ORIGINAL ARTICLE

Orexinergic innervation of the extended amygdalaand basal ganglia in the rat

Oliver Schmitt • Kamen G. Usunoff •

Nikolai E. Lazarov • Dimitar E. Itzev •

Peter Eipert • Arndt Rolfs • Andreas Wree

Received: 17 February 2011 / Accepted: 16 August 2011 / Published online: 21 September 2011

� Springer-Verlag 2011

Abstract The orexinergic system interacts with several

functional states of emotions, stress, hunger, wakefulness

and behavioral arousal through four pathways originating

in the lateral hypothalamus (LH). Hundreds of orexinergic

efferents have been described by tracing studies and direct

immunohistochemistry of orexin in the forebrain, olfactory

regions, hippocampus, amygdala, septum, basal ganglia,

thalamus, hypothalamus, brain stem and spinal cord. Most

of these tracing studies investigated the whole orexinergic

projection to all regions of the intracranial part of the CNS.

To identify the orexinergic efferents at the subnuclear level

of resolution, we focussed on the orexinergic target in the

amygdala, which is substantially involved in the LH output

and contributes mostly to the functional outcome of the

orexinergic system and the basal ganglia. Immunohisto-

chemical identification of axonal orexin A and orexin B in

male adult rats has been performed on serial sections. In

the extended amygdala many new orexinergic targets were

found in the anterior amygdaloid area (dense), anterior

cortical nucleus (moderate), amygdalostriatal transition

region (moderate), basolateral regions (moderate), baso-

medial nucleus (moderate), several bed nucleus of the

stria terminals regions (few to dense), central amygdaloid

subdivisions (dense), posteromedial cortical nucleus

(moderate) and medial amygdaloid subnuclei (dense).

Furthermore, the entopeduncular nucleus has been newly

identified as another target for orexinergic fibers with a

high density. These results suggest that subdivisions and

subnuclei of the extended amygdala are specific targets of

the orexinergic system.

Keywords Orexin � Hypocretin � Hypothalamus �Amygdala � Striatum � Globus pallidus � Substantia nigra �Bed nucleus of stria terminalis

Abbreviations

3 Oculomotor nc.

3V Third ventricle

4 Trochlear nc.

6 Abducens nc.

7 Facial nc.

10 Dors. motor nc. vagus

12S Hypoglossal nc.

AAA Ant. amygdaloid area

AAD Ant. amygdaloid area dors. p.

AASh Ant. amygdaloid area shell area

AAV Ant. amygdaloid area vent. p.

ABC Avidin–biotin–horseradish peroxidase

Ac Accumbens nc.

AcbC Accumbens nc. core

AcbSh Accumbens nc. shell

ACo Ant. cortical amygdaloid nc.

Abbreviations used within the abbreviation list: ant.: anterior,

div.: division, dors: dorsal lat.: lateral, med.: medial, p.: part,

post.: posterior, nc.: nucleus.

O. Schmitt (&) � P. Eipert � A. Wree

Institute of Anatomy, University of Rostock,

P. O. Box 100888, 18055 Rostock, Germany

e-mail: schmitt@med.uni-rostock.de

K. G. Usunoff � N. E. Lazarov

Department of Anatomy and Histology, Faculty of Medicine,

Medical University, 1431 Sofia, Bulgaria

K. G. Usunoff � D. E. Itzev

Institute of Neurobiology, Bulgarian Academy of Sciences,

1113 Sofia, Bulgaria

A. Rolfs

Albrecht Kossel Institute for Neuroregeneration,

University of Rostock, 18147 Rostock, Germany

123

Brain Struct Funct (2012) 217:233–256

DOI 10.1007/s00429-011-0343-8

AD Anterodors. thalamic nc.

AHAA Ant. hypothalamic area ant. p.

AHA Ant. hypothalamic area

AHi Amygdalohippocampal area

AHiAL Amygdalohippocampal area anterolateral p.

AHn Ant. hypothalamic nc.

AI Agranular insular cortex

Am Amygdala

AM Anteromed. thalamic nc.

Amb Ambiguus nc.

AON Ant. olfactory nc.

AP Area postrema

APT Ant. pretectal nc.

Arc Arcuate nc.

AStr Amygdalostriatal transition area

ATg Ant. tegmental nc.

AV Anterovent. thalamic nc.

AVPe Anterovent. periventricular nc.

B Bregma

B9 B9 serotonin cells

BAC Bed nc. ant. commissure

Bar Barringtons nc.

BIC Nc. brachium inferior colliculus

Ll Lat. lemniscus

BLA Ant. basolat. nc.

BLP Posterior basolat. nc.

BLV Ventral basolat. nc.

BL Basolat. nc.

BMA Ant. basomed. nc.

BMP Posterior basomed. nc.

BM Basomed. nc.

BST Bed nc. stria terminalis

BSTAL BST ant. lat. area

BSTAM BST ant. med. p.

BSTIA BST intraamygdaloid div.

BSTLD BST lat. div. dors. p.

BSTLI BST lat. div. intermediate p.

BSTLJ BST lat. div. juxtacapsular p.

BSTLP BST lat. div. posterior p.

BSTLV BST lat. div. vent. p.

BSTL BST lat. div.

BSTMA BST med. div. ant. p.

BSTMPI BST med. div. posterointermediate p.

BSTMPL BST med. div. posterolat. p.

BSTMPM BST med. div. posteromed. p.

BSTMV BST med. div. vent. p.

BSTM BST med. div.

BSTP BST posterior div.

BSTV BST posterior div. vent. nc.

BSTd BST dors. nc.

BSTrL BST lat. div. rostral p.

BSTrm BST med. div. rostral p.

CA1 Field CA1 hippocampus

CA2 Field CA2 hippocampus

CA3 Field CA3 hippocampus

CAn Cortical amygdaloid nc.

CCL1 Cerebral cortex layer 1

CCL2 Cerebral cortex layer 2

CCL3 Cerebral cortex layer 3

CCL4 Cerebral cortex layer 4

CCL5 Cerebral cortex layer 5

CCL6 Cerebral cortex layer 6

Ce Central amygdaloid nc.

CeC Capsular p.

CeL Central amygdaloid nc. lat. div.

CeM Central amygdaloid nc. med. div.

CERC Cerebellar cortex

Cg Cingulate cortex

CG Central gray

CIC Central nc. inferior colliculus

Cl Claustrum

CL Centrolat. thalamic nc.

CM Central med. thalamic nc.

CnF Cuneiforme nc.

COS Cochlear system

cp Cerebral peduncle

CPu Caudate putamen

cpv3 choroid plexus 3rd ventricle

cpv4 choroid plexus 4th ventricle

CVLM Caudal ventrolat. medulla

D3V Dorsal 3rd ventricle

DA Dors. hypothalamic area

DCIC Dors. cortex inferior colliculus

DC Dors. cochlear nc.

DEn Dors. endopiriform nc.

DG Dentate gyrus

Dk Nc. Darkschewitsch

DLGl Dors. geniculate nc. lat. p.

DM Dorsomed. hypothalamic nc.

DMDM Dorsomed. hypothalamic nc. dorsomed. p.

DNC Deep cerebellar nuclei

DPGi Dors. paragigantocellular nc.

DpMe Deep mesencephalic nc.

DPPn Dors. peduncular pontine nc.

DR Dors. raphe nc.

DRC Dors. raphe nc. caudal p.

DRcep Dors. raphe nc. central p.

DRI Dors. raphe nc. interfascicular p.

DRlw Dors. raphe nc. lat. wing

DRr Dors. raphe nc. rostral p.

DTg Dors. tegmental nc.

DTgC Dors. tegmental nc. central p.

E Ependyma and subependymal layer

ECIC External cortex inferior colliculus

234 Brain Struct Funct (2012) 217:233–256

123

EnN Endopiriform system

EP Entopeduncular nucleus

Ent Entorhinal cortex

f Fornix

F Nc. fields Forel

Fl Flocculus

FrA Frontal association cortex

FS Fundus striatum

G Gelatinosus thalamic nc.

Ge5 Gelatinous layer caudal spinal trigeminal nc.

Gi Gigantocellular reticular nc.

GI Granular insular cortex

GiA Gigantocellular reticular nc. alpha p.

GiV Gigantocellular reticular nc. vent. p.

GN Geniculate nucleus

GP Globus pallidus

Gra Gracile nc.

GraD Gracile nc. dors. p.

HDB Nc. horizontal limb diagonal band

HIPP Hippocampus

I Intercalated nc. amygdala

IAM Interoanteromed. thalamic nc.

Ilc Internal capsule

ICj Islands Calleja

ICOL Inferior colliculus

IF Interfascicular nc.

IGL Intergeniculate leaf

IG Indusium griseum

ILN Intralaminar nuclei

IMD Intermediodors. thalamic nc.

IM Intercalated amygdaloid nc. main p.

InC Interstitial nc. Cajal

i.p. intra peritoneal

IP Interpeduncular nc.

IPA Interpeduncular nc. apical subnc.

IPDM Interpeduncular nc. dorsomed. subnc.

IPI Interpeduncular nc. intermediate subnc.

IPL Interpeduncular nc. lat. subnc.

IPR Interpeduncular nc. rostral subnc.

IPRc Interpeduncular nc. central subnc.

IRt Intermediate reticular nc.

KF Koelliker Fuse nc.

LA Lat. amygdaloid nc.

LAH Lateroant. hypothalamic nc.

LC Locus coeruleus

LD Laterodors. thalamic nc.

