molecular characterization of the subnuclei in rat habenula

Title:

Molecular characterization of the subnuclei in rat habenula

Authors and author addresses:

Hidenori Aizawa1,2, Megumi Kobayashi1, Sayaka Tanaka3, Tomoki Fukai3,4 and Hitoshi

Okamoto1,4

1 Laboratory for Developmental Gene Regulation, RIKEN Brain Science Institute, 2-1

Hirosawa, Wako, Saitama 351-0198, Japan

2 Department of Molecular Neuroscience, Medical Research Institute, Tokyo Medical and

Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510 Japan

3 Laboratory for Neural Circuit Theory, RIKEN Brain Science Institute, 2-1 Hirosawa, Wako,

Saitama 351-0198, Japan

4 Core Research for Evolutional Science and Technology, Japan Science and Technology

Agency, Sanbancho Bldg., 5, Sanbancho, Chiyoda-ku, Tokyo 102-0075, Japan

Abbreviated title:

Gene expression in the rat habenula subnuclei

Associate Editor to whom the manuscript is being submitted:

John L. R. Rubenstein, University of California–San Francisco

Keywords:

Habenula, Interpeduncular nucleus, Monoamines, Acetylcholine, Glutamate

Corresponding author:

Research Article The Journal of Comparative NeurologyResearch in Systems Neuroscience

DOI 10.1002/cne.23167

© 2012 Wiley-Liss, Inc.Received: Dec 29, 2011; Revised: Jun 05, 2012; Accepted: Jun 06, 2012

This article has been accepted for publication and undergone full peer review but has not beenthrough the copyediting, typesetting, pagination and proofreading process which may lead todifferences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1002/cne.23167

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Hitoshi Okamoto, [email protected] (E-mail), +81 48 467 9712 (phone), +81 48 467 9714

(fax)

Any conflict of interest:

The authors declare no conflict of interest.

Authors contributions: to the work

HA and HO designed the experiments and wrote the manuscript. HA performed most of the

experiments with MK and ST and analyzed data with TF.

Grant information:

This research was supported by grants from RIKEN BSI, Core Research for Evolutional

Science and Technology of Japan Science and Technology Agency, and a Grant-in-Aid for

Scientific Research on Priority Areas (KAKENHI20021034), Scientific Research on

Innovative Areas (KAKENHI21115521) and Scientific Research (B) (KAKENHI19300115)

from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) to HO and

by Grant-in-Aid for Young Scientists (B) (KAKENHI24700350) from MEXT to HA.

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Abstract

The mammalian habenula is involved in regulating the activities of serotonergic and

dopaminergic neurons. It consists of the medial and lateral habenulae, with each subregion having

distinct neural connectivity. Despite the functional significance, manipulating neural activity in a

subset of habenular pathways remains difficult because of the poor availability of molecular

markers that delineate the subnuclear structures. Thus, we examined the molecular nature of

neurons in the habenular subnuclei by analyzing the gene expressions of neurotransmitter markers.

The results showed that different subregions of the medial habenula (MHb) use different

combinations of neurotransmitter systems and could be categorized as either exclusively

glutamatergic (superior part of MHb), both substance P-ergic and glutamatergic (dorsal region of

central part of MHb), or both cholinergic and glutamatergic (inferior part, ventral region of central

part, and lateral part of MHb). The superior part of the MHb strongly expressed interleukin-18 and

was innervated by noradrenergic fibers. In contrast, the inferior part, ventral region of the central

part, and lateral part of the MHb were peculiar in that acetylcholine and glutamate were

cotransmitted from the axonal terminals. On the other hand, neurons in the lateral habenula (LHb)

were almost uniformly glutamatergic. Finally, the expressions of Htr2c and Drd2 seemed

complementary in the medial LHb division, while they coincided in the lateral division, suggesting

that the medial and lateral divisions of LHb show strong heterogeneity with respect to monoamine

receptor expression. These analyses clarify molecular differences between subnuclei in the

mammalian habenula that support their respective functional implications.

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Introduction

Habenula is an epithalamic structure comprising the medial and lateral habenulae in mammals, with

each having distinct neural connectivity. Specifically, the medial habenula (MHb) mainly projects

to the interpeduncular nucleus in the midbrain, while the lateral habenula (LHb) projects to diverse

structures in the midbrain and hindbrain including the raphe nuclei, substantia nigra, ventral

tegmental area (Herkenham and Nauta, 1979), rostromedial tegmental nucleus (Jhou et al., 2009;

Kaufling et al., 2009), and nucleus incertus (Goto et al., 2001; Olucha-Bordonau et al., 2003). We

recently showed that these two distinct neural circuits are highly conserved from fish to mammals

by identifying the fish brain regions homologous to the mammalian medial and lateral habenulae

(Aizawa et al., 2005; Amo et al., 2010).

Previous studies revealed that the LHb acts as a regulatory center for the dopaminergic and

serotonergic activities in the central nervous system, since stimulation of the LHb results in the

inhibition of monoaminergic activities (Christoph et al., 1986; Wang and Aghajanian, 1977). More

recently, a series of studies using behaving monkeys revealed the LHb neurons acting in an opposite

way to the dopaminergic neurons when the animal experienced the conditioned stimuli predicting

the aversive unconditioned stimuli or in the absence of the conditioned stimuli predicting reward

(Matsumoto and Hikosaka, 2007; Matsumoto and Hikosaka, 2009). These results implicated the

LHb as a possible source of negative reward signal.

Habenula consists of a heterogeneous population of neurons and was previously subdivided

into 15 subnuclei according to their morphological features such as cell density and cellular

morphology as well as their immunoreactivities against neuropils and cell bodies (Andres et al.,

1999; Geisler et al., 2003). Despite a recent surge of interest in the role of the habenula in cognitive

behaviors (Hikosaka, 2010), genetic manipulation of a subpopulation of the habenular neurons

remains difficult due to the paucity of molecular markers to delineate the habenular subnuclei. To

overcome this difficulty, the molecular nature of the habenular neurons should be defined based on

mRNA rather than protein expression, since localization of mRNA in the perikaryal region of

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neuronal cell bodies enables us to determine the molecular nature of the neurons in the habenula. In

contrast, proteins could be expressed in the afferent axons from other brain regions, and thus not

necessarily reflect the molecular nature of the habenular neurons.

The present study therefore sought to further characterize the rat habenula subnuclear

organization defined in a previous study (Andres et al., 1999) by examining the combinatorial

expression of marker genes for known neurotransmitters such as substance P, acetylcholine, and

glutamate. We also focused on the neurotransmitter markers for incoming fibers from the brain stem

such as dopaminergic, serotonergic, and noradrenergic inputs to identify a subpopulation of neurons

that could monitor these monoaminergic activities as feedback, since the habenula in turn regulates

the activities of the monoaminergic neurons (Hikosaka, 2010).

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Materials and Methods

Animals

All experiments were carried out in accordance with the Animal Experiment Plan approved by the

Animal Experiment Committee of RIKEN. Adult Long-Evans rats (250-350 g, male; Japan SLC,

Hamamatsu) were maintained throughout the experiments on a 12/12-h schedule with lights on

during the day. We confirmed the expression of marker mRNAs or proteins in at least two animals

for the neurotransmitter systems examined in the following experiments to test the reproducibility

of the findings.

Fixation and slice preparation

Under deep anesthesia by intraperitoneal administration of urethane (1.5 g/kg), animals were

perfused intracardially with cold saline followed by 4% paraformaldehyde in 0.1 M phosphate

buffer, pH 7.0 (PB). Postfixed brains were sliced coronally into 50 µm-thick serial sections using a

vibrating microtome (DTK-3000W, Dosaka EM, Kyoto) for in situ hybridization or into 25 µm-

thick serial sections using a freezing microtome (HM500, Microm, Walldorf) for

immunohistochemistry following cryoprotection with 30% sucrose in PB.

