptor-rich dorsal and ventral portions (Elmquist
et al., 1997, 1998a; Elias et al., 2000). To compare gene
expression profiles between the leptin receptor express-
ing portion of the DMH and the adjacent metabolically
important regions of the hypothalamus, LCM was used to
isolate RNA from the PVH, dmVMH, ARC, and ventral
DMH (Fig. 1). Hypothalamic nuclei were collected from
two male C57Bl/6 mice fed on standard chow ad libitum.
Unpooled RNA from each nuclei was subjected to two
rounds of amplification, biotin-labeled, and hybridized to
the Affymetrix GeneChip 430 2.0 Array, consisting of over
To confirm the specificity of the dissectio ns and the fi-
delity of the RNA amplification, we first screened the
data generated from the microarrays for genes known to
be specifically or preferentially expressed in each of the
dissected nuclei. We confirmed the exclusive and robust
expression of Npy, Pomc, and Agrp in the ARC (Elmquist
et al., 1998c); Sf-1 and Cbln1 in the dmVMH (Segal
et al., 2005); Sim1 in the PVH (Michaud et al., 1998);
and the preferential expression of orexin (Ox) (Chou
et al., 2001) and neurotensin (Nts) (Watts et al., 1999) in
the DMH as compared to the other nuclei examined
(data not shown).
After normalization, 67 genes were found to be
expressed at least 2-fold above background levels in the
DMH. Many of the 67 genes were also coexpressed at
high levels in the ARC. To restrict the population to DMH-
specific genes, we selected genes that were expressed at
least 2-fold higher in the DMH as compared to the PVH,
dmVMH, and ARC. We found that 36 genes met this initial
criteria for DMH enrichment. Next, we removed genes
that were previously described to be strongly expressed
in regions adjacent to the DMH, but which were restricted
to only a few cells within the ventral DMH; these included
melanin-concentrating hormone (Mch) (Nahon et al.,
1989) and Ox (de Lecea et al., 1998; Chou et al., 2001),
which are predominantly expressed in the lateral hypo-
thalamic area (LHA), and histidine decarboxylase (Hdc)
(Castren and Panula, 1990), which is expressed in the
medial tuberal nucleus (MTu). These exclusions produced
a list of 33 genes preferentially expressed in the DMH
and 14 of these were selected for further analysis by
ISHH and/or qPCR (Table 2). We then eliminated those
genes whose expression could not be confirmed by both
in situ hybridization and qPCR, or which were found to
have high levels of expression outside of the DMH. Using
these criteria, we selected six genes: G-protein-coupled
receptor 50 (Gpr50), clone 4930511J11Rik, proprotein
convertase subtilisin/kexin type 5 (Pcsk5), gastrin releas-
ing peptide (Grp), sulfatase 1 ( Sulf1), and retinoid-related
orphan nuclear receptor b (Rorb) for further expression
analysis (Fig. 2). RNA in situ hybridization showed the
Gpr50 and 4930511J11Rik were most strongly expressed
in the DMH, with limited expression in other brain sites.
Pcsk5, Grp, Sulf1, and Rorb; were moderately expressed
in the DMH and also showed restricted expression in
other sites. The gene distribution patterns within the
hypothalamus were confirmed and quantified using real-
time qPCR on additional LCM samples taken from
broader sampling of hypothalamic nuclei, including the
RCN, SCN, PVH, dmVMH, ventrolateral portion of the ven-
tromedial hypothalamus (vlVMH), ARC, and DMH (Fig. 3).
qPCR analysis allowed us to determine the relative
expression levels of these genes across the hypothala-
mus and confirmed the robust expression of all six genes
within the DMH. Furthermore, our qPCR findings also con-
firmed the previously described expression of Grp (Aida
et al., 2002) and Rorb (Schaeren-Wiemers et al., 1997;
Andre et al., 1998; Sumi et al., 2002) in the SCN.
Characterization of DMH-enriched genes
To further map the distribution of these genes within
the DMH, we subdivided the DMH into rostral, central,
and caudal levels, and within each level, into dorsal, ven-
tral, and compact regions based on previously described
distributions of leptin-responsive cells (Elias et al., 2000).