LDTg Laterodors. tegmental nc.

LG Lat. geniculate complex

LGP Lat. globus pallidus

LH Lat. hypothalamic area

LHb Lat. habenular nc.

LIR Linear nc. raphe

LM Lat. mammillary nc.

LOT Nc. lat. olfactory tract

LP Lat. posterior thalamic nc.

LPB Lat. parabrachial nc.

LPGi Lat. paragigantocellular nc.

LPN Lat. preoptic nc.

LPO Lat. preoptic area

LRt Lat. reticular nc.

LS Lat. septal nc.

LSD Lat. septal nc. dors. p.

LSI Lat. septal nc. intermediate p.

LSV Lat. septal nc. vent. p.

LV Lateral ventricle

MCPO Magnocellular preoptic nc.

MDC Mediodors. thalamic nc. central p.

MDL Mediodors. thalamic nc. lat. p.

MDM Mediodors. thalamic nc. med. p.

MD Mediodors. thalamic nc.

MdD Medullary reticular nc.

MdDd Medullary reticular nc. dors. p.

MdV Medullary reticular nc. vent. p.

MED Med. group dors. thalamus

Me Med. amygdaloid nc.

ME Median eminence

Me5 Mesencephalic trigeminal nc.

MeAD Med. amygdaloid nc. anterodors. p.

MePD Med. amygdaloid nc. posterodors. p.

MePV Med. amygdaloid nc. posterovent. p.

MGP Med. globus pallidus

MG Med. geniculate nc.

MHb Med. habenular nc.

MM Med. mammillary nc. med. p.

MMn Med. mammillary nc.

MMnm Med. mammillary nc. median p.

MnPO Median preoptic nc.

MnR Median raphe nc.

Mo5 Motor trigeminal nc.

MOB Olfactory bulb A16

MPA Med. preoptic area

MPB Med. parabrachial nc.

MPO Med. preoptic nc.

MPT Med. pretectal nc.

MRF Mesencephalic reticular formation

MS Med. septal nc.

mt Mammillothalamic tract

NADPHd Nicotinamide adenine dinucleotide hydrogen

phosphate diaphorase

NGS Normal goat serum

NLL Nuclei lat. lemniscus

ON Olivary nc.

ONn Olfactory nuclei

opt Optic tract

Brain Struct Funct (2012) 217:233–256 235

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OrC Orbital cortex

OT Nc. optic tract

OX Orexin, hypocretin

OX-A Orexin A (Hypocretin 1)

OX-B Orexin B (Hypocretin 2)

OX1R Orexin 1 receptor

OX2R Orexin 2 receptor

Pa Paraventricular nc.

PaAM Paraventricular hypothalamic nc.

magnocellular p.

PAG Periaqueductal gray

PaPc Paraventricular hypothalamic nc.

parvicellular p.

PB Parabrachial nc.

PBG Parabigeminal nc.

PBP Parabrachial pigmented nc.

PBS Phosphate buffered saline

PC Paracentral thalamic nc.

PCom Nc. posterior commissure

PCRt Parvicellular reticular nc.

PDP Posterodors. preoptic nc.

Pe Periventricular hypothalamic nc.

PeF Perifornical area

PeP Posterior periventricular nc.

PePO Preoptic periventricular nc.

Pir Piriform cortex

PF Parafascicular thalamic nc.

PFx Lat. hypothalamic area perifornical p.

PG Pontine gray

PHA Posterior hypothalamic area

PLCo Posterolat. cortical nc.

PLH Posterolat. hypothalamus

PMCo Posteromed. cortical nc.

PMn Paramedian reticular nc.

PN Paranigral nc.

PnO Pontine reticular nc. oral p.

PnR Pontine raphe nc.

Po Posterior thalamic nuclear group

PP Peripeduncular nc.

PPit Posterior lobe pituitary

PPN Peduncularpontine nc.

PPT Posterior pretectal nc.

PPTg Pedunculopontine tegmental nc.

Pr Prepositus nc.

Pr5 Principal sensory trigeminal nc.

PRN Pontine reticular nc.

PS Parastriatal nc.

PSTh Parasubthalamic nucleus

PT Paratenial thalamic nc.

PtA Parietal association cortex

PV Paraventricular thalamic nc.

PVHd Paraventricular hypothalamic nc. descending

div.

R Red nc.

RAN Raphe nuclei

RCh Retrochiasmatic area

Re Reuniens thalamic nc.

RETn Reticulotegmental nc.

Rh Rhomboid thalamic nc.

RhN Rhomboid nc.

RLi Rostral linear nc. raphe

RMg Raphe magnus nc.

Ro Nc. Roller

ROb Raphe obscurus nc.

RPa Raphe pallidus nc.

Rt Reticular thalamic nc.

RVLM Rostral ventrolat. medulla

S Subiculum

SC Superior colliculus

SCh Suprachiasmatic nc.

SCO Subcommissural organ

SFO Subfornical organ

SFi Septofimbrial nc.

SHi Septohippocampal nc.

SHy Septohypothalamic nc.

SI Substantia innominata

sm Stria medullaris thalamus

SMT Submammillothalamic nc.

SN Substantia nigra A9

SNC Substantia nigra compact p.

SNL Substantia nigra lat. p.

SNR Substantia nigra reticular p.

SO Supraoptic nc.

Sol Nc. solitary tract

sox Supraoptic decussation

Sp5C Spinal trigeminal nc. caudal p.

Sp5O Spinal trigeminal nc. oral p.

Sp5nc Spinal trigeminal nc.

SPF Subparafascicular thalamic nc.

STh Subthalamic nc.

STLD Equivalent to BSTLD

STLJ Equivalent to BSTLJ

STLP Equivalent to BSTLP

STLV Equivalent to BSTLV

STMA Equivalent to BSTMA

STMV Equivalent to BSTMV

SubI Subincertal nc.

SuM Supramammillar nc.

SuOLi Superior olive

TC Tuber cinereum area

TGAC Central tegmental field

TM Tuberomammillary nc.

236 Brain Struct Funct (2012) 217:233–256

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TS Triangular septal nc.

TT Tenia tecta

TuLH Tuberal region lat. hypothalamus

TuO Olfactory tubercle

Tz Nc. trapezoid body

VC Ventral cochlear nc.

VDB Nc. vertical limb diagonal band

VES Vestibular system

VLG Ventral lat. geniculate nc.

VLPAG Ventrolat. periaqueductal gray

VL Ventrolat. thalamic nc.

VMHA Ventromed. hypothalamic nc. ant. p.

VMHP Ventromed. hypothalamic nc. posterior p

VMH Ventromed. hypothalamic nc.

VPPn Ventral peduncular pontine nc.

VP Ventral pallidum

VR Visual regions

VTA Ventral tegmental area A10

VTg Ventral tegmental nc.

Vnc Vestibular nuclei

ZI Zona incerta

ZID Zona incerta dors. p.

ZIV Zona incerta vent. p.

Introduction

Early in 1998, two independently working research teams

(de Lecea et al. 1998 and Sakurai et al. 1998) reported two

novel excitatory neuropeptide hormones in the hypothala-

mus. De Lecea et al. (1998) named them ‘‘hypocretins’’

(hypothalamic members of the incretin family, reflecting

their structural similarity to the hormone secretin, i.e.,

hypothalamic secretin), whereas Sakurai et al. (1998)

named them ‘‘orexins’’ (after the Greek word ‘‘orexis’’,

which means appetite). We chose to use orexin (OX).

There are two distinct OXs: OX-A and OX-B. The former

contains 33 amino acid residues whereas the latter 28,

OX-A binds to both OX receptors OX1R and OX2R with

similar affinity; OX-B on the other hand mainly binds to

OX2R receptors and has a five times lower affinity for

OX1R (Marcus et al. 2001; Bayer et al. 2002, 2004;

Langmead et al. 2004). Postsynaptic actions of OXs on

their numerous neuronal targets throughout the CNS are

excitatory (Horvath et al. 1999; Selbach and Haas 2006).

A central administration of OX-A increases food intake

(increase of arousal, alertness, attention and muscle tone).

Peripheral application of OX-A (nasal sprays) stimulates

wakefulness and energy expenditure.

The OX-containing neuronal perikarya are located

exclusively in the hypothalamus (caudal aspect of the lat-

eral hypothalamic area (lateral hypothalamus consisting of

peduncular (PLH), the tuberal (TuLH), the perifornical

(PeFLH) and the juxtapara-ventricular part (JPLH)), peri-

fornical area, dorsomedial hypothalamic nucleus). These

locations have been immunohistochemically characterized

in rodents (Broberger et al. 1998; Elias et al. 1998; Peyron

et al. 1998; Chen et al. 1999; Nambu et al. 1999;

McGranaghan and Piggins 2001; Mintz et al. 2001; Novak

and Albers 2002; Khorooshi and Klingenspor 2005) among

other species. The hypothalamus of the rat contains

approximately 6,400–6,700 OX-expressing neurons

(Modirrousta et al. 2005; Henny and Jones 2006).

Published reports from tracing, ultrastructural and

chemoarchitectonic studies have shown that the small OX

neuronal population emits numerous projections through-

out the neuraxis from the olfactory bulb and cerebral cortex

to the spinal cord (Broberger et al. 1998; Elias et al. 1998;

Peyron et al. 1998; Cutler et al. 1999; Date et al. 1999,

2000; Horvath et al. 1999; van den Pol 1999; Nambu et al.