In situ hybridization

In situ hybridization was carried out as described previously (Amo et al., 2010). RNA probes for rat

dopamine type 2 receptor (Drd2) (Bunzow et al., 1988), rat G protein-coupled receptor 151

(Gpr151) (Berthold et al., 2003), murine serotonin type 2c receptor (Htr2c) (Lubbert et al., 1987),

rat µ-opioid receptor (Oprm) (Chen et al., 1993), murine protocadherin 10 (Pcdh10) (Hirano et al.,

1999), rat tachykinin 1 (Tac1) (Kawaguchi et al., 1986), rat vesicular glutamate transporter 1

(Vglut1) (Ni et al., 1994), and rat vesicular glutamate transporter 2 (Vglut2) (Aihara et al., 2000)

were used for the hybridization reaction at 55oC. For double hybridization, a digoxigenin-labeled

probe for Vglut2 or Tac1 and fluorescein-labeled probe for Vglut1 or Vglut2 were visualized with

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the TSA Plus System (Perkin Elmer) and FastRed (Roche), respectively.

For probe synthesis, fragments containing nucleotide (nt) 1528-2408 of rat Drd2 mRNA

(GenBank accession number NM_012547.1), nt 360-1313 of rat Gpr151 (AJ564168.1), nt 978-

1976 of murine Htr2c (NM_008312.4, 96.8% nucleotide identity to rat Htr2c), nt 713-1221 of rat

Oprm (U35424.1), nt 2087-3005 of murine Pcdh10 (NM_001098170, 96.5% identity to rat

Pcdh10), nt 494-1011 of rat Tac1 (NM_012666.1), nt 1528-1977 of rat Vglut1 (NM_053859.1), and

nt 2366-3215 of rat Vglut2 (NM_053427.1) were PCR-amplified using rat genomic DNA (Drd2,

Oprm, Tac1, Vglut1, Vglut2) or murine cDNA (Pcdh10, Htr2c) as the templates. The amplified

fragments were subcloned into the pCRII-TOPO vector (Invitrogen), and the recombinant plasmids

were used as templates to synthesize the antisense cRNA probes (digoxigenin and fluorescein

labeling kit, Roche).

Immunohistochemistry

Sections were blocked for 30 min in 10% normal donkey serum (D9663, Sigma-Aldrich, St. Louis,

MO) in 0.1 M phosphate-buffered saline (PBS) (pH 7.4) containing 0.3% Triton X-100 (PBST),

followed by overnight incubation (16-17 hours) at 4oC with the primary antibodies (see Table 1 and

the section below for characterization of the primary antibodies used) in PBST containing 10%

normal donkey serum. After three washes in PBST (20 min each), sections were incubated

overnight with the appropriate secondary antibodies (Table 2) at 4oC. Sections were then washed

with PBST, wet-mounted using Permafluor (Thermo Scientific), and then coverslipped.

Anterograde tracing experiments

Rats were anesthetized with 2% isoflurane and placed in the stereotaxic apparatus (SR-8N,

Narishige, Japan). A tiny hole was then made in the skull and dura mater (3.6 mm posterior and 1.2

mm lateral of bregma). A glass electrode filled with 2.5% biotinylated dextran amine, molecular

weight 10,000 (BDA-10000, Invitrogen) in 0.1 M PB was inserted through the hole to the MHb

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using a 10o-tilted micromanipulator (Model 1760-61, David Kopf Instruments; 4.8 mm in depth

from the pial surface). BDA was applied iontophoretically at a current of 5 µA for 30 min (0.5 s on

and 0.5 s off). After 7 days, the animals were put under deep anesthesia with urethane as described

above, and then perfused intracardially with cold saline followed by 4% paraformaldehyde in 0.1 M

PB. The BDA was visualized by streptavidin-Alexa Fluor 488 (1:500, S11223, Invitrogen) or

Avidin-Biotin Peroxidase Complex kit (Vector Laboratories) with diaminobenzidine-nickel

substrate, and some sections were counterstained with Neutral Red.

Primary antibody characterization

All of the antibodies used in the present study are commercially available and have been tested in

previous studies as follows.

1. Goat anti-choline acetyltransferase (ChAT) polyclonal antibody (AB144P, Millipore) recognizes

a single band of 68-70 kDa on western blots of mouse brain (according to manufacturer’s

technical information). In our samples, the antibody produced a staining pattern identical to that

shown in a previous report of ChAT in the rat habenula stained with a different antibody

(Contestabile et al., 1987).

2. Mouse anti-dopamine β-hydroxylase (DBH) monoclonal antibody (MAB308, Millipore) was

tested previously for specificity by western blot analysis (Russo et al., 2010) and preabsorption

with excessive adrenal DBH (Rinaman, 2001).

3. Mouse anti-HuC/D monoclonal antibody (A21271, clone 16A11, Invitrogen) recognizes a

protein of approximately 40 kDa in molecular size on immunoblots of human cortical neurons.

The antibody specificity was confirmed by blocking antibody binding with the HuD peptide

(Marusich et al., 1994). Immunoreactivity with this antibody was observed in neurons only from

the time when they begin to differentiate (Wakamatsu and Weston, 1997).

4. Goat anti-interleukin 18 (IL-18) polyclonal antibody (AF521, R&D Systems) detects a band of

24 kDa, which corresponds to the molecular size of pro-IL-18 on mouse western blots (Hedtjarn

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et al., 2002). This antibody stained MHb with the same pattern as the other antibody used in a

previous study (Sugama et al., 2002).

5. Rat anti-substance P monoclonal antibody (MAB356, Millipore) labels substance P, but not β-

endorphin, somatostatin, leu-enkephalin, or met-enkephalin by radioimmunoassay (Cuello et al.,

1979). No labeling was found when the antibody was preadsorbed with the synthetic substance P

(Cuello et al., 1980). This antibody stained MHb (Contestabile et al., 1987) in the same manner

as the other antibody used in a previous study (Shinoda et al., 1984).

6. Rabbit anti-tyrosine hydroxylase (TH) polyclonal antibody (AB152, Millipore) detects a single

band of 62 kDa on western blots of lysate from PC12 cell lines expressing TH (manufacturer’s

technical information). We observed that this antibody produced intense staining in neuropils and

cell bodies in the midbrain dopaminergic neurons as described previously (Horger et al., 1998).

7. Goat anti-vesicular acetylcholine transporter (VAChT) polyclonal antibody (G4481, Promega)

labels the neuronal varicosities of the ventral motor neurons in rat spinal cord (manufacturer’s

technical information) and all pig paracervical ganglion neurons expressing ChAT (Podlasz and

Wasowicz, 2008). However, this labeling was abolished if the antibody was pre-incubated with

immunogenic peptides (amino acid 511-530 of the rat VAChT) (manufacturer’s technical

information).

8. Rabbit anti-VAChT polyclonal antibody (V5387, Sigma) labels a 67-70-kDa band on western

blotting, and it is specifically inhibited by the immunizing peptide, rat VAChT (amino acid 512-

530 with N-terminally added lysine). This antibody stained neurons corresponding to the known

distribution of cholinergic cell groups and terminal fields identified by numerous ChAT

antibodies, and that staining was abolished when blocked with the peptide used for immunization

(Arvidsson et al., 1997). In our sample, staining with this antibody showed punctate

immunoreactivity in the interpeduncular nucleus in a pattern similar to that observed with the

above-described antibody (G4481) from Promega.

