Available online at www.sciencedirect.com

www.elsevier.com/locate/brainres

b r a i n r e s e a r c h 1 4 9 7 ( 2 0 1 3 ) 7 3 – 8 4

0006-8993/$ - see frohttp://dx.doi.org/10

Abbreviations (ac

enzyme; DMX, do

GRN, gigantocellu

IRN, intermediate

reticular nucleus; L

PAG, periaqueduct

PPN, pedunculopo

RVLN, rostroventr

nucleus; TH, tyros

XII, hypoglossal n�Corresponding au

E-mail address:

Research Report

Pattern of tau hyperphosphorylation and neurotransmittermarkers in the brainstem of senescent tau filamentforming transgenic mice

Kerstin Morcineka,�, Christoph Kohlera, Jurgen Gotzb, Hannsjorg Schrodera

aDepartment of Anatomy II (Neuroanatomy), University of Cologne, Kerpener Strabe 62, 50924 Cologne, GermanybCentre for Ageing Dementia Research (CADR), Queensland Brain Institute (QBI), The University of Queensland, St Lucia Campus (Brisbane),

QLD 4072, Australia

a r t i c l e i n f o

Article history:

Accepted 12 December 2012

The early occurrence of brainstem-related symptoms, e.g. gait and balance impairment,

apathy and depression in Alzheimer’s disease patients suggests brainstem involvement in

Available online 20 December 2012

Keywords:

Alzheimer0s disease

Tau hyperphosphorylation

Neurotransmitter

Brainstem

Transgenic mouse

Tauopathy

nt matter & 2013 Elsevie.1016/j.brainres.2012.12.0

cording to Hof et al., 200

rsal motor nucleus of the

lar reticular nucleus; IC,

reticular nucleus; ISN,

VN, lateral vestibular n

al gray matter; PB, para

ntine nucleus; PRN, pon

olateral reticular nucleus

ine hydroxylase; V, mot

ucleusthor. Fax: þ49 221 478 531Kerstin.morcinek@uk-ko

a b s t r a c t

the initial pathogenesis. To address the question whether tau filament forming mice

expressing mutated human tau mirror histopathological changes observed in Alzheimer

brainstem, the degree and distribution of neurofibrillary lesions as well as the pattern of

cholinergic and monoaminergic neurons were investigated. The expression of the human

tau transgene was observed in multiple brainstem nuclei, particularly in the magnocellular

reticular formation, vestibular nuclei, cranial nerve motor nuclei, sensory trigeminal nerve

nuclei, inferior and superior colliculi, periaqueductal and pontine gray matter, and the red

nucleus. Most of the human tau-immunoreactive cell groups also showed tau hyperpho-

sphorylation at the epitopes Thr231/Ser235 and Ser202/Thr205, while abnormal tau

phosphorylation at the epitope Ser422 or silver stained structures were almost totally

lacking. We found no obvious differences in distribution and density of cholinergic and

monoaminergic neurons between tau-transgenic and wild type mice. Although numerous

brainstem nuclei in our model expressed human tau protein, the development of

neurofibrillary tangles, neuropil threads and ghost tangles was rare and likewise its

distribution differed largely from Alzheimer0s disease pattern. The number of monoami-

nergic neurons remained unchanged in the transgenic mice, while monoaminergic nuclei

in Alzheimer brainstem showed a distinct neuronal loss. However, the distribution of

r B.V. All rights reserved.16

0): AD, Alzheimer0s disease; AMB, ambiguous nucleus; ChAT, choline acetyltransferase

vagus; DR, dorsal nucleus of the raphe; ECU, external cuneate nucleus;

inferior colliculus; III, oculomotor nucleus; ir/ IR, immunoreactive/ immunoreactivity;

inferior salivatory nucleus; IV, trochlear nucleus; LC, locus coeruleus; LRN, lateral

ucleus; MVN, medial vestibular nucleus; NFTs, neurofibrillary tangles;

brachial nucleus; PG, pontine gray matter; PGRNl, lateral paragigantocellular nucleus;

tine reticular nucleus; PSP, progressive supranuclear palsy; RN, red nucleus;

; SC, superior colliculus; SPV, spinal trigeminal nucleus; SVN, superior vestibular

or trigeminal nucleus; VII, facial nucleus; VLL, ventral nucleus of the lateral lemniscus;

8.eln.de (K. Morcinek).

b r a i n r e s e a r c h 1 4 9 7 ( 2 0 1 3 ) 7 3 – 8 474

pretangle-affected neurons in the tau-transgenic mice partly resembled those seen in

progressive supranuclear palsy, presenting these animals as a model to examine brainstem

pathogenesis of progressive supranuclear palsy.

& 2013 Elsevier B.V. All rights reserved.