1999; Bayer et al. 2001; Baldo et al. 2003; Caillol et al.

2003; Guan et al. 2003; Sakamoto et al. 2004; Espana et al.

2005; Fadel et al. 2005; Kirouac et al. 2005; Lee et al.

2005; Stoyanova and Lazarov 2005; Balcita-Pedicino and

Sesack 2007; Oldfield et al. 2007; Shibata et al. 2008; Shin

et al. 2008). These findings were corroborated by evalua-

tions of development (Steininger et al. 2004; Steininger and

Kilduff 2005) neurophysiological investigations (Bayer

et al. 2002, 2004; Fadel and Deutch 2002; Martin et al.

2002; Zheng et al. 2005; Bisetti et al., 2006) and com-

parative approaches (Iqbal et al. 2001; McGranaghan and

Piggins 2001; Mintz et al. 2001; Moore et al. 2001; Novak

and Albers 2002; Horowitz et al. 2005; Takakusaki et al.

2005; Su et al. 2008; Hsu and Price 2009). In addition, the

orexinergic neurons raise the release of major neurotrans-

mitters like acetylcholine (Fadel and Deutch 2002) and

noradrenaline (Hirota et al. 2001).

The far-scattered projections of OX and expression of

OX1R and OX2R (Marcus et al. 2001) suggest multiple

regulatory pathways that interact mutually to control

complex physiological functions. The OX-neurons receive

numerous afferent projections that in some cases recipro-

cate their efferent connections (Winsky-Sommerer et al.

2004; Sakurai et al. 2005; Henny and Jones 2006; Yoshida

et al. 2006, and references therein).

Aims

A detailed evaluation of OX-A- and OX-B-immunoreac-

tive neurons in the hypothalamus of the rat, and their axons

in the extended amygdala as well as the basal ganglia has

been performed. The scope of the study is a semiquanti-

tative analysis with atlas-based 2D- and 3D-visualization

of OX-fiber terminations, focussing on the amygdala,

components of the basal ganglia and regions where new

Brain Struct Funct (2012) 217:233–256 237

123

findings were made or obvious differences in publications

occur. The results from studies on OX-projections by

Peyron et al. (1998); Cutler et al. (1999); Nambu et al.

(1999) have also been compared with the findings from the

current study.

Materials and methods

Animal and tissue preparation

The experiments were performed on adult male Wistar rats,

weighing 250–300 g. All housing facilities were supervised

and approved by the Ethic Commissions at the Medical

University of Sofia, Bulgaria, and the University of

Rostock, Germany, and were consonant with the guidelines

established by the National Institute of Health (NIH). The

animals were deeply anesthetized with Ketanest (50 mg/

kg, i.p.) and transcardially perfused first with 150 ml cold

0.05 M phosphate buffered saline (PBS), followed by

500 ml 4% paraformaldehyde in 0.1 M phosphate buffer

(PB), pH 7.4. After perfusion, the brains were removed and

postfixed in the same fixative solution for several hours.

Afterward, the brains were blocked in the coronal plane

and then cryoprotected in 20% sucrose in PB overnight at

4�C. Serial 30 lm thick sections were cut on a Reichert

Jung freezing microtome at -20�C and were collected in a

free floating state in PB. After rinsing in 0.1 M PBS, the

sections were separated into four series and then processed

for OX-immunohistochemistry.

Immunohistochemistry

The immunohistochemical staining procedure was per-

formed applying the ABC (avidin–biotin–horseradish per-

oxidase) method (Hsu et al. 1981). The sections were

washed in PBS/Triton X-100, treated with hydrogen per-

oxide (1.2% in absolute methanol; 30 min) to inactivate

endogenous peroxidase, and the background was blocked

with 2% normal goat serum (NGS) in PBS for 30 min. The

sections were incubated for 24 h at room temperature with

the polyclonal primary antibodies, rabbit anti-OX-A

(Oncogene, Cambridge, MA, USA); diluted 1:2,000 in a

solution of 0.1% bovine serum albumin, 10% NGS and

0.01% sodium azide), first and third groups, and rabbit anti-

OX-B (Oncogene; 1:500), second and fourth groups. In one

experiment, all sections through the amygdala (Am) and

bed nucleus of stria terminalis (BST) were immunostained

only with anti-OX-B antibody (see ‘‘Discussion’’). After

rinsing in 0.1 M PBS containing 0.025% Triton X-100

(3 9 10 min), sections were incubated with the secondary

antibody, biotinylated goat anti-rabbit IgG (Dianova,

Hamburg, Germany) at a dilution of 1:500 for 2 h at room

temperature. Following washing in PBS 0.1 M with 0.05%

Triton X-100, the ABC complex (Vector Laboratories,

Burlingame, CA, USA; 6.25 ll/ml of each compound in

PBS) was applied for 2 h at room temperature. After a final

rinsing, peroxidase activity was visualized using 2.4% SG

substrate kit (Vector Laboratories) for 5 min. The sections

were then mounted on chrome alum gelatin slides, air

dried, dehydrated in a graded series of ethanols, cleared in

xylene, and coverslipped with Entellan (Merck, Darmstadt,

Germany). Every third from the immunostained sections

was counterstained with 1% Neutral Red (Sigma, St. Louis,

MO, USA) to reveal the precise cytoarchitectonic orien-

tation of the stained neuronal population. The counter-

staining was especially useful for delineation of small

structures (bed nucleus of the anterior commissure), that

also display variability in their position (intercalated nuclei

of the amygdala, islands of Calleja). Negative controls

included omission of the primary antibody and/or its

replacement with non-immune normal serum as well as

antigen–antibody preabsorption experiments with the

respective native antigens.

Stereology, image analysis and visualization

The atlases of Swanson (1999), Paxinos and Watson (2007)

and Paxinos et al. (1999) were consulted for the precise

delineation of the investigated structures. The samples

were observed with a Zeiss Axioplan 2 research micro-

scope (Carl Zeiss MicroImaging GmbH, Gottingen, Ger-

many). Photomicrographs of selected fields were taken

with an AxioCam MRc digital camera and saved in TIF

format. Overview images were generated with the Mirax-

Scan virtual microscope (Carl Zeiss MicroImaging GmbH,

Gottingen, Germany). Measurements of the size of

immunoreactive perikarya in the lateral hypothalamus were

performed. We used Stereo Investigator (v8) (Micro-

Brightfield, Williston, VT, USA) for size measurements.

Regions that contained orexinergic terminals were traced

using a 3-axes motorized Olympus BX51 (Olympus

Europa Holding GmbH, Hamburg, Germany) videomicro-

scope (CX9000 videocamera, Microbrightfield, Williston,

VT, USA) with a 209 objective (UPlanFL N) and digital

images of the traced regions were exported for further

processing. Semiquantification was performed by classi-

fying the OX-densities (see Table 1). The densities of

terminals were calculated as the area covered by orexin

immunoreactivity divided by the total area of tissue within

the boundary of a region (area fraction) using Matlab

scripts (version 2008a) (MathWorks, Natick, MA, USA).

The minimum and maximum area fractions of all regions

were normalized and devided equally in the three ranges

that were named few, moderate and dense. 2D- and 3D-

visualizations were realized with JAVATM

(version

238 Brain Struct Funct (2012) 217:233–256

123

Table 1 The relative density of OX-positive axons in various regions of the rat brain