9. Guinea pig anti-Vglut1 polyclonal antibody (AB5905, Millipore) detects the expected protein of

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60 kDa in molecular size on western blotting, and staining of the rat brain with this antibody and

other Vglut1 antibodies showed complete overlap (Melone et al., 2005). Preabsorption with the

immunogen peptide eliminates all staining (manufacturer’s technical information).

10. Rabbit anti-Vglu2 polyclonal antibody (MAB5504, Millipore) labels the expected 56 kDa band

on western blots of rat brain lysates (Griffin et al., 2010). Double labeling of the mouse retina

showed that this antibody labeled the same structures as the other two antibodies raised against

Vglut2 (guinea pig anti-Vglut2 polyclonal antiserum from Chemicon Cat. No. AB5907 and rabbit

anti-Vglut2 polyclonal antiserum from Synaptic Systems Cat. No. 134-3) (Wassle et al., 2006).

Microscopy

Fluorescent labeling was observed by confocal laser scanning microscopy (Zeiss LSM510 Meta),

and some images were reconstructed using ImageJ software (Abramoff et al., 2004). Confocal

images for the synaptic proteins in the interpeduncular nucleus were obtained through a 63 x oil

immersion objective lens (NA = 1.40; Zeiss, Plan-Apochromat) attached to an Axioplan2 compound

microscope (Zeiss) under the following conditions: the focal plane was set to 2-5 mm beneath the

surface of sections, where the highest immunofluorescence was observed, with a pinhole size of 1.0

airy unit and line averaging of 4 times. With this optics, the limits of spatial resolution for Alexa

Fluor 488, Alexa Fluor 594, and Alexa Fluor 647 dyes were theoretically 0.61 x 519/1.40 = 226.14

nm, 0.61 x 617/1.40 = 268.84 nm, and 0.61 x 668/1.40 = 291.06 nm, respectively. Analysis of the

fluorescent signals from these dyes should represent the frequency of coincidence between each

synaptic proteins at a single axonal terminal, since the resolution was sufficiently high to determine

the size of axonal terminals, but not high enough for detecting synaptic vesicles (Nakamura et al.,

2007). Emission filter windows used for Alexa Fluor 488, 594, and 647 were 500-530 nm, 565-615

nm, and 650-704 nm, respectively. The images were stored in an 8-bit format at 1024 x 1024 pixels.

Conventional microscopic images were taken on a Zeiss AxioCam HRc camera attached to a

Axoplan2 compound microscope.

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For visualization of Vglut1 and VAChT immunoreactivities only in the axons labeled with

BDA, we first made binary images from a channel for BDA and applied it as a mask to the other

two channels for Vglut1 and VAChT to remove the fluorescence outside the axons labeled with

BDA by ImageJ software.

Figures were prepared using Adobe Photoshop CS5 (Adobe Systems, San Jose, CA). Image

brightness and contrast were adjusted.

Quantitative analysis of colocalization of the synaptic proteins

We adopted Pearson’s correlation coefficient (CC) as an indicator for the colocalization frequency

among fluorescence signals from Vglut1, Vglu2, and VAChT at single axon terminals, as described

previously (Manders et al., 1993; Nakamura et al., 2007). Briefly, after removal of a small number

of pixels in which the signal intensity were 0 or 255, we subtracted the background signals from the

images containing the axonal terminals and calculated CC with Mander's coefficient plug-in of

ImageJ. We defined thresholds according to the background calculated as the mean value of the

signal intensity in the area of the neuronal cell bodies that lacked axonal terminals.

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Results

Andres and colleagues (1999) previously proposed a subdivision of the rat habenula into

fifteen subnuclei based on light and electron microscopic findings. This scheme was later supported

by immunohistochemical (Geisler et al., 2003) and electrophysiological studies (Kowski et al.,

2009). We used this same subdivision definition and nomenclature in the present study to identify

the neurotransmitter systems used by habenular neurons by mapping the mRNA and protein

expression of marker genes for neurotransmitters and their receptors on each subnucleus (Fig. 1).

Specifically, MHb (pink in Fig. 1) comprises a superior part (MHbS), inferior part (MHbI), central

part (MHbC), lateral part (MHbL), and commissural part, while LHb comprises the medial division

(LHbM, light green in Fig. 1), which is further subdivided into superior part (LHbMS)

parvocellular part (LHbMPc), central part (LHbMC), magnocellular part (LHbMMg), and anterior

part (LHbMA, not shown Fig. 1), and the lateral division (dark green in Fig. 1), which is further

subdivided into parvocellular part (LHbLPc), magnocellular part (LHbLMc), oval part (LHbLO),

basal part (LHbLB, not shown in Fig. 1), and marginal part (LHbLMg, not shown in Fig. 1).

Since we frequently use the abbreviations both for mRNA and protein names below, we

intentionally described the abbreviated form of mRNA and protein names with the italic and regular

forms, respectively.

Combinatory gene expression of the neurotransmitter markers delineates the subnuclear

structure in rat medial habenula

Despite the regional expression of substance P-ergic and cholinergic neurotransmission

markers (Contestabile et al., 1987), neurotransmission between MHb and the interpeduncular

nucleus (IPN) also involves glutamate as shown by anatomical (Qin and Luo, 2009) and

electrophysiological studies (Brown et al., 1983; McGehee et al., 1995). More recently, an

electrophysiological study confirmed that the mouse MHb co-releases glutamate and acetylcholine

in the IPN, at least in vitro (Ren et al., 2011). To see how these three types of neurotransmission

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characterize the neuronal subtypes in MHb, we examined the distribution of markers for substance

P-ergic, cholinergic, and glutamatergic neurons and compared it with that of the neuronal cell

bodies visualized by the pan-neuronal marker HuC/D (Marusich et al., 1994).

Tachykinin 1 mRNA (Tac1), a precursor for substance P, was detected in the dorsal region of

MHbC (MHbCd, magenta in Fig. 2A and 2C). On the other hand, distribution of the cholinergic

neurons expressing ChAT was restricted to MHbI, the ventral region of MHbC (MHbCv), and

MHbL (green in Fig. 2B and 2C), and was complementary to that of Tac1. Intriguingly, MHbS at

the dorso-medial edge of MHb expressed neither Tac1 nor ChAT (asterisk in Fig. 2C).

Next, to address where glutamate acts as a neurotransmitter in the habenula, we examined

the distribution of mRNA for the type 1 and 2 vesicular glutamate transporters (Vglut1 and Vglut2)

as markers of glutamatergic transmission (Fremeau et al., 2001; Varoqui et al., 2002). Vglut2

mRNA was found in almost all habenular neurons, with stronger expression in MHbS and weaker

expression in MHbCd (Fig. 2D-F). Double labeling experiments revealed that MHbS expressing

stronger Vglut2 mRNA (magenta in Fig. 2G and 2I) lacked Tac1 mRNA (green in Fig. 2H and 2I).

In contrast to the ubiquitous expression of Vglut2 mRNA both in MHb and LHb, Vglut1 mRNA was

restricted to MHbS, MHbI, and MHbCv regions (Fig. 2J-L), and was not expressed in the other

subnuclei of the habenula such as MHbCd and MHbL (arrowheads in Fig. 2L), or LHb (bracket in

Fig. 2L). The fact that MHbS expressing both Vglut1 and Vglut2 lacked the expression of Tac1

mRNA and ChAT protein indicated that this small subnucleus used glutamate, but not substance P

or acetylcholine, as a neurotransmitter.