To visualize the distribution of the six DMH-enriched
genes relative to leptin responsive cells we performed
dual-label ISHH for the genes of interest with IHC for p-
STAT3, a protein downstream of leptin receptor that is
commonly used as a marker of leptin signaling (Figs. 4, 5)
(Vaisse et al., 1996). All of the genes examined were
Dmh-enriched genes identified by LCM
The Journal of Comparative Neurology | Research in Systems Neuroscience 3621
Figure 1. Isolation of specific hypothalamic nuclei from mouse brain by laser-capture microdissection. Left: Hypothalamic sections stained
with thionin allow visualization of the neuroanatomical subdivisions. Right: The remainder of the hypothalamus after selective capture and
removal of the cells of interest. 3V, third ventricle; Arc, arcuate nucleus; dmVMH, dorsomedial ventromedial hypothalamic nucleus; PVH,
paraventricular nucleus; vlVMH, ventrolateral ventromedial hypothalamic nucleus. Scale bar ¼ 200 lm.
Lee et al.
3622 The Journal of Comparative Neurology | Research in Systems Neuroscience
expressed throughout the rostral, central, and caudal lev-
els of the DMH. Interestingly, Gpr50, 4930511J11Rik,
Pcsk5, and Sulf1 were distributed in a pattern similar to
those seen for leptin responsive cells, with greatest
expression in the dorsal and ventral portions of the DMH,
and limited and scattered expression within the compact
DMH. Despite this general similarity, there were only lim-
ited occurrences of colocalization between the RNA in
situ silver grains and cells immunopositive for p-STAT3
(Figs. 4, 5). In contrast, while silver grains representing
Grp mRNA were observed throughout the DMH, they
were concentrated in the rostral and compact DMH, in
regions that were devoid of leptin responsive cells. While
Rorb had the most extensive distribution of silver grains
throughout the brain, with especially robust signal within
the DMH, there was only moderate colocalization with
neurons immunopositive for p-STAT3. Genes expressed
primarily in nonneuronal cells would also show limited
colocalization with p-STAT3 immunostaining. However,
careful examination of dual-label ISHH/IHC sections
showed silver grain deposition patterns consistent with
that seen for neurons, with no histological evidence of
gene expression in glial cells (Fig. 4E,F).
The DMH has been shown to innervate the ARC and
PVH (Gautron et al., 2010), sites critical for the control of
energy balance, glucose homeostasis, and food intake
(Balthasar et al., 2005; Coppari et al., 2005). To assess
the potential role of the DMH-enriched genes on metabo-
lism we performed real-time qPCR assays from LCM dis-
sected samples to assess gene expression levels in mice
fed a standard maintenance chow, a high-fat and high-
cholesterol diet (HFD), or mice that underwent an
extended fast for 24 hours (Fig. 6). Of the genes assayed,
moderate and statistically significant changes in
response to fasting were seen in the expression of Gpr50
(0.8-fold), 4930511J11Rik (1.2-fold), and Grp (0.6-fold).
Sulf1 showed moderate upregulation (1.4-fold) in
response to HFD. LCM dissected samples obtained from
leptin-deficient, ob/ob mice did not show significant
changes in gene expression between those that were in-
traperitoneally injected with leptin and those treated with
saline (data not shown).