A1 A2 A3 A4 # A1 A2 A3 A4 # A1 A2 A3 A4 # A1 A2 A3 A4 #

3 1 3 2 BSTLI 2 1 DLGl 2 1 IPI 1 1

6 1 1 2 BSTLJ 1 1 DM 2 2 3 3 IPL 3 1

7 2 2 2 BSTLP 3 1 DMDM 2 1 IPR 1 1

10 2 3 2 BSTLV 1 2 2 DNC 1 1 IPRc 1 1

12S 1 1 1 3 BSTM 3 1 DPGi 2 1 IRt 2 2 2

AAA 2 2 2 BSTMA 3 1 DpMe 1 1 KF 3 1

AAD 3 1 BSTMPI 2 1 DPPn 1 1 LA 1 1 1 3

AASh 3 1 BSTMPL 2 2 2 DR 3 3 2 LAH 3 1

AAV 3 1 BSTMPM 3 3 2 DRC 2 1 LC 3 2 2

Ac 1 1 BSTMV 3 1 DRcep 2 1 LD 1 1 2

AcbC 1 1 2 BSTP 2 1 DRI 3 1 LDTg 3 3 2 3

AcbSh 2 2 2 BSTrL 2 1 DTg 3 2 2 LG 1 1 2

ACo 2 1 BSTrm 3 1 DTgC 2 1 LGP 2 2 2

AD 1 1 2 BSTV 3 1 E 2 1 LH 3 3 3 3

AHA 1 1 CA1 1 1 2 ECIC 3 1 LHb 1 1 2

AHAA 2 1 CA2 1 1 2 EnN 2 2 2 LIR 2 1

AHi 2 2 2 3 CA3 1 1 2 Ent 1 1 ll 3 1

AHn 3 1 CAn 2 2 2 EP 3 1 LM 3 3 2

AI 1 1 CCL1 2 2 2 F 1 1 LOT 2 2 2

AM 1 1 CCL2 1 1 2 Fl 2 1 LP 1 1 2

Amb 1 1 CCL3 1 1 2 FrA 1 1 LPB 3 3 2

AON 2 2 2 CCL4 1 1 2 FS 2 2 2 LPGi 2 3 2

AP 2 3 2 CCL5 1 1 2 G 1 1 LPN 3 1

APT 1 2 2 CCL6 2 2 2 Ge5 3 1 LPO 3 3 2

Arc 3 3 2 Ce 3 3 2 GI 2 1 LRt 2 2 2

AStr 3 3 2 3 CeC 3 1 Gi 2 2 2 LS 3 1

ATg 1 1 CeL 3 1 GiA 3 1 LSD 3 1

AV 1 1 2 CeM 3 1 GiV 3 1 LSI 2 1

AVPe 2 1 CERC 1 1 GP 1 1 2 LSV 3 1

B9 3 1 CG 3 1 Gra 1 1 MCPO 3 1

BAC 3 1 2 Cg 1 1 GraD 1 1 MD 1 1

Bar 3 3 2 CIC 2 1 HDB 2 1 MDC 1 1

bic 1 1 Cl 3 3 1 3 HIPP 1 1 MdD 1 1

BL 1 1 2 3 CL 2 2 2 I 1 2 2 MdDd 3 1

BLA 2 1 CM 3 3 3 3 IAM 1 3 2 MDL 1 1

BLP 2 1 CnF 1 1 2 ICj 1 1 MDM 1 1

BLV 2 1 COS 1 1 ICOL 1 1 MdV 2 1

BM 1 1 2 CPu 1 1 1 3 IF 2 1 ME 3 2 2 3

BMA 2 1 cpv3 1 1 IG 3 3 2 Me5 2 2 2

BMP 2 1 cpv4 1 1 IGL 2 1 MeAD 3 1

BST 2 1 CVLM 2 1 ILN 3 1 MePD 3 1

BSTAL 2 1 DA 1 1 IM 1 1 MePV 3 1

BSTAM 3 1 DC 3 3 2 IMD 3 3 2 MG 1 1 2 3

BSTd 2 1 DCIC 2 1 InC 1 1 MGP 1 3 2

BSTIA 3 1 DEn 3 1 IP 2 1 MHb 1 1

BSTL 3 1 DG 1 1 2 IPA 3 1 MM 1 1

BSTLD 2 1 Dk 2 2 2 IPDM 3 1 MMn 1 1

MMnm 1 1 PePO 2 1 RETn 1 1 SPF 3 1

MnPO 2 2 2 PF 1 1 Rh 3 1 STh 1 2 3 3

MnR 3 3 2 PG 1 1 RhN 3 2 2 SubI 3 1

Mo5 1 2 2 3 PHA 2 3 2 RLi 1 3 2 SuM 1 3 2

Brain Struct Funct (2012) 217:233–256 239

123

1.6.0_20) (Oracle Corporation, Redwood Shores, CA,

USA) and VTK (version 5.6.1) (Kitware, New York, USA)

within neuroVIISAS (Schmitt et al. 2011).

Results

Cytoarchitecture of the OX-A-

and OX-B-immunoreactive neurons

No differences were found in the regional distribution of

OX-A- and OX-B-immunoreactive cells. The immuno-

positive OX-A fibers were analyzed semiquantitatively

whereas OX-B immunostains were examined without fur-

ther analysis. The following descriptions of fibers are

related to OX-A immunopositive fibers. The OX-immu-

nopositive cells are distributed exclusively in the tuberal

part of the hypothalamus. The rostrocaudal dimension of

the OX neuronal group is approximately 1.6 mm, from

Bregma (B) -2.3 to 2.4–Bregma -3.9 to 4.0, according to

the stereotaxic atlases of Paxinos and Watson (2007) and

Paxinos et al. (1999) (Figs. 1, 2). Rostrally the border of

the OX-immunopositive neuronal group corresponds to the

caudal border of the paraventricular hypothalamic nucleus,

and caudally it reaches the initial (ventrally located) por-

tion of the mamillothalamic tract (mt). The majority of

the immunoreactive neurons are located between planes

B -2.7 and B -3.5 (Figs. 1, 2, and 9). Here, the OX-cells

are comprised of a prominent group stretched mediolater-

ally that might be subdivided in central, lateral and medial

divisions. The central division located dorsal to the fornix

column builds the perifornical nucleus (interperifornical

nucleus, lateral hypothalamus perifornical area) (PeF) and

contains densely packed neurons (Fig. 1). A considerably

lower number of immunostained neurons are located

immediately ventral to the fornix. However, OX-positive

Table 1 continued

A1 A2 A3 A4 # A1 A2 A3 A4 # A1 A2 A3 A4 # A1 A2 A3 A4 #

MOB 2 2 2 Pir 2 2 2 3 RMg 1 2 2 SuOLi 1 1

MPA 3 3 2 PLCo 2 1 Ro 1 1 TC 3 1

MPB 2 3 2 PLH 3 1 ROb 2 1 TGAC 2 1

MPO 3 3 2 PMCo 2 1 RPa 1 1 2 TM 3 3 2

MPT 1 1 PMn 2 1 Rt 1 1 TS 1 1

MRF 2 1 PN 2 1 RVLM 3 1 TT 2 2 2

MS 2 1 PnO 1 1 S 1 1 TuLH 2 1

NLL 1 1 PnR 1 1 2 SC 2 1 2 TuO 1 1

ON 1 1 2 Po 2 1 SCh 2 1 2 3 Tz 1 1 1 3

ONn 1 1 PP 2 2 2 SCO 1 1 VC 2 2 2

OrC 1 1 PPit 1 1 SFi 1 1 2 VDB 3 1

OT 1 1 2 PPN 3 1 SFO 2 2 2 VES 1 3 2

Pa 2 1 PPT 1 1 SHi 1 1 VL 1 1

PaAM 1 1 PPTg 1 1 SHy 1 1 VLG 2 1

PAG 2 1 Pr 1 1 2 SI 2 3 2 VLPAG 2 1

PaPc 3 1 Pr5 2 2 2 sm 2 1 VMH 3 2 2

PB 2 1 PRN 1 1 SMT 3 1 VMHA 2 1

PBG 1 1 2 PS 2 1 SN 1 1 VMHP 3 1

PBP 2 1 PT 1 1 2 SNC 1 2 3 3 Vnc 1 1

PC 2 1 PtA 1 1 SNL 3 1 VP 1 1

PCom 1 1 PV 3 3 3 3 SNR 1 1 2 VPPn 3 1

PCRt 1 2 2 PVHd 3 1 SO 2 3 2 3 VR 1 1

PDP 2 1 R 1 1 2 Sol 2 3 2 VTA 2 1 2

Pe 3 1 RAN 2 1 Sp5C 1 1 VTg 1 1

PeF 2 3 2 RCh 2 2 2 Sp5nc 1 3 2 ZI 3 3 2

PeP 2 1 Re 3 3 2 Sp5O 2 1 ZID 3 1

ZIV 2 1

The abbreviations are sorted and regions are listed continous from top to bottom proceeding to the right of each gray column. #, number of observed connections: 1, low; 2,

moderate; 3, high relative density of OX-positive terminals. A1, Cutler et al. (1999) describe densities using terms and symbols—(absent), ?, ??, ???; A2, Nambu et al. (1999)

use fiber drawings for semiquantification and symbols—(very sparse), ?, ??, ???, A3, Peyron et al. (1998) use fiber drawings for semiquantification and symbols—

(insignificant number of fibers), ?, ??, ???, ????, ?????; A4, results of this study. To facilitate comparison with the study of Cutler et al. the relative densities ????

and ????? of Nambu et al. and Peyron et al. were summarized into density group 3

240 Brain Struct Funct (2012) 217:233–256

123

neurons are also seen more ventrally in the tuber cinereum

within the tuberal region of LH (TuLH) (Fig. 1). The lat-

eral division is located in the dorsal part of the LH and the

peduncular part of LH (PLH) (Figs. 1, 2). It comprises a

substantial number of OX-positive cells that are, however,

less densely arranged than the neurons in the perifornical

nucleus. Sagittaly the lateral division reaches the internal

capsule. The medial division contains a smaller number of

OX-neurons. They are distributed on the territory of the

dorsomedial hypothalamic nucleus (DM) (Figs. 1, 2).

Some of the immunostained neurons are located close to

the ependyma of the third ventricle.

In rostral direction, the number of OX-neurons dimin-

ishes gradually. Near the rostral pole of the OX-population

the OX-neurons do not form a compact group, but disperse

in lateral, dorsal and ventral direction (Fig. 1a). The lateral

group is more prominent. Its neurons are scattered in the

LH and reach the medial border of the entopeduncular

Fig. 1 a Overview of OX-B

immunoreactivity in the tuberal

part of the lateral hypothalamus.

In the rostral region of the LH

(B -2.0) the OX-B-containing

perikarya have a low-density.

b Detail of a left LH with

immunoreactive perifornical

perikarya. c The diffuse and

dense pattern of

immunoreactive perifornical

OX-B nerve fibers and fiber

origins of the perikarya are

visible. d Overview at a more

caudal level of LH at B -3.0.

OX-B-containing perikarya are

located in the dorsomedial

hypothalamic nucleus (DM), the

perifornical area (PF), the LHand the tuberal region of LH

(TuLH). e Low magnification at

the caudal level of LH at B =

-3.96. At the caudal border of

the LH around the subthalamic

nucleus (STh) and the

parasubthalamic nucleus (PSTh)

OX-B-containing neurons are

less dense distributed within the

LH. f, g Fusiform OX-B-

containing neurons with large

elongated primary dendrites.

h The arrow points to an axon

originating from a dendritic

stem. Scale bars = 200 lm in

a, d and e; 100 lm in b, 50 lm

in f, h, 30 lm in g

Brain Struct Funct (2012) 217:233–256 241

123

nucleus (EP). The dorsal group is located in the territory of

the dorsal hypothalamic area. Occasionally, ventral neu-

rons are found in the tuber cinereum, lateral to the ven-

tromedial hypothalamic nucleus.