Neurons expressing Vglut1 and Vglut2 localize differentially in the adult brain, i.e., Vglut1

was expressed in the cerebral cortex and hippocampus, while Vglut2 has been localized in the

subcortical structures such as thalamus (Fremeau et al., 2001; Varoqui et al., 2002). Habenula is one

of the exceptional regions expressing both Vglut1 and Vglut2 mRNA (Fremeau et al., 2001), and

indeed we observed the colocalization of these two markers in MHbS, MHbI, and MHbCv (Fig

2M-O). For MHbL, instead of Vglut1, we observed the strong expression of µ-opioid receptor

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(Oprm) mRNA (Fig. 2P-R).

Taken together, our results indicated that MHb subnuclei use different combinations of

neurotransmitter systems. Thus, the subnuclei were categorized as exclusively glutamatergic

(MHbS), both substance P-ergic and glutamatergic (MHbCd), or both cholinergic and glutamatergic

(MHbI, MHbCv, and MHbL).

Cotransmission of glutamate and acetylcholine from the medial habenula to the

interpeduncular nucleus

Since the marker genes for glutamatergic (Vglut1 or Vglut2) and cholinergic (ChAT) neurons were

expressed in MHbI, MHbCv, and MHbL, we suspected that the same neurons in this subnucleus

might use both glutamate and acetylcholine for neurotransmission to the IPN, which predominantly

receives MHb inputs (Groenewegen et al., 1986). To test this hypothesis, we first checked whether

the markers for glutamatergic and cholinergic neurons were co-expressed in the same MHb

neurons. Double-labeling experiments revealed that neurons expressing Vglut1 (magenta in Fig.3A

and 3C) in MHbI and MHbCv or Vglut2 (magenta in Fig. 3D and 3F) in MHbI, MHbCv, and MHbL

co-expressed ChAT (green in Fig. 3B, 3C, 3E and 3F), suggesting that the cholinergic neurons in

MHb could have glutamatergic properties at their synapses.

To address the possibility that the axonal terminals from MHb could co-release glutamate

and acetylcholine, we examined the distribution of synaptic proteins specific for glutamatergic or

cholinergic transmission in the IPN, which is the major source of afferents from the MHb

(Groenewegen et al., 1986). Triple-immunolabeling experiments revealed the abundant expression

of glutamatergic (Vglut1, red in Fig. 3G and Vglut2, blue in Fig. 3G) and cholinergic (vesicular

acetylcholine transporter, VAChT, green in Fig. 3G) markers in IPN. Analysis of colocalization

using Pearson’s correlation coefficient indicated the frequency of colocalization at single axonal

terminals based on the fluorescent signals for each synaptic marker, as described previously

(Manders et al., 1993; Nakamura et al., 2007). This analysis suggested that VAChT was more

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frequently present with Vglut1 than with Vglut2 in IPN (Fig. 3H). The IPN receives afferents not

only from MHb, but also to a lesser extent from the other brain regions such as median raphe and

dorsal tegmental regions (Groenewegen et al., 1986). Therefore, to exclude the possibility that the

colocalization of Vglut1/2 and VAChT immunoreactivities in IPN might represent axonal terminals

originating from the other brain regions, we labeled the MHb-projected axons by injecting the

anterograde tracer, BDA (molecular weight 10,000) into MHb (arrowhead in the inset of Fig. 3J),

and then examined the distribution of Vglut1, Vglut2, and VAChT (Fig. 3J-L). Labeled axons

traversed horizontally in the central subnucleus of the IPN (Fig. 3J) and frequently showed bouton-

like morphology suggesting the presence of synapses (arrowheads in Fig. 3K). To check whether

these labeled axons contained both glutamatergic and cholinergic synapses, we localized the

immunoreactivities of Vglut1 and VAChT in the MHb-derived axons. We extracted the signals for

Vglut1 and VAChT only when those signals were colocalized with BDA visualized by streptavidin-

Alexa 633 and found colocalization of Vglut1 and VAChT at the same boutons even in the

reconstructed images containing only the extracted signals (arrows in Fig. 3L).

These results suggested that MHb could corelease glutamate and acetylcholine from axonal

terminals in the IPN.

Specific innervation of the superior part of the medial habenula expressing interleukin-18 by

noradrenergic fibers

We next examined the neurochemical nature of the MHbS regions that we defined by stronger

Vglut2 mRNA expression and the absence of markers for substance P-ergic and cholinergic

neurons. A study previously reported that proinflammatory cytokine interleukin 18 (IL-18) was

strongly expressed in MHbS (Sugama et al., 2002). Consistent with this, we observed stronger

expression of IL-18 in the MHbS (magenta in Fig. 4A and 4C), although a weaker signal for IL-18

was also found in MHbCd expressing substance P (green in Fig. 4B and 4C). This suggested that

MHbS produces IL-18 in addition to conventional neurotransmitters such as glutamate.

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IL-18 production can be induced by noradrenalin through the activation of β2-adrenergic

receptors in immune cells such as monocytes (Takahashi et al., 2004). We therefore postulated that

the production of IL-18 in MHbS might be regulated by noradrenergic inputs, even in the central

nervous system. To address this, we examined the distribution of noradrenergic fibers marked by

dopamine β-hydroxylase (DBH) immunoreactivity and compared it with that of tyrosine

hydroxylase (TH), a rate-limiting enzyme for catecholamine synthesis. We found that DBH-

expressing fibers were specifically localized in MHbS, which was consistent with a previous report

(Swanson and Hartman, 1975) (magenta in Fig. 4D and 4F), although fibers expressing only TH

were also found in MHbL and medial half of LHb (green in Fig. 4E and 4F). Double-labeling

experiments showed that innervation of the habenula by DBH-expressing fibers (green in Fig. 4H

and 4I) was largely restricted to MHbS expressing IL-18 (magenta in Fig. 4G and 4I). Intriguingly,

we also observed a small number of cells showing strong immunoreactivity for IL-18 in the region

lateral to the MHbS, and DBH-expressing fibers also terminated on these neurons (arrowheads in

Fig. 4G-I), further indicating a correlation between noradrenergic innervation and IL-18 expression.

These results suggested that MHbS could be delineated by the presence of Vglut2-, IL-18-,

and DBH-positive fibers, and the production of IL-18 in these neurons might occur under the

influence of noradrenergic inputs.

Heterogeneous expression of the monoaminergic receptors in the lateral habenula

Previous studies reported that the subnuclei in LHb differed in the extent of dopaminergic

innervation (Geisler et al., 2003). Thus, to determine the responsiveness of the neurons in each

subnucleus of LHb to the dopaminergic neurotransmission, we first search the literatures (Ariano et

al., 1997; Bouthenet et al., 1991; Mansour et al., 1990; Meador-Woodruff et al., 1992; Weiner et al.,

1991) and Allen mouse brain atlas (Allen Brian Institute, available at http://mouse.brain-map.org)

for mRNA expression pattern of previously known five dopaminergic receptors in LHb and found

that the dopamine type 2 receptor (Drd2) was the only subtype showing the mRNA expression

17

detectable in rodent LHb. The neurotransmitter markers used to delineate MHb showed no

expression (Vglut1, Tac1 and ChAT) (Fig. 2A, 2B and 2J) or uniform distribution (Vglut2) in LHb

(Fig. 2D and 2G). Therefore, we examined Drd2 distribution (magenta in Fig. 5A and 5C) and

compared it with the position of the HuC/D-positive neuronal cell bodies (green in Fig. 5B and 5C)

to identify the subdivision postulated recently (Andres et al., 1999; Geisler et al., 2003).