Gene symbol Gene name Accession no. Normalized intensity
G-protein-coupled receptor 50 NM_010340 28.17
sulfatase 1 NM_172294 8.10
RIKEN cDNA 4930511J11 gene BC027071 7.29
gastrin releasing peptide NM_175012 5.52
RIKEN cDNA 4930547N16 gene XM_125861 4.67
Lypd6* LY6/PLAUR domain containing 6 NM_177139 4.55
G protein-coupled receptor 64 NM_178712 4.29
proprotein convertase subtilisin/kexin type 5 BC013068 3.58
Neuropilin 1 NM_008737 3.24
Synpr* synaptoporin NM_028052 3.00
Trhr* thyrotropin releasing hormone receptor NM_013696 2.81
RAR-related orphan receptor beta NM_146095 2.80
2310010M24Rik RIKEN cDNA 2310010M24 gene NM_027990 2.76
Cbln2 cerebellin 2 precursor protein NM_172633 2.68
Met met proto-oncogene NM_008591 2.68
Erbb4 v-erb-a erythroblastic leukemia viral oncogene homolog 4 NM_010154 2.55
Rgs16* regulator of G-protein signaling 16 NM_011267 2.54
Ccnd2 cyclin D2 NM_009829 2.47
zinc finger, MIZ-type containing 1 NM_183208 2.34
4921525O09Rik RIKEN cDNA 4921525O09 gene AV312506 2.31
3110001A13Rik RIKEN cDNA 3110001A13 gene NM_025626 2.31
Transcribed locus AV133559 2.27
Dlx6os2 Dlx6 opposite strand transcript 2 BB023120 2.25
C130034I18Rik RIKEN cDNA C130034I18 gene NM_177233 2.23
Cachd1 cache domain containing 1 NM_198037 2.22
Galr1 galanin receptor 1 NM_008082 2.21
Nts neurotensin NM_024435 2.21
Lbxcor1 ladybird homeobox 1 homolog corepressor 1 NM_172446 2.15
kin of IRRE like 3 NM_026324 2.13
EG226654 predicted gene, EG226654 XM_129558 2.10
hect domain and RLD 4 NM_026101 2.09
Ankrd38 ankyrin repeat domain 38 NM_172872 2.08
Vwf Von Willebrand factor homolog NM_011708 2.08
Dmh-enriched genes identified by LCM
The Journal of Comparative Neurology | Research in Systems Neuroscience 3623
A number of the newly identified genes have been
shown to be expressed in a circadian manner in other tis-
sues. For instance, Gpr50 expression oscillates in tany-
cytes lining the third ventricle (Kamphuis et al., 2005),
Rorb in retina and peripheral white adipose tissue (Kam-
phuis et al., 2005; Yang et al., 2006), and Grp in the SCN
(Zoeller et al., 1992). Despite these reports and the inner-
vations between the DMH and the SCN and SPZ, we did
not detect any day–night expression differences in the six
DMH-enriched genes (data not shown).
Historically, hypothalamic gene expression studies
were performed on samples from homogenized whole
hypothalamus or from tissue punches of the approximate
Figure 2. A series of low-power darkfield photomicrographs summarizing the distribution of the candidate genes in the mouse brain. Arc,
arcuate nucleus; BMP, posterior basomedial amygdaloid nucleus; CA3, field CA3 of hippocampus; chp, choroid plexus; Cx, cortex; DEn,
dorsal endopiriform nucleus; DG, dentate gyrus; DMH, dorsal medial hypothalamus; MD, mediodorsal thalamus; ME, median eminence;
MePV, posteroventral medial amygdaloid nucleus; Pir, piriform cortex; PV, paraventricular thalamic nuclei; STh, subthalamic nucleus; Th,
thalamus; VEn, ventral endopiriform nucleus. Scale bar ¼ 2 mm.
Lee et al.
3624 The Journal of Comparative Neurology | Research in Systems Neuroscience
region of interest. These methods were imprecise and
limited to hypothalamic regions that could be grossly
identified. Using LCM, we are able to obtain pure, nuclei
specific samples with minimal cross-contamination from
adjacent tissues. This is particularly important because of
the spatial and functional organization within the hypo-
thalamus and the sensitivity of the microarray and qPCR
expression assays that we used.
We identified six genes that are strongly enriched in
the ventral portion of the DMH relative to other areas of
the hypothalamus. Five of these genes (Gpr50, Pcsk5,
Grp, Sulf1, and Rorb) can be seen to be expressed in the
DMH in the Allen Mouse Brain Atlas (Lein et al., 2007)
when sections at the level of the DMH are individually
analyzed, but the overall expression levels were too
restricted to be included on their annotated list of hypo-
Other groups have also used microarray analysis to
investigate the molecular makeup of the DMH. Draper
et al. (2010) examined differential gene expression
between NPY neurons in the DMH and the ARC in juvenile
mice. While they identified 41 genes that were preferen-
tially expressed in the DMH, there was no overlap with
the genes from our assay. These differences are most
likely due to differences between juvenile and adult mice,
and between isolated NPY neurons and the heterogene-
ous population of cells found in the ventral DMH.