The OX-population also diminishes in caudal direction

(Fig. 1e). Most of the immunostained cells are distributed

laterally in the dorsal portion of the lateral hypothalamus

with many of the lateral cells abutting the parasubthalamic

nucleus (PSTh). Few cells are located in the posterior

hypothalamic area although occasional immunoreactive

neurons can be found ventrally, lateral to the caudal pole of

the ventromedial hypothalamic nucleus. Follow the caudal

direction, the OX-population disappears abruptly which

could be seen in several consecutive serial sections.

The labeled neurons are medium in size (25.4 ± 2.3 lm

maximal diameter and 16.2 ± 1.6 lm in minimal diame-

ter, mean ± SEM, n = 120). The cells are fusiform or

multipolar in shape (Figs. 1f–h, 2b–f). The poles of the

fusiform neurons emit two robust dendrites that often

extend for a considerable distance without branching

Fig. 2 a Most OX-B

immunoreactive perikarya are

located in the central part of the

LH around B -3.0. b, c Here,

we found clustering of 2–10

Orexin-B immunoreactive

perikarya (arrows). d Overview

of the LH at B = -3.0 where

disseminated cells indicate a

heterogeneous distribution in

DM and in the direction to the

internal capsule. e Some of OX-

B immunoreactive neurons form

small clusters (arrow) in LH.

f Between multipolar OX-B

immunoreactive cells some are

bipolar (arrows) only. Scalebars = 200 lm in a, e; 100 lm

in b, c, e and f

242 Brain Struct Funct (2012) 217:233–256

123

although occasionally some dendrites bifurcate close to the

cell body have been noted. 3–4 dendrites with a few sec-

ondary branches arise from the multipolar perikarya. Some

are also thin in the proximal portions but frequently a short,

thick dendritic trunk is formed that bifurcates in two

slender dendrites. Among the multipolar neurons, second-

ary dendritic branches are common; tertiary branching is

only observed sporadically. The dendrites appear to be

aspiny or bear only occasional spines with extremely rare

dendritic appendages. The axon arises from a perikaryal

pole and sometimes from a dendrite (Fig. 1h).

The neuropil surrounding the OX-neurons consists of a

dense network of labeled axons containing OX-immuno-

reactive varicosities (Fig. 2b–f). Almost all hypothalamic

nuclei and areas are richly innervated. Few OX-axons enter

the suprachiasmatic and supraoptic nuclei, and only occa-

sional axons are seen in the mammillary bodies.

Orexin pathways and thalamic projections

OX-immunoreactive axons extend to numerous regions of

the rat brain comparable to the observations in the mouse

brain. These axons are organized in four major pathways, as

originally described by Peyron et al. (1998): (1) dorsal

ascending, (2) ventral ascending, (3) dorsal descending, and

(4) ventral descending pathways. In addition, on coronal

sections, two compact efferent bundles that appear to be

components of the dorsal and ventral ascending pathways

would be distinguished (Peyron et al. 1998). The first bundle

ascends from the dorsal hypothalamus to the thalamus

(Fig. 3), coursing consecutively through nucleus reuniens,

nucleus rhomboideus, and nucleus interanteromedialis. All

nuclei of the midline are strongly innervated, especially the

paraventricular thalamic nucleus (Fig. 3b, and c). The cen-

tral medial nucleus also contains a substantial number of

OX-positive fibers, whereas the paracentral and central lat-

eral (centrolateral) nuclei (CL) receive a moderate number

of labeled axons. Most of the relay thalamic nuclei (medial

geniculate nucleus (GN), lateral GN, dorsal lateral GN,

ventral lateral GN, intergeniculate leaflet, ventral posterior

complex, posterior nucleus, gustatory and visceral nuclei)

are only sparsely innervated, and the reticular thalamic

nucleus contains practically no OX-axons. In contrast, all

regions of zona incerta contain numerous OX-positive

axons. The density of OX fibers are described as follows

and summarized in Table 1.

Projections to the amygdala

The second bundle runs in lateral direction (Fig. 4a). The

initial part of this bundle runs intermingled with the

axons coursing in the supraoptic decussation and more

laterally the labeled axons traverse the thin layer of the

ventrocaudolateral substantia innominata located ventral to

the entopeduncular nucleus (EP) and globus pallidus (LGP)

(Fig. 4a–c). Reaching the dorsal aspect of the amygdaloid

complex, numerous labeled axons bend ventrally and enter

the amygdala through the central amygdaloid nucleus. All

three subdivisions of the central nucleus, medial, central

lateral, and central capsular contain a substantial to mod-

erate number of labeled axons. Most of the axons are thin

and long with relatively few varicosities. However, short

segments of thicker axons with densely arranged varicos-

ities are occasionally seen.

From the central nucleus, the labeled axons proceed

ventromedially in the medial nucleus of the amygdala,

running parallel to the brain surface. The most prominent

labeling is observed in the posterodorsal part of the medial

nucleus (MePD), followed by the anterodorsal (MeAD) and

posteroventral (MePV) portions. Most of the axons ramify

in the deeper zones of these subnuclei, although some

axons are also seen superficially in the molecular layer.

In the cortical nuclei (anterior, posteromedial and pos-

terolateral) the density of OX-axons is moderate. Most

axons are thin, with sparse and small varicosities. They

have a straight course and branch infrequently. Occasion-

ally coiled segments of thicker axons with more densely

arranged varicosities are seen, especially in the posterior

corticomedial nucleus.

In the second main group of amygdala nuclei (lateral,

basolateral and basomedial nuclei), the number of OX-

positive axons is lower than in the corticomedial group.

The lateral nucleus contains the smallest number of OX-

axons in the amygdaloid complex. In its lateral part, which

is close to the external capsule, only occasional segments

of twisted, fine OX-positive axons are seen. In the anterior

part of the lateral nucleus, there are a few, scattered and

infrequently branched OX-axons, but the tendency for

concentration of such axons (not shown) is extremely rare.

The number of OX-axons in the basolateral nucleus is

somewhat larger. In the posterior part of the basolateral

nucleus the labeled axons are scant. However, grouping of

such fibers in discrete patches has been observed. In the

anterior part of the basolateral nucleus, the density of OX-

positive axons is larger. Most axons are thin and bear few

varicosities. However, axons with prominent varicosities

also exist. The ventral part of the basolateral nucleus

contains a broad variety of labeled axons. Most of OX-

fibers are very thin, straight, with a few varicosities. Oca-

sionally, the presence of twisted, robust axonal fragments

with densely arranged large varicosities can be found.

The basomedial nucleus contains labeled axons in all

subdivisions. A dense accumulation is present in its lateral

sector of the central portion which is immediately medial

to the anterior part of the basolateral nucleus. A moderate

number of labeled axons traverse the medio-lateral

Brain Struct Funct (2012) 217:233–256 243

123

extension of the anterior part of the basomedial nucleus.

Along with the thin and straight axons, thicker labeled

axons are seen. These axons exhibit a wavy course and

more densely arranged varicosities. Rarely a web of OX-

positive tortuous terminal axons is also seen. In the pos-

terior part of the basomedial nucleus, mainly thin, straight,

and sparsely branching axons are seen. They run predom-

inantly in mediolateral direction.

In the amygdaloid complex, the area most heavily

innervated by OX-axons is the anterior amygdaloid area.

The ventral part of the anterior amygdaloid area contains

patches of densely arranged OX-positive axonal fragments.

Both thin and thick OX-positive axons are convoluted and

displayed numerous varicosities. In the shell region of the

anterior amygdaloid area located dorsal to the ventral part,

most of the OX-axons run in a ventromedial-dorsolateral

direction and branch infrequently. The distribution of OX-

axons in the dorsal amygdaloid area is similar to the ventral

area. The amount of thick fibers in this area is, however,

less prominent.

Fig. 3 a Overview of the

midline thalamus at B = -2.16

showing the dorsal third

ventricle (D3V) and the medial

habenular nuclei (MHb). The

paraventricular thalamic

nucleus (PV), the central medial

thalamic nucleus (CM) and the

interanteromedial thalamic

nucleus (IAM) contain OX-A

fibers of different orientations.

In the PV and CM fibers are

ascending and in the IAM they

cross the midline. The twoarrows are indicating the

principal pathway of ascending

OX-A fibers. b Detail from the

superior part of PV from

a showing a dense fiber

distribution as well as in

c. Scale bars 200 lm in a,

100 lm in b and c

244 Brain Struct Funct (2012) 217:233–256

123

The distribution of OX-positive axons in the nucleus of

the lateral olfactory tract is uneven. In layer 1 there are

only a very few positive fibers but in layer 2 their number is

substantial. Their number diminishes in dorsal direction

resulting in a moderate amount of OX-axons in layer 3.

All sectors of the intraamygdaloid portion of the bed

nucleus of stria terminalis contain a substantial number of

OX-axons (Fig. 5a, b) that are, however, unevenly dis-

tributed. Dense terminal fields are adjacent to regions

containing very scant immunolabeled axons (Fig. 5a). The

axons are generally thin but some of them bare prominent

varicosities (Fig. 5b). The intercalated nuclei of the

amygdala (I) contain a moderate number of OX-immuno-

positive axons, mainly located in their periphery (Fig. 5c–

f). Some of the labeled axons are markedly convoluted

(Fig. 5c, c0), while others represent the typical thin, straight

fibers that branch infrequently (Fig. 5d, d0, e).