1. Introduction

Abnormally phosphorylated forms of the microtubule asso-

ciated protein tau - physiologically abundantly expressed in

neurons and glial cells (Lee et al., 2001) - constitute the major

component of neurofibrillary tangles (NFTs) and neuropil

threads in the brain tissue of patients suffering from

Alzheimer0s disease (AD) (Mercken et al., 1992). In addition to

telencephalic changes (Braak and Braak, 1991a) evidence has

been presented for the early appearance of tau pathology in

brainstem nuclei of AD subjects (Simic et al., 2009). In the

midbrain, the oral raphe nuclei, i.e. central linear, central

superior and dorsal raphe nucleus (DR) show tau deposition

in correlation with the staging system of Braak and Braak (Rub

et al., 2000). During the disease process the supratrochlear

subunit of the DR is already affected by tangle formation prior

to the transentorhinal stage (Grinberg et al., 2009). The peria-

queductal gray matter (PAG), the pedunculopontine nucleus

(PPN), the parabrachial nucleus (PB) (German et al., 1987; Parvizi

et al., 2001) and the locus coeruleus (LC) (Hirano and

Zimmerman, 1962; Ishii, 1966; Parvizi et al., 2001) – inclusive

the oral raphe nuclei constituting the main components of the

brainstem ascending arousal system – exhibit insoluble fila-

mentous tau during isocortical stages. Within the reticular

formation – containing several control centers of vitally impor-

tant functions, e.g. cardiovascular, respiratory and visceral

regulation - the intermediate reticular nucleus (IRN) develops

NFTs (Parvizi et al., 2001; Rub et al., 2001). In particular the

recent finding of intraneuronal pretangle material in the LC of

children and young adults may indicate that the pathologic

process leading to abnormal tau pathology begins in selected

subcortical nuclei (Braak and Del Tredici, 2011b).

Along with tau pathology, several neurotransmitter systems

in AD brains are affected by dysfunction, loss of neurons and

reduced transmitter concentrations (Palmer and DeKosky,

1993). Particularly the cholinergic basal nucleus of Meynert

and septal nuclei with projections to almost all cortical areas,

the hippocampus and the thalamus are severely impaired in a

subset of AD brains (Herholz, 2008; McGeer et al., 1984). The

loss and functional disturbance of cholinergic forebrain neu-

rons is associated with a reduction of cholinergic neurotrans-

mission, choline acetyltransferase and acetylcholinesterase

enzyme activity (Francis et al., 1999; Herholz, 2008; Procter

et al., 1988). Likewise, the content of the catecholamine

noradrenaline is reduced in AD temporal and frontal cortices

(Palmer and DeKosky, 1993). The neuron density in the pontine

LC – major nucleus of origin of corticopetal noradrenergic

fibers - is significantly diminished (Zweig et al., 1988). Also

the concentration of the neurotransmitter serotonin and the

number of serotonergic nerve endings and receptors is sig-

nificantly reduced in cortical areas of the temporal lobe

and the prefrontal cortex in AD (Gottfries, 1990; Perry, 1988;

Procter et al., 1988). The number of serotonergic neurons in the

DR - source of the ascending serotonergic transmitter system

to numerous cortical and subcortical regions of the forebrain

(Rub et al., 2000) - declines to 50% of the control value in AD

subjects (Zweig et al., 1988). Dysfunction within this system

can cause disrupted sleeping pattern, depressive mood swings,

and inadequate affective control (Rub et al., 2000). Whether

these deficits in neurotransmission are linked to the occur-

rence of neurofibrillary lesions in cholinergic and monoami-

nergic nuclei, e.g. in the noradrenergic locus coeruleus or the

serotonergic oral raphe complex, is not finally clarified.

Notwithstanding the findings of NFTs and neuropil threads in

brainstem nuclei and the neuronal loss in monoaminergic nuclei

in the reticular formation, no animal model reflecting brainstem

changes that are observed in AD patients has been described yet.

Only sparse information about neurofibrillary lesions in the

brainstem of tau transgenic mice is obtainable (Delobel et al.,

2008; Dutschmann et al., 2010; Menuet et al., 2011; Overk et al.,

2009; Probst et al., 2000). Therefore the objective of the present

study was to investigate the degree and localization of neurofi-

brillary lesions as well as the distribution of cholinergic, cate-

cholaminergic and serotonergic neurons throughout the

brainstem of senescent P301L tau transgenic pR5 mice.

2. Results

2.1. Location of anti-tau antibodies in the brainstemof P301L tau transgenic pR5 mice

2.1.1. HT7-immunoreactivity (IR)The phosphorylation-independent anti-tau antibody HT7

specifically recognizing the human tau protein was used to

detect neurons expressing the transgene construct.