While we initially predicted that genes highly expressed
in the DMH would be strongly regulated in response to di-
etary manipulations, we detected modest changes in
gene expression in only four of the genes. It is possible
that the technical limitations of profiling gene expression
from minute cell populations, which requires cDNA ampli-
fication, may mask or suppress differences in gene
expression. However, our initial tests suggest that the
expression levels of most genes are faithfully and repeat-
edly assayed after amplification. It is also possible that
these genes are important for DMH-associated physiolog-
ical processes other than metabolism, such as reproduc-
tion, body temperature, or corticosterone secretion.
While p-STAT3 immunoreactivity is commonly used to
identify leptin-responsive neurons, it has recently been
shown that the assessment of p-STAT3 immunoreactivity
shortly after leptin administration may underrepresent
the total number of leptin-responsive cells in certain
areas of the brain, including the DMH (Scott et al., 2009).
Thus, while we found a limited degree of colocalization
between our DMH-specific genes and p-STAT3 immunore-
activity, a more detailed study following prolonged lep tin
treatment, or using one of the newly described LepRb re-
porter mice may be necessary (Scott et al., 2009). Alter-
natively, it is possible that the DMH-enriched genes
respond to HFD and fasting through leptin-independent
pathways. It has recently been shown that NPY neurons
Figure 3. Relative distribution of candidate genes across the hypothalamus. Graphs are representative of duplicate experiments. Values
are plotted as the mean of triplicate measurements 6 standard deviation error bars. Arc, Arcuate nucleus; DMH, dorsomedial hypothala-
mus; dmVMH, dorsomedial ventromedial hypothalamic nucleus; PVH, paraventricular nucleus; RCN, retrochiasmatic nucleus; SCN, supra-
chiasmatic nucleus; vlVMH, ventrolateral ventromedial hypothalamic nucleus.
Dmh-enriched genes identified by LCM
The Journal of Comparative Neurology | Research in Systems Neuroscience 3625
Figure 4. Distribution of p-STAT3 immunoreactivity and Grp50 and 4930511J11Rik mRNA in the DMH. A,C,E: Gpr50 ISHH with p-STAT3
IHCC. B,D,F: 4930511J11Rik ISHH with p-STAT3 IHCC. mRNA hybridization is represented by silver grains, while p-STAT3 immunoreactivity
is represented by brown staining. A,B: Darkfield image. C–F: Brightfield images. The DMH regions of C,D are magnified in E,F. Black arrows
indicate examples of neurons doubly labeled with either Gpr50 or 4930511J11Rik riboprobe and anti-p-STAT3 antisera. Scale bars ¼ 100
Lee et al.
3626 The Journal of Comparative Neurology | Research in Systems Neuroscience
of the DMH are not leptin-responsive (Draper et al.,
2010), suggesting that there is indeed a leptin-insensitive
population of DMH neurons that responds to metabolic
Food entrainment or food anticipatory activity
describes the range of physiological behaviors that are
associated with expected mealtimes (Bolles and Stokes,
1965; Krieger, 1974; Herzog and Muglia, 2006). In
rodents, this includes increased wakefulness, activity
such as running, eating, licking, body temperature,
hormone secretion, digestive activity, and hepatic gene
transcription. Furthermore, these anticipatory behaviors
persist during prolonged fasts (Boulos et al., 1980). The
existence of a food entrainable oscillator, similar to the
light entrainable oscillator located in the SCN, has been
postulated since the late 1970s (Krieger et al., 1977). It
is unclear whether this oscillator exists as a unique clus-
ter of cells within the DMH, is distributed among various
hypothalamic nuclei and/or the brainstem (Mistlberger,
2011), or if it requires a functional interaction between
the SCN and DMH (Acosta-Galvan et al., 2011). It is in-
triguing to note that Grp and Rorb are also expressed in
the SCN, while Grp, Gpr50, and Rorb have been reported
to have oscillating patterns of expression in other tissues
(Zoeller et al., 1992; Kamphuis et al., 2005; Yang et al.,
2006). While our preliminary analyses did not reveal
Figure 5. Camera lucida drawings of dual-label ISHH/IHC showing the relative distribution of p-STAT3 and the DMH-enriched genes.