OX-immunoreactive axons are also present in the tran-

sitional areas of the amygdaloid complex. In the amygd-

alostriatal transitional area (AStr), a substantial number of

thin axons, coursing in different directions are seen,

although in rare occasions, segments of thicker axons have

been noted (Fig. 5e). In the amygdalohippocampal transi-

tional area, a moderate number of OX-positive axons are

visualized. Some of them appear tortuous (Fig. 5g). Some

nuclei of the amygdaloid complex that contain OX-fibers

are summarized in the atlas image in Fig. 6c.

The stria terminalis contains no OX-immunopositive

axons in its posterior (retrothalamic, ascending) part and

supracapsular part. However, in its anterior (prethalamic,

descending) part, some immunolabeled axons are observed

(Fig. 7a and inset). Practically all sectors of the bed

nucleus of stria terminalis (BST) contain a significant

number of OX-positive fibers (for an overview see Fig. 6)

The medial division of this region appears to be among the

most significantly innervated structure of the limbic fore-

brain in particular (Fig. 7b–h). The lateral division of BST

receives a substantial number of OX-immunolabeled ax-

ons. At more caudal BST levels including the lateral

division, the dorsal part (BSTLD) (Fig. 7b), and posterior

part (BSTLP) (Fig. 7c) the OX-fiber density is moderate to

dense. At the small juxtacapsular part (BSTLJ) only a faint

OX-innervation is observed (Fig. 7b). A large amount of

immunolabeled axons is present in the BST medial divi-

sion, posteromedial part (BSTMPM). These axons form a

loosely arranged bundle that are oriented dorsoventrally

and are surrounded by terminal arborizations (Fig. 7c). In

addition, some robust tortuous fibers are present (Fig. 7d).

The OX-immunoreactive axons in the BST medial divi-

sion, posterointermediate part (BSTMPI) are less numerous

and build an irregular meshwork (Fig. 7e). The fornix

axons are encircled by a web of numerous OX-immuno-

labeled axons. Their dorsal part is located on the territory

of BST medial division, posterolateral part (BSTMPL)

(Fig. 7f), and the axons ventral to the fornix belong to

BSTMPM. The bed nucleus of the anterior commissure

contains a small number of immunolabeled axons (Fig. 7g,

h). They enter the scant neuropil of this small but compact

structure from the septohypothalamic nucleus (medially)

and from BSTMA (laterally).

At rostral BST levels a significant number of OX-im-

munopositive fibers are also present (Fig. 8a–d). These are

two very prominent accumulations. The dorsal one is

located in the BST medial division, anterior part (BSTMA)

Fig. 4 a Low magnification of

a mighty lateral fiber bundle

coursing from LH (arrow)

above the optic tract (opt)within the supraoptic

decussation (sox) toward the

basal ganglia. b Detail from

a showing fibers running

parallel to the optic tract.

c Detail from a where fibers

(arrows) are leaving the OX-

fiber bundle to enter the

amygdala. Scale bars 200 lm in

a, 100 lm in b, c

Brain Struct Funct (2012) 217:233–256 245

123

(Fig. 8a, b), whereas the ventral one in the BST medial

division, ventral part (BSTMV) (Fig. 8a, c). Moving lat-

erally, within the subcommissural portion of the BST lat-

eral division, ventral part (BSTLV), an appreciable number

of immunolabeled axons are also observed (Fig. 8d).

Projections to the basal forebrain and basal ganglia

Practically all sectors of substantia innominata contain a

significant number of immunopositive axons (Fig. 4b, c).

At more rostral levels, dense accumulations of immuno-

labeled axons are seen in the dorsal sector of substantia

innominata and in its ventral sector. Along the thin OX-

immunopositive axons, a significant number of thick ax-

ons with numerous prominent varicosities are present. The

substantia innominata regions that contain the cholinergic

neurons of the basal magnocellular nucleus of Meynert

also comprise a dense web of OX-immunoreactive axons,

like the region immediately ventral to the internal capsule,

and immediately medial to the external globus pallidus

(LGP).

The OX-fiber projections to the basal ganglia spread

from the LH in lateral and rostral direction. They enter the

entopeduncular nucleus through its ventral aspect, from the

Fig. 5 a Dense terminal field in

the posterior part of the bed

nucleus of the stria terminalis

intraamygdaloid division

(BSTIA). b Thin OX-A axons in

the BSTIA with varicosities.

c The intercalated nuclei of the

amygdala (I) contain a moderate

number of OX-A axons. The

OX-A immunoreactivity is

predominantly located in the

periphery of I (see also d–e).

c0 Detail form c showing a

strongly convoluted axon.

d OX-positive axons at the

periphery of I. d0 An OX-

positive axon leaving I and

roping into BLA. e Massa

intercalata (I) with a OX-

positive thin axon. f Segments

of thin axons in the

amygdalostriatal transition area

(AStr) are more frequent than

thick OX-axons. g In the

amygdalohippocampal area

anterolateral part (AHiAL) a

moderate number of OX-axons

with varicosities are found.

Some tortuous axons with

varicosities are indicated by

arrows. Scale bars 50 lm in a,

b, c0, d0, e–g. 100 lm in c, d

246 Brain Struct Funct (2012) 217:233–256

123

directed, laterally oriented main OX-immunopositive

bundle. Other axons run in the most medial part of the

internal capsule and enter the entopeduncular nucleus via

its medial border. The great majority of these axons

resemble passing fibers, e.g., they are straight, long and

with only a few varicosities. Rarely clusters of short,

curved immunolabeled axonal segments are noted, sug-

gesting the presence of terminal fields.

A clear part of projections to the ventral striatal region

terminate in the accumbens nucleus at different levels of

intensity. Only a few fibers were found in the accumbens

core whereas in the accumbens shell a moderate amount is

visible. More ventrally in the fundus of the striatum, the

densities of OX-projections increase. A fine OX-axonal

network intermingled with thick axonal terminals was

found. Likewise the islands of Calleja receive some OX-

projections. OX-fibers rise up through the external capsule

to the lateral caudate putamen and claustrum. In the lateral

extension of the dorsal endopirifom nucleus, a dense OX-

fiber distribution has been found. These OX-fibers are

partially tortuous and possess varicosities.

The subthalamic nucleus receives strong OX-immuno-

positive afferents composed of thin axons and thick axon

with varicosities.

Projections to the midbrain

In the midbrain, dense OX-fibers were found in the ventral

tegmental area. Because most of these fibers are elongated,

they possess features of passing fibers. The OX-fibers have

varicosities and exhibit axonal convolutions. In the raphe

nuclei a dense OX-innervation is found. Especially, the

rostral linear nucleus of the raphe offers a very dense

network of OX-fibers with varicosities and convolutions.

Similarly, the dorsal raphe nucleus interfascicular part

contains a comparable OX-fiber network as the rostral

linear nucleus. In the paranigral nucleus, an OX-fiber net-

work of moderate density was detected. The parabrachial

pigmented nucleus also receives a moderate number of

OX-fibers and terminal patches with varicosities.

The compact part of the substantia nigra exhibits a very

high OX-fiber density, whereas in the reticular part only a

few OX-fibers are visible. In particular, we observed sev-

eral terminal fields of OX-axons in SNc possessing vari-

cosities and exhibiting tortuous fragments.

In the pedunculopontine nucleus (peduncular pontine

nucleus, PPN) a high density of OX-fibers exists. These

fibers possess many varicosities and exhibit convolutions.

In the more dorsal part of the PPN, the OX-immunoposi-

tive fibers become thin and only a few thick axonal frag-

ments with varicosities are visible. In contrast, in the

ventral PPN, the density of OX-fibers is obviously higher

and more varicosities can be observed. Besides small

convoluted OX-fibers, long-passing axons with varicosities

have been found.

Discussion

To facilitate the functional and connectional subsumption,

the principal known afferents and efferents are summarized

and reconstructed spatially (Fig. 9). However, we are aware

that at a more precise level of connectivity presentation—

only relevant superior regions were analyzed here—we are

confronted with thousands of differentially weighted

afferents and efferents of ipsi-, contra- and bilaterally

projections. An analysis of these high-resolution connec-

tivity data will be presented soon from the network systems

point of view.

Technical considerations

OX is synthesized in neurons of several LH subnuclei:

perifornical lateral hypothalamus (PF), dorsomedial hypo-

thalamus (DM), anterior lateral hypothalamic area anterior,

medial subfornical part, posterior subfornical part, tuberal

subfornical part (tuberal part), and lateral hypothalamic

area proper. Further parts of the lateral hypothalamus exist

where OX-immunoreactive perikarya were not described:

LH perisupraoptic region, LH premammillary subfornical

region, juxtaparaventricular part of the LH, peduncular part

of the LH, and LH retinoceptive region. Since we focused

on OX-fibers that derived from the perikarya of the OX-

population of neurons, we cannot provide any evidence for

the subregional origin of OX-immunoreactive fibers found

as an afferent in target regions.

Terminating and passing OX-fibers are differentiated

only by approximate characterization of the morphology of

fiber courses. Hence, it is necessary to validate and enrich

the data of this study with retrograde tracings. Furthermore,

subregional anterograde tracings may help to differentiate

specific subregional contributions of orexinergic efferents.