Distribution of Drd2 mRNA in LHb appeared to be “inverse C-shaped” lacking signal in the

parvocellular subnucleus of the medial division (LHbMPc) and central subnucleus of the medial

division (LHbMC) of LHb (Fig. 5A-C). Expression of Drd2 mRNA was observed in the marginal

subnucleus of the medial division (LHbMMg), magnocellular subnucleus of the lateral division

(LHbLMc) and oval subnucleus of the lateral division (LHbLO) of LHb as well as MHbS (Fig. 5A-

C). Intriguingly, TH-expressing fibers (green in Fig. 5E and 5F) was primarily observed in

LHbMPc and LHbMC that contained few Drd2-positve neuronal cell bodies (magenta in Fig. 5D

and 5F), suggesting that the dendrites of the Drd2-expressing habenular neurons receiving the

dopaminergic inputs might be concentrated in LHbMPc and LHbMC.

For the serotonergic receptors, serotonin type 2c receptor (Htr2c) is peculiar among the

diverse serotonergic receptor subtypes in that the activation of this receptor by systemic injection of

the Htr2c agonist inversely correlated with the firing rate of the serotonergic neurons in the dorsal

raphe (Sharp et al., 2007) and the dopaminergic neurons in the ventral tegmental area (Gobert et al.,

2000). Since Htr2c was expressed much more specifically and strongly in LHb than the other

subtypes (Mengod et al., 1990), activation of the habenula-raphe pathway might contribute to the

inhibition of firing in the serotonergic neurons evoked by Htr2c receptor activation. Therefore, to

identify the LHb nucleus which is under the influence of this specific receptor subtype, Htr2c

mRNA expression in LHb was examined (magenta in Fig. 5G and 5I) and compared with the

position of the neuronal cell bodies detected by HuC/D (green in Fig. 5H and 5I). We observed that

Htr2c was predominantly found in the superior subnucleus of LHbM (LHbMS) ventral half of

LHbMPc, LHbMC, parvocellular subnucleus of LHbL (LHbLPc), LHbLMc and LHbLO (Fig. 5G-

18

I). The distribution of neurons expressing Htr2c was overlapped with that of TH-expressing fibers

at the ventral half of LHbMS and LHbMPc (Fig. 5J-L).

Accordingly, expression of Drd2 and Htr2c genes coincided in some LHbL subnuclei such

as LHbLO and the dorsal part of LHbLMc, but showed rather complementary pattern with each

other in LHbM such as LHbMS, LHbMC, LHbMPc, and LHbMMg.

These results suggested that subnuclei in LHb showed neuronal heterogeneity with regard to

the monoaminergic receptors, and differential distribution of dopaminergic and serotonergic

receptor indicated that neuronal activity in the medial and lateral division of LHb might be

regulated in a distinct manner by these monoamine neurotransmitters.

Habenular neurons show heterogeneity among the subnuclei

Finally, we further tested whether the expression pattern of the other genes in the habenula are

consistent with the subdivision which we proposed above or not by examining the mRNA

distribution of the known marker gene for LHb or the gene expressed exclusively in the habenula

but not in the other brain regions.

We previously showed that protocadherin 10 (Pcdh10) was specifically expressed in LHb,

but not in MHb across species (Amo et al., 2010), suggesting that we might be able to introduce our

transgene of interest to express exclusively in LHb by inserting it into the Pcdh10 locus in the

genome. To determine the subnuclear localization of this gene, we examined the distribution of

Pcdh10 mRNA (magenta in Fig. 6A and 6C) and compared it with the position of the neuronal cell

bodies expressing HuC/D (green in Fig. 6B and 6C). We found that Pcdh10 mRNA were more

abundant in the medial division of LHb such as LHbMS, LHbMPc and LHbMC than the subnuclei

in the lateral division of LHb such as LHbLO. Intriguingly, Pcdh10 mRNA was found only in the

medial part of the LHbL subnucleus such as LHbLMc suggesting further heterogeneity within the

habenular subnuclei (Fig. 6A-C).

Interestingly, expression of the G-protein coupled orphan receptor gene called Gpr151

19

(synonymous with PGR7, nGPCR-2037, Galanin-receptor like) was virtually restricted to the

habenula (Berthold et al., 2003; Ignatov et al., 2004). Gpr151 mRNA expression was abundantly

found in MHbCv and MHbL, but not in MHbI (Fig. 6D-F). In the LHb, Gpr151 showed stronger

expression in the particular subnuclei such as LHbMS and LHbLPc, and weaker expression in a

subpopulation of neurons in all the other LHb subnuclei (Fig. 6D-F), suggesting that manipulating

the neurons expressing Gpr151 might involve a subpopulation of neurons in all habenular subnuclei

except MHbCd and MHbS.

LHb with neuronal heterogeneity across the boundaries of the subnuclei is in clear contrast

to the MHb, where the combinatorial patterns of gene expression delineate the boundaries between

subnuclei more clearly.

20

Discussion

The present study examined the subnuclear organization of MHb and LHb based on the expression

patterns of marker genes and proteins, and revealed differential expression of a combination of

neurotransmitter marker genes in MHb (Fig. 7). Unlike MHb, the majority of neurons in LHb were

uniformly glutamatergic (Fig. 2D-F) except for some GABAergic neurons scattered in the LHbLO

(Brinschwitz et al., 2010). However, the LHb showed heterogeneity in neuronal character including

monoamine receptor expression even among the specific subnuclei postulated by previous studies

(Fig. 7). Below, we discuss how the subnuclear organization proposed herein is implicated in the

functional diversity of habenula.

The differential expression of neurotransmitter markers reveals a novel subdivision of the rat

medial habenula

Since neurotransmitter expression is a critical factor in determining neuronal character, we first

divided MHb into three subnuclei according to the gene expressions of specific marker

neurotransmitters as follows: MHbS showed a strong glutamatergic character, MHbCd showed a

strongly substance P-ergic and weakly glutamatergic character, and MHbI, MHbCv, and MHbL

showed a strongly cholinergic and glutamatergic character. The present results are largely consistent

with a previous study (Contestabile et al., 1987) showing the topographical projections from the SP-

expressing dorsal region of MHb to rostral and lateral subnuclei of the IPN and from the ChAT-

expressing ventral two-thirds of MHb to the central and intermediate subnuclei of IPN. Identifying

MHbS in addition to these two subnuclei might also be in agreement with their experiments, since

Contestabile et al. (1987) actually presented an image showing that the dorsomedial end of MHb

lacked SP expression (see their Figure 4), although without a relevant description in the text.

Intriguingly, we observed that Vglut1, Oprm, and Gpr151 were expressed differentially in

the cholinergic regions in MHb such as MHbI, MHbCv, and MHbL. It was previously suggested

that the ventrolateral part of MHb, which is rich in β4/α4 subunit-containing nicotinic acetylcholine

21

receptor, is primarily connected with the caudal/lateral part of the IPN, while the ventromedial part

is primarily connected with the central parts of the IPN (Contestabile and Flumerfelt, 1981; Fonck

et al., 2009). Therefore, it is worth examining whether the differential expression of the genes we

examined in each subdivision of MHb may reflect different neural connectivities with IPN.

Based on a recent study that conducted a comprehensive search for genes enriched in the

mouse medial and lateral habenulae (Quina et al., 2009), mapping the expression pattern of these

genes to the subnuclei proposed in the present study should provide further insights into how each

subnucleus in the habenula might contribute to specific animal behaviors.

Medial habenula could corelease glutamate and acetylcholine at the synaptic terminals in the

interpeduncular nucleus

Recent advances in neuroscience revealed that the co-release of several transmitters from the same

neurons is not exceptional, but rather is a prevalent feature of the neurons (Trudeau and Gutierrez,

2007). Our results substantiated that glutamatergic neurons also have the ability to synthesize

acetylcholine in MHb as reported in the other brain regions such as pedunculopontine tegmental

nucleus (Lavoie and Parent, 1994), developing spinal cord (Li et al., 2004; Nishimaru et al., 2005),

and basal forebrain (Allen et al., 2006). Indeed, a recent study showed that the optogenetic

stimulation of cholinergic axons from MHb induced a neural response that was sensitive to both

glutamatergic and cholinergic receptor blockers in vitro (Ren et al., 2011). Our finding that the

MHb-derived axons contained the vesicular transporters for both glutamate and acetylcholine (Fig.