Drawings were made from representative sections from three rostral-caudal levels of the DMH, and only include signals detected within
the DMH. Sections are organized from rostral (left) to caudal (right). Each circle represents one neuron. White circles, neurons with silver
grains (ISHH); black circles, neurons with DAB (p-STAT3 IHC), red circles, double stained. Arc, Arcuate nucleus; 3V, third ventricle; ARC,
arcuate nucleus; DMHc, compact subdivision of the DMH, DMHd, dorsal subdivision of the DMH, DMHv, ventral subdivision of the DMH;
VMH, ventromedial nucleus.
Dmh-enriched genes identified by LCM
The Journal of Comparative Neurology | Research in Systems Neuroscience 3627
circadian expression in the DMH, these genes remain in-
triguing candidates for mediators of circadian and meta-
Gpr50 was initially cloned from the pituitary but has
since been shown to be expressed in the DMH and tany-
cytes lining the third ventricle (Reppert et al., 1996; Drew
et al., 1998, 2001; Sidibe et al., 2010). Despite high
sequence similarity to the melatonin receptor family,
Gpr50 does not bind to radiolabeled melatonin (Reppert
et al., 1996); however, recent work has shown that it
does form heterodimers with the melatonin receptor MT1
to reduce the activity of MT1 itself (Levoye et al., 2006).
Furthermore, Gpr50 expression is decreased in short-day
photoperiods (Barrett et al., 2006). Interestingly,
sequence variants in humans have been associated with
circulating triglyceride levels, high-density lipoprotein
(HDL) levels, bipolar affective disorder, and autism
(Thomson et al., 2005; Bhattacharya et al., 2006). Most
recently, a genetically modified mouse lacking Gpr50 was
shown to have decreased body weight accompanied by
increased activity and basal metabolic rate (Ivanova
et al., 2008). The authors examined the regulation of
Gpr50 expression levels using radioactive RNA in situ
hybridization, and found that Gpr50 was strongly
decreased by a 36-hour fast, or after a 5-week exposure
to a high-calorie diet. In our experiments, we found Gpr50
expression in the DMH to be only moderately regulated
by fasting, but did not show regulation in response to
HFD. These differences could be due to die t formulation,
sample collection, or the assay methodology. It should
also be noted that we also detected very low levels of
Gpr50 expression in the RCN and ARC, two metabolically
significant regions and thus potentially important sites of
action for Gpr50.
Clone 4930511J11Rik has recently been identified by
sequence homology as claudin 26, a member of the clau-
din protein family that is important for tight junction for-
mation (Mineta et al., 2011). Claudin 26 was shown to be
expressed in mouse embryo and in adult brain and intes-
tine. In the brain, claudins are known to be important for
the maintenance of the blood–brain barrier, and more
recent work suggests that they may have broader func-
tions, such as stress response and embryonic morpho-
genesis (Cardoso et al., 2010). The expression of
4930511J11Rik/claudin 26 in the DMH is puzzling, but
may reflect a role for these proteins outside of tight junc-
tions. Indeed, the expression of several claudins have
recently detected in the oligodendrocytes, neurons, and
astrocytes of the cortex in humans (Romanitan et al.,
Pcsk5 is a proprotein convertase involved in the proc-
essing of secretory proteins (Lissitzky et al., 2000). Pcsk5
has been previously shown to be expressed in the rodent
brain, where it is speculated to be involved in processing
of the neuropeptides, including cholecystokinin and
Figure 6. Metabolic regulation of candidate genes. Values are plotted as the mean of triplicate measurements 6 standard deviation error
bars. Fold change is compared to standard chow, expressed as 1. Only values less than 0.8 or greater than 1.2 fold change are reported
as significant. ***P < 0.001.
Lee et al.