Several results on the distribution of OX-positive

neurons reported in the previous studies could not be

confirmed. Chen et al. (1999) reported that ‘‘…Isolated

OX-like immunoreactive neurons were occasionally found

in the median eminence and arcuate nucleus’’. We found

no immunoreactive neurons in the region representing the

interface between the hypothalamus and the anterior pitu-

itary. Instead, as already observed by many other authors,

we encountered densely arranged OX-positive fibers in the

median eminence running vertically from the internal and

external zone to the ependymal cell layer. Sometimes these

fibers form discrete fascicles that indeed resemble neuronal

perikarya, but a reliable evidence for OX-immunoreactive

neurons in this region was never detected.

Brain Struct Funct (2012) 217:233–256 247

123

Sakurai et al. (1998) plotted some OX-positive neurons

in the subthalamic nucleus but this was not confirmed in

the parallel investigation of Peyron et al. (1998) and in

several consecutive studies, including the team of Sakurai

(Nambu et al. 1999). In our study, we regularly found that

the most caudolateral OX-cells are located immediately

248 Brain Struct Funct (2012) 217:233–256

123

medial to the parasubthalamic nucleus, stereotaxic level

Bregma -3.8 to 4.0 but the OX-positive neurons did not

invade the parasubthalamic and subthalamic nuclei.

An unexpected finding was reported by Ciriello et al.

(2003). They observed OX-B-immunoreactive neurons in

CeL (Fig. 4) and BSTLD (Fig. 7) in rats. Even in a very

dense series of our sections stained exclusively for OX-B,

immunoreactivity in Am and BST neurons could not be

displayed.

Interestingly, from the study of Espana et al. (2005) it is

known that some contralateral projections from MS, MPO,

SI and LC also exist. Furthermore, Balcita-Pedicino and

Sesack (2007) have found that a few OX-terminals in the

VTA are located at dopamine and GABA neurons; how-

ever, the majority of OX-fibers are passing the VTA.

Basal ganglia

Our data of low density or OX-fibers in the CPu are in line

with the observations of Date et al. (1999), Nambu et al.

(1999), Cutler et al. (1999) with regard to density and OX-

fiber location. In contrast, Peyron et al. (1998) did not find

OX-fibers in the CPu. By retrograde fluorogold tracing,

Duva et al. (2005) showed that the CPu projects moderately

to the orexinergic anterior and posterior part of the lateral

hypothalamic area (LHAa, LHAp) as well as to the tuberal

part of the lateral hypothalamus (TuLH). In conjunction

with Peyron et al. (1998), we found a moderate to strong

projection of orexinergic fibers to the fundus of the

striatum.

The claustrum can be subdivided into two cortico-rela-

ted zones: the medial sensorimotor zone and the lateral

visuoauditory zone (Sadowski et al. 1997; Sloniewski et al.

1986). Peyron et al. (1998) reported a moderate OX-fiber

density in the claustrum. However, Nambu et al. (1999)

described a high OX-fiber density, which is largely in

agreement with our own findings. More specifically, we

observed more OX-fibers in the medial sensorimotor zone

than in the lateral visuoauditory zone. In contrast to fiber

densities reported by Peyron et al. (1998) and Nambu et al.

(1999), we found a moderate-to-low OX-fiber density in

the claustrum.

As a new observation, we found a high density of OX-

fibers in the entopeduncular nucleus (EP). The lateral

globus pallidus (LGP) receives direct inhibitory (Park et al.

1982) input from the striatum (Hedreen and DeLong 1991)

and excitatory input from the subthalamic nucleus (Kita

and Kitai 1987). Feedback output of the LGP converges on

the striatum (Beckstead 1983). We found a moderate

density of OX-fibers in the LGP thus confirming the

observation of Cutler et al. (1999).

Amygdala

Anterior amygdala

The anterior amygdaloid area (AAA) is part of the olfac-

tory amygdala and of the superficial cortical-like nuclei and

has an abundant number of intrinsic amygdaloid afferents

and efferents (de Olmos et al. 2004). Besides these massive

intraamygdaloid connections, afferents from the thalamus,

hypothalamus and cerebral cortex occur. Peyron et al.

(1998) and Nambu et al. (1999) found a moderate to strong

density of OX-immunopositive fibers in the AAA. How-

ever, due to these data it was unclear whether the OX-fiber

distribution is inhomogeneous and if certain subregions of

the AAA are targets of the orexin projection. As a new

finding, we observed dense OX-terminals in the dorsal

(AAD), ventral (AAV) and shell parts (AASh) of the AAA.

The anterior cortical amygdaloid nucleus (ACo) occu-

pies the rostral third of the cortical amygdaloid area with

numerous intrinsic amygdaloid afferents and efferents (de

Olmos et al. 2004). The extrinsic efferents proceed to the

thalamus, cerebral cortex, and hippocampus. The extrinsic

afferents to the ACo originate in CA1, infra- and prelimbic

cortex, entorhinal cortex, ventral tegmental area and locus

coeruleus as well as the hypothalamus. To our knowledge,

no reports have documented the occurrence of OX-fibers in

ACo as described here.

Amygdalostriatal transition area (AStr)

The AStr encompasses a zone of the striatum that is

wedged between the central amygdaloid nucleus and the

laterobasal nuclear complex of the deep amygdaloid

complex. The AStr sends efferents to the basomedial

nucleus, the dorsolateral part of the lateral nucleus, the

posteromedial cortical nucleus, the medial part of the

medial geniculate nucleus, the suprageniculate thalamic

nucleus, the posterior intralaminar thalamic nucleus and

the suprafascicular thalamic nucleus (de Olmos et al. 2004;

Groenewegen and Witter 2004). Afferents to the AStr

originate in the agranular insular, infralimbic, prelimbic

and perirhinal cortex, auditory regions, the temporal asso-

ciation areas, thalamic nuclei, midbrain and brainstem

Fig. 6 The unilateral distribution of OX-fibers from bregma 0.27 to

-5.2 with the anterior BST-nuclei: STLJ, STLD, STLP, STLV,

STMV, STMA (in these images from the rat brain atlas of Paxinos

and Watson (2007) nuclei abbreviations do not have a prefix ‘‘B’’) the

abbreviations are equivalent with BSTLJ, BSTLD, BSTLP, BSTLV,

BSTMV, BSTMA. From bregma -1.2 to -4.36 OX-perikarya are

indicated by small dots. The color of the dots corresponds to the

density of terminals in the target regions. Regions that receive

OX-fibers are marked by a colored contour and Y symbols indicating

fiber terminals of a certain density as shown by the three colors in the

upper left. Regions and projections are visualized in 3D in Fig. 9

b

Brain Struct Funct (2012) 217:233–256 249

123

regions (A1, A2, A4, A5, A6, A7, A8, A10). In the AStr,

we found a moderate OX-fiber density which has not been

reported before.

Basal amygdala

The efferents of the BLA reach the cingulate, medial

agranular prefrontal, agranular insular, infra- and prelimbic,

lateral entorhinal cortex, the anterior amygdalopiriform

transition area, BST lateral division, dorsomedial hypo-

thalamic nucleus posterior part, and the medial amygdaloid

nucleus (de Olmos et al. 2004; Santiago and Shammah-

Lagnado 2005; Jasmin et al. 2004; Conde et al. 1995;

Hoover and Vertes 2007; Onat et al. 2002).

The BLV has about a twofold of known efferents in

comparison to BLP or BLA. It has efferents to different

Fig. 7 a The anterior part of

the stria terminalis contains

some OX-A axons (LV lateral

ventricle). b Overview of BST

anterior medial part (BSTMA),

BST lateral division, dorsal part

(BSTLD) and BST lateral

division, juxtacapsular part

(BSTLJ) around B -0.12. c The

BST lateral division, posterior

part (BSTLP) receiving a

substantial number of

OX-axons. d A large amount of

OX-fibers were found in the

BST medial division,

posteromedial part (BSTMPM).

e The BST medial division,

posterointermediate part

(BSTMPI) contains a few

OX-immunoreactive fibers. f In

the BST medial division,

polsterolateral part (BSTMPL)

around the fornix a moderate

amount of OX-fibers appears.

g The bed nucleus of the

anterior commissure (BAC)

possesses few OX-fibers which

enters from the

septohypothalamic nucleus

(SHy) and BSTMA (f fornix, acanterior commissure). h Detail

of g. Arrows are arranged in

parallel to fibers arriving from

the locations of SHy and

BSTMA. Scale bars 100 lm in

a, b, g, 50 lm in c–f, h

250 Brain Struct Funct (2012) 217:233–256

123

subregions of the striatum and accumbens nucleus, the

nucleus of the horizontal limb of the diagonal band, to CA

regions of the hippocampus and to several BST subregions.

For each BLA, BLP and BLV subnucleus, there are about

30 afferents known. These afferents can be summarized

with regard to their origins in the cortical and subcortical

nuclei, amygdala, thalamic and hypothalamus. In the three

subnuclei BLA, BLP and BLV of the basolateral amygdala,

a homogeneous moderate OX-fiber density was identified

that has not been published elsewhere.

The basomedial amygdaloid nucleus is formed by two

succeeding nuclei in the rostrocaudal direction: the anterior

and posterior subdivisions of the basomedial amygdaloid

nucleus. The connectivity of the basomedial amygdaloid

nucleus is complex because about 40 afferents and 33

efferents are known. We found a homogeneous moderate

OX-fiber density in the two subnuclei of the basolateral

amygdaloid nucleus that was not reported before.