In the reticular formation from caudal to rostral a moderate

number of HT7-positive neurons was observed in the lateral

reticular nucleus (LRN) and GRN (Fig. 1a), fewer neurons were

stained in the IRN (Fig. 1b), lateral part of paragigantocellular

(PGRNl), parvocellular, mesencephalic and pontine (PRN)

reticular nucleus (Fig. 2, Bregma �7.5 mm to �4.4 mm). In

two tau transgenic mice a solitary HT7-labeled perikaryon

was located in the rostroventrolateral reticular nucleus

(RVLN). No expression of human tau was detected in nuclei

of the raphe in the median region and in the LC and PB in the

lateral region of the formatio reticularis. The nucleus raphe

obscurus showed a single labeled neuron in two individuals.

In the caudal medulla oblongata one to five human tau-

positive neurons per section were found in the external

cuneate nucleus (ECU) and in the more medially located

spinal vestibular nucleus (Figs. 1c and 2, Bregma �7.5 mm),

as well as in the lateral (LVN) and superior (SVN) vestibular

nucleus (Figs. 1d and 2, Bregma �6.1 mm) in the rostral

Fig. 1 – Expression of human tau protein throughout the brainstem of P301L tau transgenic pR5 mice within different

functional pathways. Coronal brainstem sections (5 lm thick) counterstained with nuclear fast red. HT7-IR in (a) the

gigantocellular and (b) the intermediate reticular nucleus, (c) the external cuneate nucleus dorsomedial of the inferior

cerebellar peduncle (icp), (d) the lateral and the superior vestibular nucleus medial of the inferior cerebellar peduncle, (e) the

motor trigeminal nucleus, (f) the oculomotor nucleus lateral of the medial longitudinal fasciculus (mlf), (g) the inferior

colliculus, (h) the red nucleus and (i) in fibers in the medial geniculate nucleus (MG) lateral of the mesencephalic reticular

nucleus (MRN) and the nucleus of the brachium of the inferior colliculus (NB). Bar¼200 lm.

b r a i n r e s e a r c h 1 4 9 7 ( 2 0 1 3 ) 7 3 – 8 4 75

medulla oblongata and the pons. Also in the area of the

medial vestibular nucleus (MVN) a few HT7-immunoreactive

(ir) perikarya were noticed in three animals, but because of

the close neighborhood and difficult delineation of LVN and

SVN an unequivocal assignment of the labeled neurons was

not possible. In the dorsal cochlear nucleus, located ventro-

lateral to the vestibular nuclei, one HT7-labeled neuronal cell

body was observed in a single case.

In cranial nerve motor nuclei a dense cluster of large HT7-ir

neurons was found in the motor nucleus of the trigeminal

nerve (V) (Figs. 1e and 2, Bregma �5.2 mm). In the hypoglossal

nucleus (XII), the ambiguous nucleus (AMB) and the nucleus

of the facial nerve (VII) (Fig. 2, Bregma �6.1 mm) sporadic

HT7-labeled neurons were detected. Three of the five exam-

ined pR5 mice also showed moderate expression of human

tau in the trochlear (IV) and the oculomotor (III) nucleus

(Figs. 1f and 2, Bregma �4.4 mm to �3.6 mm), whereas the

abducens nucleus was devoid of immunostaining. In the pons

a low density of HT7-positive cell bodies was present in the

inferior salivatory nucleus (ISN) in two animals (Fig. 2,

Bregma �6.1 mm). In the spinal (SPV) and the principal

sensory trigeminal nerve nuclei six to ten HT7-labeled neu-

rons per section were observed (Fig. 2, Bregma �7.5 mm to

�5.2 mm), just as in the pontine gray matter (PG) (Fig. 2,

Bregma �3.6 mm). In the ventral nucleus of the lateral

lemniscus (VLL) a moderate number of HT7-ir perikarya was

discovered (Fig. 2, Bregma �4.4 mm).

In the midbrain, small HT7-labeled neurons were observed

in moderate density in the inferior (IC) and superior (SC)

colliculi (Figs. 1g and 2, Bregma �6.1 mm to �3.6 mm). The

red nucleus (RN) showed a moderate number of HT7-

stained cells as well (Figs. 1h and 2, Bregma �3.6 mm).

Fewer human tau containing perikarya were found in the

nucleus of the brachium of the IC (Figs. 1i and 2, Bregma

�3.6 mm). In a single case the expression of human tau

was noticed in the PAG (Fig. 2, Bregma �4.4 mm). All deep

cerebellar nuclei, including the fastigial, the interposed and

the dentate nucleus (Fig. 2, Bregma �6.9 mm to �6.1 mm)

showed a low density of HT7-ir neurons in all tau transgenic

animals.

In addition to the occurrence of labeled fibers in nuclei

containing HT7-positive neuronal perikarya, numerous

intensely stained fibers were observed from caudal to rostral

in the pyramidal and ventral spinocerebellar tract, the infer-

ior and middle cerebellar peduncle, the medial longitudinal

fasciculus, the cerebellar white matter and the medial geni-

culate nucleus (Fig. 1i).

In the brainstem of the non-transgenic littermates there

was no expression of the human tau protein at all (Supple-

mentary data, Fig. 1l).