3L) added morphological evidence for the hypothesis that glutamate and acetylcholine could be

released from the same axonal terminals in the IPN. Since acetylcholine mediates the slower

response to tetanic stimulation in the neurotransmission between MHb and IPN (Ren et al., 2011),

and the efficiency of glutamatergic transmission is facilitated by activation of nicotinic

acetylcholine receptor at presynaptic axonal terminals in habenulo-interpeduncular transmission

(McGehee et al., 1995), acetylcholine released from the axonal terminals of MHb expressing

22

Vglut1 may act upon the adjacent presynaptic site to adjust the release probability for glutamatergic

synaptic vesicles.

Cytokine production in the medial habenula and its regulation by noradrenergic inputs

IL-18 is a pro-inflammatory cytokine expressed in a variety of tissues including immune cells,

adrenal gland, and brain, and its production could be induced in the adrenal gland following

activation of the hypothalamus-pituitary-adrenal axis (Sugama and Conti, 2008). It is likely that

MHbS neurons expressing IL-18 act as a sensor in the central nervous system to monitor stressful

stimuli, since these neurons increased IL-18 production in response to restraint stress (Sugama et

al., 2002). However, it remained unclear how MHbS neurons received such information while the

animals are in the stressed condition. In the present study, we observed the coincidence of IL-18-

expressing neurons and DBH-expressing fibers in the MHbS, suggesting that production of this

particular cytokine might be under control of the noradrenergic inputs. A previous report indicated

that the noradrenergic inputs to putative MHbS originated from the locus coeruleus rather than

peripheral sympathetic ganglion (Bjorklund et al., 1972). Since this cytokine is implicated in the

modulation of glutamatergic neural transmission efficiency (Takahashi et al., 2004), IL-18 in MHbS

might modulate efficiency in the habenulo-interpeduncular transmission according to the central

noradrenergic activation under stressful conditions.

Expression of monoamine receptors in the lateral habenula as a feedback control to regulate

monoaminergic activities

Based on the findings in the present studies and previous literature (Ariano et al., 1997; Bouthenet

et al., 1991; Mansour et al., 1990; Meador-Woodruff et al., 1992; Weiner et al., 1991), Drd2 is the

only one among five types of dopaminergic receptors that is expressed in LHb, of which the medial

division receives denser innervation by the dopaminergic axons. Complementary distribution of

dopaminergic fibers and Drd2-positive cell bodies indicated that the dopaminergic inputs were

23

terminated more specifically on the dendrites rather than the cell bodies. This finding is consistent

with a previous view that the majority of dopaminergic axons terminate at the dendrites, as reported

in the medium spiny neurons of rat striatum (Levey et al., 1993) and the pyramidal cells of primate

cerebral cortex (Goldman-Rakic et al., 1989). Examination of the area containing the neuronal

dendrites in each subnucleus will be helpful in determining which subnuclei are under the influence

of the dopaminergic inputs (Kowski et al., 2009).

Serotonergic activity in the brain is controlled by feedback inhibition through presynaptic

serotonin autoreceptors on the serotoninergic neurons in the raphe (Blier et al., 1998) as well as by

postsynaptic serotonin receptors (Sharp et al., 2007). According to the abundant expression of Htr2c

in LHb, it is suggested that LHb might be a source of serotonergic feedback through this receptor

subtype (Sharp et al., 2007). Since the activation of LHb inhibits serotonergic neurons in the raphe

(Wang and Aghajanian, 1977), the present results support the possibility that the inhibitory control

of serotonergic cell firing might be mediated by neurons in the specific subnuclei that showed

strong Htr2c expression such as LHbMS, LHbMPc, and LHbLPc.

Heterogeneity of the habenular neurons between subnuclei

Gene expression is useful for defining a specific population of neurons that shares character

similarities within the central nervous system, and recent progress in genetic manipulation by

bacterial artificial chromosome (BAC) transgenesis enables us to target those specific populations

of neurons that express the same set of genes (Lee et al., 2001). Since the habenula consists of MHb

and LHb, each of which has distinct neural connectivity and neurotransmitter expression, it would

be highly useful to identify the genes differentially expressed in either MHb or LHb, or the genes

expressed only in the habenula for genetic dissection of the habenular functions in animal

behaviors. Indeed, we recently revealed that zebrafish had the medial and lateral habenula

homologues according to gene expressions and neural connectivity (Aizawa et al., 2005; Amo et al.,

2010), and that genetic inactivation of the neural transmission between a subnuclei of the medial

24

habenula homologue and IPN results in experience-dependent enhancement of the fear response

(Agetsuma et al., 2010).

Differences in the neurotransmitter systems between subnuclei support the view that each

subnucleus in the habenula impacts distinctly on behaviours in the animal. Conventional lesion or

pharmacological treatment of the entire habenula structure could therefore preclude us from

properly deciphering the physiological functions of the habenula subregions, provided that

subpopulations of neurons in the habenula act on the same animal behavior in a different way.

Examining specific behavioral changes after activation or inactivation of particular habenular

subnuclei by genetic manipulation would be useful to unravel the specific function of a

subpopulation of the habenular neurons that share common molecular characteristics.

.

25

Acknowledgments

We thank the members of our laboratories for valuable discussions related to this work and Toshio

Miyashita for the murine Htr2c probe.

26

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Varoqui H, Schafer MK, Zhu H, Weihe E, Erickson JD. 2002. Identification of the differentiation-

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Wakamatsu Y, Weston JA. 1997. Sequential expression and role of Hu RNA-binding proteins

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34

Figure legends

Figure 1. Subnuclear organization of the rat habenula postulated by previous studies. Schematic

diagram showing the subdivision of the medial habenula (MHb) (pink), medial division of the

lateral habenula (LHb) (light green), and lateral division of the LHb (dark green), according to

Andres et al. (1999). MHbC, central part of MHb; MHbI, inferior part of MHb; MHbL, lateral part

of MHb; MHbS, superior part of MHb; LHbMC, central part of the medial division of LHb;

LHbMMg, marginal part of the medial division of LHb; LHbMPc, parvocellular part of the medial

division of LHb; LHbMS, superior part of the medial division of LHb; LHbLMc, magnocellular

part of the lateral division of LHb; LHbLO, oval part of the lateral division of LHb; LHbLPc,

parvocellular part of the lateral division of LHb.

Figure 2. Delineation of the medial habenular subnuclei by neurotransmitter gene expression

analysis. Coronal sections of rat habenula showing the mRNA expression of tachykinin 1 (Tac1)

(magenta in A, C and green in H, I), glutamatergic markers vesicular glutamate transporter 2

(Vglut2) (magenta in D, F, G, I and green in N, O) and vesicular glutamate transporter 1 (Vglut1)

(magenta in J, L, M, O), and µ-opioid receptor (Oprm) (magenta in P, R) relative to the

immunostaining for choline acetyltransferase (ChAT) (green in B, C) and pan-neuronal marker

HuC/D (green in E, F, K, L, Q, R). Dotted lines in panels C, F, I, L, O, and R indicate the

boundaries between the subnuclei in the medial habenula. Asterisk in C indicates MHbS. Arrows in

L indicate the Vglut1-negative region in MHbL. A bracket in L indicates LHb. Insets in M, N, and O

are higher magnifications of the boxed areas in each panel. Arrowheads in R indicate neurons

expressing Oprm in MHbL. Scale bars, A, D, G, J, M, P, 200 µm. Scales for the panels in the middle

and right columns are the same as in the left column.