Bed nucleus of the stria terminalis (BSTIA, BSTLJ, BSTLP,

BSTMPi, BSTMV)

The subdivisions of the bed nucleus of the stria terminalis

are parts of the extended amygdala with a central division

(CEXA) and a medial division (MEXA) (Dong et al. 2001;

de Olmos et al. 2004). MEXA-regions containing OX-

fibers are the BSTIA, BSTMPi and BSTMV which are not

known so far to receive orexinergic projections. CEXA-

members are the BSTLJ and BSTLP nuclei that also

receive orexinergic projections.

The efferents of the MEXA-member BSTIA (intra-

amygdaloid division) are proceeding to the anterior

amygdaloid area, amygdalohippocampal area, accessory

olfactory bulb, BSTL, BSTM, central amygdaloid nucleus

lateral and medial divisions, medial amygdaloid nucleus,

medial septal nucleus, peripeduncular nucleus and the

dorsomedial hypothalamic nucleus anterior part (de Olmos

et al. 2004). The afferents to the BSTIA originate in cor-

tical (insular, hippocampal) areas, amygdaloid, hypotha-

lamic and thalamic subnuclei.

The BSTMV sends specific efferents to the medial and

perifornical part of LH (Yoshida et al. 2006). Most affer-

ents to the BSTMV come from amygdaloid subnuclei and

LH whereby Peyron et al. (1998) have shown OX-fibers in

the BSTM. High OX-fiber density was observed in the

BSTIA, the BSTLP and the BSTMV whereas a moderate

OX-fiber density was found in the BSTLJ and BSTMI.

These orexinergic innervations of the five BST subnuclei

have not yet been reported to date.

Central amygdaloid nucleus (Ce)

The Ce in the dorsal central part of the amygdaloid com-

plex is bordered dorsolaterally by the striatum. It consists

of the central medial (CeM), central lateral (CeL), and

central capsular (CeC) subnuclei that contain a substantial-

Fig. 8 a In the BST medial

division, anterior part (BSTMA)

dense OX-A positive patches of

fibers. b Detail from a showing

numerous varicosities. c Second

detail from a with few thick and

many thin OX-axons in the BST

medial division, ventral part

(BSTMV). d The

subcommissural portion of the

BST lateral division, ventral

part (BSTLV) has a moderate

orexinergic innervation. Scalebars 100 lm in a, b, 50 lm

in c, d

Brain Struct Funct (2012) 217:233–256 251

123

Fig. 9 a All regions that are mapped in the rat brain atlas of Paxinos

and Watson (2007) are reconstructed in combination with the

transparent CNS to visualize the 3D-location of OX-terminals. b To

obtain an optimal projection the reconstruction has been rotated as

indicated by stereotactic axes. This projection is used in c–f. c Shows

the LH (transparent) and regions with OX-terminals in a compact

visualization without the CNS envelope. d A 3-axes expansion view

to separate the amygdaloid subregions and in e the abbreviations of all

regions are shown. f Provides a visualization of our observed

connections in terms of three classes of weights

252 Brain Struct Funct (2012) 217:233–256

123

to-moderate number of OX-labeled axons not described

elsewhere.

Intercalated nuclei of the amygdala

The intercalated nuclei of the amygdala receive input from

the basolateral amygdala and inhibit the central nucleus of

the amygdala known as a main output for conditioned fear

responses. We can confirm the findings of Nambu et al.

(1999) and Peyron et al. (1998) of a low-to-moderate OX-

fiber density in the intercalated nucleus. Our findings of the

presence of low-to-moderate OX-fiber density in the

intercalated nuclei confirms the previous findings of

Nambu et al. (1999) and Peyron et al. (1998).

Nucleus of the lateral olfactory tract (LOT)

The LOT is part of the corticomedial nuclear complex und

consists of three layers: the molecular layer, the pyramidal

layer and layer III (Millhouse and Uemura-Sumi 1985).

The LOT is integrated in a complex local, interregional,

ipsi- and bilateral efferent network to the olfactory bulb,

anterior olfactory nucleus, bilaterally to the anterior piri-

form cortex, dwarf cell cap regions of the olfactory

tubercle, the lateral shell of the accumbens, and contra-

laterally to the lateral part of the interstitial nucleus of the

posterior limb of the anterior commissure. We found a

moderate OX-fiber intensity in the LOT that was also

reported by Nambu et al. (1999).

Medial amygdaloid nuclei

The MeA is one of the central members of the superficial

amygdala, respectively, the extended amygdala and occu-

pies its rostromedial side. Cytoarchitectonically, it can be

subdivided into a principal, an anterodorsal (MeAD) and a

posterodorsal (MePD) part. These three subdivisions have

further subparts as described by de Olmos et al. (2004).

In the review of de Olmos et al. (2004), the MeAD is

one of the nuclei of the amygdala that has many intrinsic

input and output connections (efferents to subnuclei of the

anterior amygdaloid area, about 10 BST subnuclei, cen-

tromedial subnuclei, ventrolateral, posteromedial, lateral

entorhinal cortex subparts, and agranular cortex). Interest-

ingly, OX-fibers in the MeAD have not been described

elsewhere. We found dense OX-immunoreactive fibers in

the MeAD.

Efferents to the MePD come from the anterior and

posterior basomedial nucleus, the subnuclei of the medial

division of the BST, the amygdalopiriform transition

area, the ventrolateral subnuclei, medial amygdaloid

nucleus, the medial preoptic area, supramammillary

nucleus, and subregions of lateral entorhinal cortex. The

MePD receives afferents from the medial amygdaloid

nucleus posteroventral part and the anterior basomedial

nucleus. Here, we detected a dense OX-immunopositive

fiber distribution in MePD that has not been described

before.

Posterior amygdala

The posterolateral cortical amygdaloid nucleus (PLCo)

belongs to the superficially located amygdaloid nuclei or

the cortical-like nuclei sometimes called periamygdaloid

cortex. The PLCo can be further subdivided into five cell

groups as described by de Olmos et al. (2004). The PLCo

receives about 40 known afferents from cortical regions

and subcortical nuclei (de Olmos et al. 2004). Just like

Cutler et al. (1999), we found a direct orexinergic project-

ion to the ICj.

The posteromedial cortical nucleus of the amygdala

(PMCo) belongs to the vomeronasal system implicated in

the control of reproductive behavior. From rostrally, it

extends lateral to the posteroventral medial amygdaloid

nucleus and medial to the posterolateral cortical amygda-

loid nucleus (PLCo) and continues ventral to the antero-

lateral and posteromedial (AHiPM) parts of the

amygdalohippocampal area and medial to the PLCo, until

the amygdalopiriform transition area (APir). The vomero-

nasal amygdala consists of the PMCo and the medial

amygdaloid nucleus (Me). It is integrated with the vom-

eronasal system and receives afferents from the accessory

olfactory bulb. The PMCo and other structures of the

vomeronasal system are sexually dimorphic. In the PMCo,

males show larger volumes and a greater number of neu-

rons than females. We investigated male rats only and

observed a moderate OX-fiber density in the PMCo which

was unknown so far.

Other regions

We have not found publications documenting OX-fibers in

the lateral part of the ventral geniculate nucleus (VLG) and

the lateral part of the dorsal geniculate nucleus (DLGl) or

other parts of the geniculate nucleus. The OX-fiber-density

in the DLGl is moderate. In other parts of the geniculate

nucleus, we have not found any OX-immunoreactivity.

However, the intergeniculate leaf (IGL) and the central

lateral part of the geniculate nucleus (VLG) have a mod-

erate OX-fiber density described here for the first time. The

dorsal peduncular pontine nucleus (DPPn) and the ventral

part of the peduncular pontine nucleus (VPPn) have a low

OX-fiber density that was not described elsewhere. The

interfascicular part of the dorsal raphe nucleus (DRI)

possesses a strong OX-fiber density, described here for the

first time.

Brain Struct Funct (2012) 217:233–256 253

123

Conclusions

The first part of new findings concerns the amygdaloid

complex where new orexinergic targets in the dorsal,

ventral and shell parts of the anterior amygdaloid area

(dense), anterior cortical nucleus (moderate), amygdalo-

striatal transition region (moderate), basolateral regions

(moderate) and the basomedial nucleus (moderate) were

detected. In addition, dense orexinergic projections were

observed in the divisions of the medial amygdaloid

nucleus. At least the central amygdaloid nuclei receive a

dense orexinergic projection. Second, new moderate-to-

dense orexinergic projections were found in several bed

nuclei of the stria terminals: BSTLJ, BSTLP, BSTMA,

BSTMPI, BSTMV, BSTAM, BSTIA and BSTLD. The

third important outcome of this study was the detection of a

high density of OX-fibers in the entopeduncular nucleus

(EP). Besides the description of further new scattered o-

rexinergic projections (e.g., interfascicular part of the

dorsal raphe nucleus, dorsal and ventral peduncular pontine

nucleus), we could confirm most of the relative densities of

known orexinergic projections.

Acknowledgments This article is dedicated to the memory of our

friend and colleague, Prof. Dr. Kamen Usunoff, who sadly passed

away on February 28, 2009. He was an outstanding personality with

an enormous experience in neuroanatomy and the whole field of

neuroscience. Kamen has provided substantial work to this study. We

thank Frauke Winzer for expert technical assistance and Alexander

Hawlitschka for sharing his knowledge on immunohistochemical

methods.

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