Bregma - 3.6 mm

ENTAMYG

CA1

CA3

DG

NB

SC

RNIII

RN

PG

PG

SC

CA1

AMYG

SUB

CA3

DG

1 mmBregma - 4.4 mm

VIS

SUB

ENT

ICIC

SCSC

PAG

IV

PRNVLL

VLL

PRNVLL

ENT

SUB

VIS

1 mm

Bregma - 6.1 mm 1 mm

AT8HT7

DN

IP

SPVSPV

SVNSVN

LVN

ISNGRN

ICIC

VII

Bregma - 5.2 mm

VIS

ENT

VIS

ENT

MEV

PRNPRN

PSV

V

V

ICIC

1 mm

Bregma - 6.9 mm

DNIP

FN

SPV

SPV

SPV

IRN

GR N GRN

1 mmBregma - 7.5 mm

ECU

SPVSPVSPVN

SPV

ECU

LRN LRN LRNLRN

GRNGRNGRN

HT7 AT8HT7 AT8

HT7 AT8

HT7 AT8HT7 AT8

SPV

1 mm

Fig. 2 – Drawings of selected coronal sections (from caudal to rostral) of one representative P301L tau transgenic pR5 mouse

to illustrate the distribution of cell bodies immunoreactive for HT7 (left) and AT8 (right), with each dot representing one

labeled neuronal perikaryon. AMYG, nuclei of the amygdala; CA1, CA1 field of the Ammon0s horn; CA3, CA3 field of the

Ammon0s horn; DG, dentate gyrus; DN, dentate nucleus; ENT, entorhinal cortex; FN, fastigial nucleus; IP, interposed nucleus;

MEV, mesencephalic trigeminal nucleus; NB, nucleus of the brachium of the inferior colliculus; PSV, principal sensory

trigeminal nucleus; SPVN, spinal vestibular nucleus; SUB, subiculum; VIS, visual cortex.

b r a i n r e s e a r c h 1 4 9 7 ( 2 0 1 3 ) 7 3 – 8 476

2.1.2. Immunoreactivity pattern of hyperphosphorylationmarkers AT180 and AT8AT8 and AT180 anti-tau antibodies recognize phosphorylated

epitopes of both soluble tau aggregates and NFTs, and

thereby constitute markers of pretangle tau hyperphosphor-

ylation state, whereas AT8-IR is supposed to peak somewhat

earlier than AT180-IR (Braak et al., 1994; Goedert et al., 1994;

Matsuo et al., 1994).

From medulla oblongata to mesencephalon the brainstem

nuclei LRN, RVLN, PGRNl, the mesencephalic reticular nucleus

and PRN in the formatio reticularis showed an AT180-IR pattern

comparable to that of human tau expression regarding the

density of labeled cells and the intensity of staining. The same

holds true for the vestibular nuclei MVN and SVN, the cranial

nerve motor nuclei XII, AMB, VII, V, IV and III, ISN and the dorsal

cochlear nucleus in the pons and VLL, PG, SC and RN in the

midbrain. AT180-ir neurons outnumbered HT7-positive cells in

ECU and LVN in the hindbrain and in IC in the caudal

mesencephalon. A low density of AT180-staining was detected

in all animals in GRN in the medial reticular formation and in

b r a i n r e s e a r c h 1 4 9 7 ( 2 0 1 3 ) 7 3 – 8 4 77

the sensory trigeminal nerve nuclei, including the mesence-

phalic nucleus, as well as in PAG in three tau transgenic mice.

In individual cases (for specifics see supplementary data,

Table 1) one to five AT180-positive cell bodies were noticed in

the magnocellular reticular nucleus in the medial zone of the

formatio reticularis, in the spinal vestibular nucleus in the

hindbrain, in the pontine nucleus Barrington, the nucleus

Kolliker–Fuse (weak intensity of staining), and in the ventral

cochlear nucleus and the nucleus of the brachium of the IC in

the midbrain. The deep cerebellar nuclei (i.e. the fastigial, the

interposed and the dentate nucleus) exhibited a moderate

number of AT180-positive neurons in the majority of animals

(for specifics see Supplementary data, Table 1).