Figure 3. Medial habenula cotransmits glutamate and acetylcholine to the interpeduncular nucleus.

(A-F) Expression of the markers for glutamatergic and cholinergic neural transmissions in the

35

habenulo-interpeduncular projection. Coronal sections of rat habenula (A-F) showing the mRNA

expression of vesicular glutamate transporter 1 (Vglut1) (magenta in A, C), and 2 (Vglut2) (magenta

in D, F), with immunostaining for choline acetyltransferase (ChAT) (green in B, C, E, F). Insets in

C and F are higher magnification of the boxed areas in each panel. (G) A coronal section of the

interpeduncular nucleus showing the distributions of Vglut1 (red), vesicular acetylcholine

transporter (VAChT, green), and Vglut2 (blue) immunoreactivities. (H) Bar graph representing

Pearson’s correlation coefficients for the combination of synaptic proteins. Values are mean +

standard errors of mean. (J-L) Axonal terminals from the medial habenula co-expressed the

glutamatergic and cholinergic synapse markers. Anterograde tracer biotinylated dextran amine

(BDA, molecular weight 10,000) was injected into the medial habenula. Coronal sections of the rat

habenula (inset in J) and interpeduncular nucleus (J-L) showing the injection site (arrowhead in

inset of J), labeled axons (black in J, K, and blue in L), and immunostaining for glutamatergic

(Vglut1, red in L) and cholinergic (VAChT, green in L) synaptic markers. Arrows in K indicate the

bouton-like structure of a labeled axon. To visualize the fluorescent signals for Vglut1 and VAChT

immunoreactivities on the BDA-labeled axons, the fluorescent signals for Vglut1 or VAChT

colocalized with that for BDA was extracted and projected along X (right panel), Y (upper panel),

or Z axes (lower left panel). Arrows in L indicates the colocalization of Vglut1, VAChT, and BDA

in the same bouton-like structures. Scale bars, A, D, J, 200 µm; inset in C and F, 25 µm; G, K, 20

µm; L, 5 µm. Scales for the panels in the middle and right columns are the same as in the left

column except for the insets.

Figure 4. Superior part of the medial habenula receives noradrenergic inputs. (A-I) Coronal

sections of the rat medial habenula showing the immunostaining for interleukin-18 (IL-18)

(magenta in A, C, G, I), substance P (SP) (green in B, C), tyrosine hydroxylase (TH) (green in E, F),

and dopamine b-hydroxylase (DBH) (magenta in E, F and green in H, I). Dotted lines in C, F, and I

indicate the MHbS. Panels D, E, and F are higher magnifications of the boxed areas in the inset of

36

each panel. Arrowheads in G, H, and I indicate the colocalization of an IL-18-expressing cell

(magenta in G and I) and DBH-expressing fibers (green in H and I). Scale bars, A, G, 50 µm; inset

in D, 200 µm. Scales for the panels in the middle and right columns are the same as in the left

column except for the inset.

Figure 5. Gene expression patterns for the monoamine receptors in lateral habenular subnuclei.

Coronal sections of rat habenula showing the mRNA expression of dopamine type 2 receptor

(Drd2) (magenta in A, C, D, F) and serotonin type 2c receptor (Htr2c) (magenta in G, I, J, L)

relative to the immunostaining for tyrosine hydroxylase (TH) (green in E, F, K, L) and HuC/D

(green in B, C, H, I). Dotted lines in panels C, F, I, L indicate the boundaries between the subnuclei

in the medial and lateral habenulae. Scale bars, A, D, G, J, 200 µm. Scales for the panels in the

middle and right columns are the same as in the left column.

Figure 6. Gene expression patterns for the lateral habenula- and habenula-specific genes. Coronal

section of rat habenula showing the mRNA expressions of protocadherin 10 (Pcdh10) (magenta in

A, C) and G-protein coupled receptor 151 (Gpr151) (magenta in D, F) relative to the

immunostaining for HuC/D (green in B, C, E, F). Dotted lines in panels C and F indicate the

boundaries between the subnuclei in the medial and lateral habenulae. Scale bars, A, D, 200 µm.

Scales for the panels in the middle and right columns are the same as in the left column.

Figure 7. Gene expression profile of the habenula subnuclei. Schematic diagram summarizing the

gene expression patterns in the subnuclei of the medial habenula (pink), medial division of the

lateral habenula (light green), and lateral division of the lateral habenula (dark green).

Abbreviations are the same as in Figure 1 except for MHbCd (central part of MHb, dorsal region)

and MHbCv (central part of MHb, ventral region). The number of plus signs indicates the relative

fluorescent signal intensity. Three levels were defined within the detectable range from the lowest

37

level just above background (“weak”) to the most intense signal detected (“strong”), as follows: -,

not detectable; +, weak expression; ++, intermediate expression; +++, strong expression. Signal

intensity can be compared between brain regions, but not between probes.

1

Tables

Table 1

Details of the primary antibodies used

Antibody target Immunogen Manufacturer, catalog

number, species Dilution

Choline acetyltransferase Human placental choline

acetyltransferase enzyme

Millipore, AB144P, goat

polyclonal

1:100

Dopamine β-hydroxylase Purified bovine DBH (from adrenal

medulla)

Millipore, MAB308 clone

4F10.2, mouse

monoclonal

1:500

HuC/D antibody Synthetic peptide

QAQRFRLDNLLN from human

HuD

Invitrogen, A21271,

mouse monoclonal

1:100

Interleukin 18 E. coli-derived, recombinant rat

interleukin 18

R&D systems, AF521,

goat polyclonal

10 µg/ml

Substance P Substance P conjugated to bovine

serum albumin

Millipore, MAB356, rat

monoclonal

1:100

Tyrosine hydroxylase Denatured tyrosine hydroxylase

from rat pheochromocytoma

(denatured by sodium dodecyl

sulfate)

Millipore, AB152, rabbit

polyclonal

1:500

Vesicular acetylcholine

transporter

Carboxy-terminal peptide sequence

CSPPGPFDGCEDDYNYYSRS, aa

511-530 corresponding to cloned rat

vesicular acetylcholine transporter

protein

Promega, G4481, goat

polyclonal

1:200

Vesicular acetylcholine

transporter

Peptide sequence

SPPGPFDGCEDDYNYYSRS, aa

512-530 of cloned rat vesicular

acetylcholine transporter protein

Sigma, V5387, rabbit

polyclonal

1:10000

Vesicular glutamate

transporter 1

Synthetic peptide

GATHSTVQPPRPPPPVRDY, aa

542-560 of rat vesicular glutamate

transporter 1 protein.

Millipore, AB5905,

guinea pig polyclonal

1:2000

Vesicular glutamate

transporter 2

Recombinant protein for the whole

rat vesicular glutamate transporter 2

Millipore, MAB5504,

rabbit polyclonal

1:2000

6

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Journal of Comparative Neurology

2

Table 2

Details of the secondary antibodies used

Antibody Fluorescent label

conjugated

Manufacturer, catalog

number, species Dilution

Anti-guinea pig IgG Cy3 Jackson Immuno Research,

706-165-148, Donkey

1:200

Anti-goat IgG Alexa Fluor 488 Invitrogen, A11055, Donkey 1:200

Anti-mouse IgG Alexa Fluor 488 Invitrogen, A21202, Donkey 1:200

Anti-rabbit IgG Alexa Fluor 594 Invitrogen, A21207, Donkey 1:200

Anti-rabbit IgG Alexa Fluor 647 Invitrogen, A31573, Donkey 1:200

Anti-rat IgG Alexa Fluor 594 Invitrogen, A21209, Donkey 1:200

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Figure 1. Subnuclear organization of the rat habenula postulated by previous studies. Schematic diagram showing the subdivision of the medial habenula (MHb) (pink), medial division of the lateral habenula (LHb) (light green), and lateral division of the LHb (dark green), according to Andres et al. (1999). MHbC, central part of MHb; MHbI, inferior part of MHb; MHbL, lateral part of MHb; MHbS, superior part of MHb; LHbMC, central part of the medial division of LHb; LHbMMg, marginal part of the medial division of LHb; LHbMPc,

parvocellular part of the medial division of LHb; LHbMS, superior part of the medial division of LHb; LHbLMc, magnocellular part of the lateral division of LHb; LHbLO, oval part of the lateral division of LHb; LHbLPc,

parvocellular part of the lateral division of LHb.