3628 The Journal of Comparative Neurology | Research in Systems Neuroscience
neurotensin (Dong et al., 1995; Villeneuve et al., 1999;
Villeneuve et al., 2000; Cain et al., 2003). Pcsk5 is also
essential for development, and deletion of the catalytic
subunit is embryonic lethal at the implantation stage
(Essalmani et al., 2006, 2008). Intriguingly, variants have
been associated with HDL cholesterol levels in humans
(Iatan et al., 2009), although this is thought to be most
likely due to an effect on endothelial lipase activity rather
than a central effect of Pcsk5.
Grp is a bombesin-like peptide known for stimulating
the release of gastrin and inhibiting feeding. It is
expressed throughout the brain and periphery, especially
in the gastrointestinal tract (Aida et al., 2002; Ohki-Hama-
zaki et al., 2005; Gonzalez et al., 2008). Its receptor, Grp-
R, is expressed peripherally, as well as in the brain, with
strong expression in the hypoth alamus. Grp and its recep-
tor are involved in numerous physiological processes,
including, smooth-muscle contraction in the gastrointesti-
nal/urogenital tract, immune function, insulin secretion,
cancer, regulation of circadian rhythms, thermoregula-
tion, behavior, and satiety (reviewed in Gonzalez et al.,
2008). In fact, Grp can directly excite NPY neurons in
electrophysiological slice preparations (van den Pol et al.,
2009). While a Grp mouse has not been yet reported, sev-
eral groups have produced Grpr-deficient mice. The loss
of the receptor caused behavioral changes, including
increased locomotor activity and social responses, and
eliminated the effects of bombesin administration on
feeding suppression (Wada et al., 1997; Hampton et al.,
1998). Grp also plays a crucial role in the photic signaling
cascade in the SCN (Piggins et al., 1995; Albers et al.,
1995; Aida et al., 2002; Karatsoreos et al., 2004). Grp
cells within the SCN receive direct retinal input via the
retinohypothalamic tract, and microinjections of exoge-
nous Grp into the SCN can cause a phase shift of the cir-
Rorb is a member of the nuclear receptor family of
transcription factors (Carlberg et al., 1994). It is highly
expressed in the br ain, especially in the regions involved
in sensory processing, such as the cortex, thalamus; and
circadian timing, such as the pineal gland, retina, and the
SCN, where it oscillates with a diurnal rhythm (Schaeren-
Wiemers et al., 1997; Andre et al., 1998; Sumi et al.,
2002). Furthermore, Rorb-deficient mice are ataxic, with
decreased fertility and retinal degeneration (Andre et al.,
1998; Masana et al., 2007). Despite the retinal degenera-
tion, these mice still respond and entrain to a light-dark
cycle, but show an altered free-running period during the
Sulf1 is a heparin sulfate 6-O-endosulfatase and modi-
fies heparin sulfate proteoglycans (HSPGs) at the cell sur-
face to regulate signaling molecules, such as fibroblast
growth factor, bone morphogenic protein (BMP), wingless
(WNT), Decapentaplegic (Dpp), and hedgehog (Hh) (Perri-
mon and Bernfield, 2000; Morimoto-Tomita et al., 2002;
Ai et al., 2003; Hacker et al., 2005; Lamanna et al.,
2007). Sulf1-deficient mice have defects in neurite out-
growth, behavioral problems, and reduced spine density
in the hippocampus (Kalus et al., 2009). The loss of either
Sulf1 or the closely related Sulf2 disrupts cartilage home-
ostasis through the BMP and fibroblast growth factor
(FGF) pathways (Otsuki et al., 2010). Mice that are miss-
ing both Sulf1 and Sulf2 show increased neonatal lethal-
ity, accompanied by skeletal and renal def ects (Holst
et al., 2007). Sulf1 and Sulf2 also appear to function to-
gether for proper esophageal innervation (Ai et al., 2007).
We described six genes that are highly expressed
within the DMH. Of these genes, Gpr50, 4930511J11R ik,
Grp, and Sulf1 respond to basic metabolic manipulations,
but none of them exhibit diurnal fluctuations. Future
experiments that include gene targeting will be required
to define the role of these genes in the DMH. In addition
to extending our knowledge about the molecular makeup
of the DMH, the identification of these and other DMH-
specific genes will undoubtedly add to the molecular
tools available for studying this elusive part of the hypo-
thalamus, including the development of reporter con-
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