In comparison to HT7 and AT180 fewer AT8-ir cells per

nucleus were observed in most cases. In the reticular formation

one to five AT8-labeled neurons per section were found in LRN

(Fig. 3a), GRN and PRN (Fig. 2, Bregma �7.5 mm to �4.4 mm); in

IRN, PGRNl, and the mesencephalic reticular nucleus AT8-ir cell

bodies were detected only in individual cases (for specifics see

Supplementary data, Table 1), whereas RVLN and the parvocel-

lular reticular nucleus were devoid of staining. From the caudal

medulla oblongata to the rostral pons ECU, the spinal vestibular

nucleus, ISN, LVN, the dorsal cochlear nucleus, SVN and PG

revealed small numbers of AT8-labeled perikarya in a few

animals (for specifics see Supplementary data, Table 1). Among

the motor nuclei of the cranial nerves, V (Fig. 2, Bregma

�5.2 mm) showed a medium density of AT8-positive cells in

all tau transgenic mice. Fewer labeled neurons were noticed in

two animals in AMB (Fig. 3b) and in three mice in IV (Fig. 3c),

while in XII, VII and III AT8-staining was completely lacking. In

all sensory trigeminal nerve nuclei (Fig. 2, Bregma �7.5 mm to

�5.2 mm) solitary AT8-ir neurons were observed in the majority

of animals (for specifics see supplementary data, Table 1). In the

midbrain, small numbers of AT8-positive neuronal perikarya

Fig. 3 – Hyperphosphorylation of tau protein in P301L tau tra

subjects. Coronal brainstem sections (5 lm thick) counterstaine

reticular nucleus dorsal of the ventral spinocerebellar tract (vsc),

the medial longitudinal fasciculus (mlf), (d) the superior colliculu

Bar¼200 lm.

were seen in IC, SC, RN and PAG (Fig. 2, Bregma �6.1 mm to

�3.6 mm, 3d to 3f). In VLL a few weakly stained neurons were

detected in two individuals. In the deep cerebellar nuclei AT8-ir

cells were absent, except in the fastigial nucleus two marked

neurons were identified in one case.

In addition to AT180 and AT8-positive fibers in several

nuclei displaying AT180- and AT8-ir perikarya a distinct

AT180-ir fiber labeling was present in the inferior and middle

cerebellar peduncle, and the trapezoid body.

Neither AT180- nor AT8-IR was noticed in the brain sections

of non-transgenic littermates (Supplementary data, Fig. 2f).

2.1.3. Immunoreactivity of pS422 and Gallyas silverimpregnation in pR5 miceThe abnormal phosphorylation of tau protein at Ser422

appears to be linked with the process of tau filament forma-

tion and aggregation (Augustinack et al., 2002; Deters et al.,

2008); hence the pS422-IR was used as marker of NFT stages.

In addition, Gallyas silver impregnation was applied to

visualize argyrophilic NFTs, neuropil threads and ghost

tangles (Braak et al., 1994; Heinsen et al., 1989).

Throughout the brainstem pS422-labeling was observed in

five individual neurons. One single pS422-positive neuronal

perikaryon was detected in the reticular formation in GRN

and PRN in two transgenic mice (Supplementary data, Figs. 3a

and b) and in SPV in one animal. In tissue sections stained

with Gallyas silver impregnation only one labeled neuron was

found in GRN in the hindbrain of one animal (Supplementary

data, Fig. 3c). The cerebellum was devoid of pS422-ir cell

bodies and silver staining.

Neither pS422-ir nor silver stained fibers were present in

brainstem and cerebellum.

The brainstem of the non-transgenic littermates was

devoid of pS422-IR and Gallyas silver impregnated structures.

nsgenic pR5 mice in brainstem nuclei also affected in PSP

d with nuclear fast red. AT8-ir neurons in (a) the lateral

(b) the ambiguous nucleus, (c) the trochlear nucleus lateral of

s, (e) the red nucleus and (f) the periaqueductal gray matter.

b r a i n r e s e a r c h 1 4 9 7 ( 2 0 1 3 ) 7 3 – 8 478

2.2. Location of neurotransmitters in the brainstemof P301L tau transgenic pR5 mice

The distribution of cholinergic, catecholaminergic and sero-

tonergic neurons was in general in accordance with the

findings described in the literature (VanderHorst and

Ulfhake, 2006).

2.2.1. Choline acetyltransferase (ChAT) immunoreactivityChAT-IR was used to display the pattern of cholinergic

neurons in the murine brainstem.

From the medulla oblongata to the mesencephalon in the

brainstem of P301L tau transgenic pR5 mice a moderate

density of ChAT-positive perikarya was present in IRN, PGRNl

and PB in the reticular formation. In the hindbrain a few

cholinergic cells were observed in the area postrema, the

nucleus of the solitary tract, the prepositus hypoglossal

nucleus and MVN. Among the cranial nerve motor nuclei

XII, the dorsal motor nucleus of the vagus (DMX), AMB, VII, V

and III showed numerous intensely stained large neurons.

Fewer ChAT-labeled cell bodies were detected in ISN, the

accessory facial nucleus, the abducens nucleus and IV. In four

animals a moderate number of ChAT-ir cells was identified in

SPV of the sensory trigeminal complex. In the pons, medial to

middle cerebellar peduncle a dense cluster of weakly stained

neurons was found in the nucleus Kolliker–Fuse. A moderate

density of cholinergic neurons was noticed in the laterodorsal

tegmental nucleus and PPN in the mesopontine tegmentum,

as well as in the ventrally located periolivary nuclei. Lateral to

the laterodorsal tegmental nucleus one to five ChAT-ir neu-

rons per section were detected in the nucleus Barrington in

three mice. In the midbrain ventrolateral to IC in the area of

nucleus sagulum and parabigeminal nucleus numerous

ChAT-positive perikarya were observed, but because of the

close topographical relationship of these nuclei a definite

classification was not feasible.