197x157mm (150 x 150 DPI)

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Figure 2. Delineation of the medial habenular subnuclei by neurotransmitter gene expression analysis. Coronal sections of rat habenula showing the mRNA expression of tachykinin 1 (Tac1) (magenta in A, C and green in H, I), glutamatergic markers vesicular glutamate transporter 2 (Vglut2) (magenta in D, F, G, I and

green in N, O) and vesicular glutamate transporter 1 (Vglut1) (magenta in J, L, M, O), and µ-opioid receptor (Oprm) (magenta in P, R) relative to the immunostaining for choline acetyltransferase (ChAT) (green in B, C) and pan-neuronal marker HuC/D (green in E, F, K, L, Q, R). Dotted lines in panels C, F, I, L, O, and R

indicate the boundaries between the subnuclei in the medial habenula. Asterisk in C indicates MHbS. Arrows in L indicate the Vglut1-negative region in MHbL. A bracket in L indicates LHb. Insets in M, N, and O are

higher magnifications of the boxed areas in each panel. Arrowheads in R indicate neurons expressing Oprm in MHbL. Scale bars, A, D, G, J, M, P, 200 µm. Scales for the panels in the middle and right columns are the

same as in the left column. 130x269mm (150 x 150 DPI)

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Figure 3. Medial habenula cotransmits glutamate and acetylcholine to the interpeduncular nucleus. (A-F) Expression of the markers for glutamatergic and cholinergic neural transmissions in the habenulo-

interpeduncular projection. Coronal sections of rat habenula (A-F) showing the mRNA expression of vesicular

glutamate transporter 1 (Vglut1) (magenta in A, C), and 2 (Vglut2) (magenta in D, F), with immunostaining for choline acetyltransferase (ChAT) (green in B, C, E, F). Insets in C and F are higher magnification of the

boxed areas in each panel. (G) A coronal section of the interpeduncular nucleus showing the distributions of Vglut1 (red), vesicular acetylcholine transporter (VAChT, green), and Vglut2 (blue) immunoreactivities. (H)

Bar graph representing Pearson’s correlation coefficients for the combination of synaptic proteins. Values are mean + standard errors of mean. (J-L) Axonal terminals from the medial habenula co-expressed the glutamatergic and cholinergic synapse markers. Anterograde tracer biotinylated dextran amine (BDA,

molecular weight 10,000) was injected into the medial habenula. Coronal sections of the rat habenula (inset in J) and interpeduncular nucleus (J-L) showing the injection site (arrowhead in inset of J), labeled axons

(black in J, K, and blue in L), and immunostaining for glutamatergic (Vglut1, red in L) and cholinergic

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(VAChT, green in L) synaptic markers. Arrows in K indicate the bouton-like structure of a labeled axon. To visualize the fluorescent signals for Vglut1 and VAChT immunoreactivities on the BDA-labeled axons, the fluorescent signals for Vglut1 or VAChT colocalized with that for BDA was extracted and projected along X

(right panel), Y (upper panel), or Z axes (lower left panel). Arrows in L indicates the colocalization of Vglut1,

VAChT, and BDA in the same bouton-like structures. Scale bars, A, D, J, 200 µm; inset in C and F, 25 µm; G, K, 20 µm; L, 5 µm. Scales for the panels in the middle and right columns are the same as in the left

column except for the insets. 166x229mm (150 x 150 DPI)

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Figure 4. Superior part of the medial habenula receives noradrenergic inputs. (A-I) Coronal sections of the rat medial habenula showing the immunostaining for interleukin-18 (IL-18) (magenta in A, C, G, I),

substance P (SP) (green in B, C), tyrosine hydroxylase (TH) (green in E, F), and dopamine b-hydroxylase (DBH) (magenta in E, F and green in H, I). Dotted lines in C, F, and I indicate the MHbS. Panels D, E, and F are higher magnifications of the boxed areas in the inset of each panel. Arrowheads in G, H, and I indicate the colocalization of an IL-18-expressing cell (magenta in G and I) and DBH-expressing fibers (green in H and I). Scale bars, A, G, 50 µm; inset in D, 200 µm. Scales for the panels in the middle and right columns

are the same as in the left column except for the inset.

172x182mm (150 x 150 DPI)

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Figure 5. Gene expression patterns for the monoamine receptors in lateral habenular subnuclei. Coronal sections of rat habenula showing the mRNA expression of dopamine type 2 receptor (Drd2) (magenta in A, C, D, F) and serotonin type 2c receptor (Htr2c) (magenta in G, I, J, L) relative to the immunostaining for

tyrosine hydroxylase (TH) (green in E, F, K, L) and HuC/D (green in B, C, H, I). Dotted lines in panels C, F, I, L indicate the boundaries between the subnuclei in the medial and lateral habenulae. Scale bars, A, D, G,

J, 200 µm. Scales for the panels in the middle and right columns are the same as in the left column. 196x269mm (150 x 150 DPI)

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Figure 6. Gene expression patterns for the lateral habenula- and habenula-specific genes. Coronal section of

rat habenula showing the mRNA expressions of protocadherin 10 (Pcdh10) (magenta in A, C) and G-protein coupled receptor 151 (Gpr151) (magenta in D, F) relative to the immunostaining for HuC/D (green in B, C, E, F). Dotted lines in panels C and F indicate the boundaries between the subnuclei in the medial and lateral habenulae. Scale bars, A, D, 200 µm. Scales for the panels in the middle and right columns are the same as

in the left column. 214x153mm (150 x 150 DPI)

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Figure 7. Gene expression profile of the habenula subnuclei. Schematic diagram summarizing the gene expression patterns in the subnuclei of the medial habenula (pink), medial division of the lateral habenula (light green), and lateral division of the lateral habenula (dark green). Abbreviations are the same as in

Figure 1 except for MHbCd (central part of MHb, dorsal region) and MHbCv (central part of MHb, ventral region). The number of plus signs indicates the relative fluorescent signal intensity. Three levels were defined within the detectable range from the lowest level just above background (“weak”) to the most intense signal detected (“strong”), as follows: -, not detectable; +, weak expression; ++, intermediate expression; +++, strong expression. Signal intensity can be compared between brain regions, but not

between probes. 182x225mm (150 x 150 DPI)

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Gene expression pattern for the habenula-specific gene G-protein coupled receptor 151 (Gpr151). A coronal section of rat habenula showing the mRNA expressions of Gpr151 (magenta) relative to the immunostaining

for HuC/D (green). Dotted lines indicate the boundaries between the subnuclei in the medial and lateral

habenulae. 199x199mm (300 x 300 DPI)

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Gene expression pattern for the habenula-specific gene G-protein coupled receptor 151

(Gpr151). A coronal section of rat habenula showing the mRNA expressions of Gpr151

(magenta) relative to the immunostaining for HuC/D (green). Dotted lines indicate the

boundaries between the subnuclei in the medial and lateral habenulae.

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molecular characterization of the subnuclei in rat habenula

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