Numerous ChAT-labeled fibers were present in the cranial

nerves and corresponding motor nuclei XII, DMX, AMB, VII

and V, in the pons in the area of PG and in the midbrain in SC,

the interpeduncular nucleus and the fasciculus retroflexus.

This distribution of cell bodies and fibers ir for choline

acetyltransferase coincides with the pattern of cholinergic

neurons in the brainstem of non-transgenic littermates.

2.2.2. Tyrosine hydroxylase (TH) immunoreactivityIn the ventrolateral part of the caudal medulla oblongata six

to ten TH-ir neurons per section were noticed in RVLN (C1

group of adrenergic cells), in the dorsomedial part of the

lower brainstem a smaller number of labeled neurons was

present in the area postrema, DMX, the nucleus of the

solitary tract and the prepositus hypoglossal nucleus. In the

pons a dense cluster of TH-positive cells was localized lateral

to the fourth ventricle in LC (A6 dopaminergic cells). A few

TH-stained perikarya were found medial to V analogous to

A7 group of noradrenergic cells. In the midbrain reticular

formation a moderate number of catecholaminergic neurons

was observed in the central linear raphe nucleus, the retro-

rubral field (A8 dopaminergic cell group) and the ventral PAG

(dorsocaudal division of the A10 dopaminergic cell group),

whereas the substantia nigra (A9 dopaminergic cell group)

and the ventral tegmental area (A10 dopaminergic cells)

showed numerous TH-labeled neurons.

Distinct TH-ir fiber labeling was noticed in several animals

from caudal pons to rostral mesencephalon in LC, PPN and

the central linear nucleus of the raphe.

The findings again equate to those found in the non-

transgenic littermates.

2.2.3. Serotonin immunoreactivityA moderate number of serotonergic perikarya was present in

the nucleus raphe obscurus, nucleus raphe pallidus, nucleus

raphe magnus, central superior nucleus and dorsal nucleus

(DR) of the raphe in the median zone of the reticular

formation. In the magnocellular region of the formatio

reticularis and dorsolateral to the pyramidal tract extending

from the medulla oblongata to pons a low to moderate

density of 5-hydroxytryptamine-positive neurons was

detected within the inferior olivary complex, in PGRNl, the

peripyramidal nucleus and in two transgenic mice also in the

nucleus of the trapezoid body. In the midbrain six to 10

immunolabeled neurons per section were counted in B9

serotoninergic cell group lateral to the interpeduncular

nucleus, and in the majority of cases in PAG.

Numerous serotonin-containing fibers were found in the

inferior olivary complex and in the cranial nerve nuclei XII,

DMX, AMB, the nucleus of the solitary tract, SPV, SVN (very high

density of 5-hydroxytryptamine-positive fibers), VII and V.

Altogether we found no obvious differences in the distribu-

tion and number of neurons ir for serotonin between pR5

mice and the non-transgenic control group.

3. Discussion

3.1. Technical considerations

The immunoreactivity (IR) of the applied anti-tau antibodies

was confirmed in coronal sections of the midbrain, which

also contained temporal parts of the telencephalon. In the

entorhinal and visual cortex, the hippocampus including the

subiculum, the dentate gyrus and the CA1 and CA3 fields of

the Ammon0s horn as well as in nuclei of the amygdala the

pattern of HT7-, AT180-, AT8- and pS422-IR as well as the

distribution of Gallyas silver stained structures (Fig. 2, Bregma

�5.2 mm to �3.6 mm) equates to findings in P301L tau

transgenic pR5 mice described previously (Deters et al.,

2008; Kohler et al., 2010).

Brainstem nuclei and fiber tracts were identified using the

cytoarchitectonic mouse brain atlas by Hof et al. (2000). To

ensure the correct designation of very small structures or

those difficult to categorize unequivocally, their position was

measured and compared to stereotaxic coordinates based on

the Hof mouse brain atlas. Due to the fact that the genetic

background of the P301L tau transgenic pR5 model (C57BL/6)

as well as the applied method of tissue preparation was in

accordance with this atlas, the given dimensions could be

adopted almost one by one. In addition, the identification of

ChAT-, TH- and Serotonin-ir cell groups was performed by

means of the detailed report of the organization of the

b r a i n r e s e a r c h 1 4 9 7 ( 2 0 1 3 ) 7 3 – 8 4 79

cholinergic and monoaminergic systems within the mouse

brainstem (VanderHorst and Ulfhake, 2006).

3.2. Tau hyperphosphorylation pattern in P301L tautransgenic pR5 mice

The expression of human tau protein presently observed in

brainstem nuclei of P301L tau transgenic pR5 mice seems to

be restricted to several functional systems. The majority of

relay stations within the brainstem vestibular system showed

phosphorylation-independent HT7-labeling. In all vestibular

nuclei (i.e. the spinal vestibular nucleus, LVN, MVN, SVN),

that receive their primary input from the equilibrium organ

in the inner ear (Maklad and Fritzsch, 2002), the human tau

protein was detected. HT7-ir fibers were found within the

efferent vestibular pathways toward the ocular muscle nuclei

(Sekirnjak and du Lac, 2006) via the medial longitudinal

fasciculus and the vestibulocerebellum (Maklad and

Fritzsch, 2002) via the inferior cerebellar peduncle. Likewise,

all deep cerebellar nuclei (i.e. the dentate, the interposed and

the fastigial nucleus) as well as the PG and the RN expressed

the transgenic tau protein. In addition to the dominant input

from Purkinje cells (Chung et al., 2009), the cerebellar nuclei

receive afferents from the spinal cord, the inferior olivary

complex, the pontine nuclei, and the RN. In the rat, the deep

cerebellar nuclei were found to send projections to the

thalamus, the vestibular nuclei, the reticular formation, the

inferior olivary complex and the RN (Gonzalo-Ruiz and

Leichnetz, 1987).

Within the brainstem part of the ascending somatosensory

system, consisting primarily of the dorsal column nuclei and

sensory trigeminal nuclei (Mantle-St. John and Tracey, 1987),

human tau protein was observed in neurons of the ECU, the

SPV and the principal trigeminal nucleus, whereas the pro-

prioceptive mesencephalic trigeminal nucleus was devoid of

HT7-IR. The dominant input to the mouse somatosensory

system arises from the facial vibrissae. In the rat, projections

of the dorsal column and sensory trigeminal neurons reach

the ventrobasal thalamus and, in particular efferent fibers

from the ECU, the cerebellum (Kemplay and Webster, 1989;

Mantle-St. John and Tracey, 1987).

The trigeminal motor neurons, which exhibited a remark-

able high density of HT7-positive perikarya, innervate the

muscles of mastication (Terashima et al., 1994) and receive

subcortical projections from the reticular formation, the

principal and the mesencephalic trigeminal nucleus (data

from the rat (Travers and Norgren, 1983)).

Within the retinotectal pathway transgenic human tau was

expressed in neurons of the SC, the III and the IV. The three

superficial layers of the SC obtain substantial input from the

retina and the primary visual cortex, neurons of the deeper

layers respond to auditory and in particular somatosensory

stimuli (Draeger and Hubel, 1975; Draeger and Hubel, 1976).

The principal target regions for efferent projections of the rat

SC are the parabigeminal nucleus, the lateral posterior

pulvinar complex, the pretectum and the lateral geniculate

nucleus (Taylor et al., 1986). The murine ocular muscle nuclei

receive amongst other sources input from the vestibular

nuclei (Sekirnjak and du Lac, 2006).

Also different relay stations within the auditory system

including the IC, the VLL and the nucleus of the brachium of

the IC possessed HT7-ir neurons. The ascending input to the

IC arises from the superior olivary complex, the nuclei of the

lateral lemniscus and the cochlear nuclei (Cant and Benson,

2003; Ryugo et al., 1981), descending projections to the IC

originate from the auditory cortex (Malmierca, 2003). Further-

more, the IC receives non-auditory input from the somato-

sensory system (i.e. the spinal cord, dorsal column nuclei and

the SPV; data from opossum and guinea pig (Robards, 1979;

Zhou and Shore, 2006). Collicular efferents terminate in the

medial geniculate nucleus, which showed a high density of

HT7-labeled fibers. However, in the cochlear nuclei, the

superior olivary complex and the trapezoid body – likewise

nuclei within the auditory pathway - the human tau protein

was largely absent.

In the neuronal network of the lower brainstem, including

cardiovascular, respiratory and visceral control centers,

numerous nuclei especially within the magnocellular zone

of the reticular formation displayed HT7-ir neurons. A

detailed functional classification of the HT7-positive

reticular nuclei (i.e. LRN, RVLN, IRN, GRN, the parvocellular

reticular nucleus, PGRNl, PRN, the mesencephalic reticular

nucleus, AMB) turned out to be difficult, because only sparse

information about their ascending and descending projec-

tions is available and their circuitry is only poorly

understood yet.

To sum up, the expression of the transgenic human tau was

noticed in brainstem nuclei of the vestibular, the somatosen-

sory, the retinotectal and the auditory circuitry, parts of the

reticular formation, as well as in the V, whereas e.g. all main

components of the ascending reticular activating system

(promoting wakefulness) were devoid of human tau protein.

Accordingly, the distribution of human tau protein in tau

transgenic mice seems to be linked to functional pathways.

Moreover, the finding, that different tau transgenic models

expressing human tau protein along with P301L-mutation

offer dissimilar phenotypes with respect to the distribution of

human tau protein and consequently the location of neurofi-

brillary lesions and the kind of behavioral disturbances (Fox

et al., 2011; Lewis et al., 2000; Terwel et al., 2005) suggests an

important role of the promoter and strain background in the

determination of HT7-expression pattern.

The distribution of soluble pretangle material in the brain-

stem of pR5 mice turned out to coincide with the pattern of

human tau protein expression. Almost every HT7-ir cell

group also contained non-filamentous hyperphosphorylated

tau, whereas the density of AT180-positive neurons was

clearly higher than the density of AT8-labeled perikarya.

The occurrence of AT8 and AT180-positive neurons in

brainstem nuclei without transgenic human tau expression

in rare cases (i.e. in the magnocellular reticular, the Barring-

ton, the ventral cochlear and the mesencephalic trigeminal

nucleus) could be explained by a potential cross reactivity of

these antibodies with hyperphosphorylated endogenous tau

protein (Gotz and Ittner, 2008; Kohler et al., 2010).

PS422-IR as well as silver staining was almost totally

missing, indicating the absence of intracellular filamentous

tau deposits and ghost tangles in pR5 brainstem at the age of

20 months.

b r a i n r e s e a r c h 1 4 9 7 ( 2 0 1 3 ) 7 3 – 8 480

3.3. Comparison with findings in the brainstem of ADpatients

The comparison of the distribution of hyperphosphorylated

tau in AD patients (Supplementary data, Table 2) with the

pattern observed in P301L tau transgenic pR5 mice was

complicated by the impreciseness of the references regarding

nomenclature of affected cell groups, particularly the nuclei

of the reticular formation. Due to the lack of insoluble NFTs

within the brainstem of the tau transgenic animals we

focused on the AT8-labeled nuclei, supposing that the phos-

phorylation of Ser202/Thr205 precedes the hyperphosphory-

lation of tau protein forming the final, pathological NFTs

(Menuet et al., 2011) and therefore represents potential tangle

deposition areas.

Just as in pR5 mice, the distribution of pretangle material

in the brainstem of AD subjects seems to be confined

to functional pathways (Braak and Del Tredici, 2011a). In

humans, all cell groups constituting the major source of

brainstem input of the ascending arousal system, exhibit

AT8-IR (Parvizi et al., 2001; Rub et al., 2000; Rub et al., 2001).

This pathway, composed of two major branches ascending to

thalamic relay nuclei and the lateral hypothalamic area,

regulates sleep and produces wakefulness (Saper et al.,

2005). The first branch originates from the cholinergic PPN

and the laterodorsal tegmental nucleus, the second from the

noradrenergic LC, the serotonergic DR and central superior

nucleus of the raphe as well as from the dopaminergic

ventral PAG (Saper et al., 2005). Furthermore, pretangle

material is present in several nuclei of the lower brainstem

respiratory, cardiovascular and swallowing regulation center,

namely the DMX, the AMB, the nucleus of the solitary tract,

the IRN and the PB (Parvizi et al., 2001; Rub et al., 2001).

Evidence of the existence of reciprocal connections between

these nuclei was provided by means of animal tracing

experiments (data from the rat (Yamada et al., 1988)).

Also several relay stations of the spinomesencephalic path-

way within the somatosensory system (Yezierski, 1988) con-

tain AT8-positve neurons, including the cuneiform nucleus,

the Edinger–Westphal nucleus and the PAG (Parvizi et al.,

2001). Due to the imprecise description regarding the affected

layers a functional classification of the AT8-positive SC

turned out to be difficult. While the superficial layers of the

SC are mainly concerned with visually related reflex mechan-

isms, the deeper layers receive a predominant somatosensory

input (data from the monkey (Wiberg et al., 1987)).

Within the auditory circuitry pretangle material is present

in the central subnucleus of the IC (Parvizi et al., 2001),

obtaining input from the cochlear nuclei, the superior olivary

complex, and nuclei of the lateral lemniscus (Huffman and

Henson, 1990). Likewise, neurons of the ventral tegmental

area – constituting the mesolimbic dopaminergic system

together with the telencephalic nucleus accumbens (Nicola

et al., 2005) – show tau hyperphosphorylation at the AT8

epitopes (Parvizi et al., 2001).

The pR5 transgenic model approximates the distribution of

AT8-phosphorylated tau protein observed in the human AD

brainstem concerning four different nuclei (i.e. AMB, IRN, IC,

and SC). To conclude, the pattern of brainstem tau pathology

in pR5 mice differs largely from those in AD patients and

therefore this model seems to be less convenient to examine

brainstem etiopathology of AD.

3.4. Comparison of neurotransmitter expression pattern

The distribution of cholinergic and monoaminergic brain-

stem nuclei in P301L tau transgenic pR5 mice was found to be

consistent with that detected in the non-transgenic litter-

mates and other rodents (Armstrong et al., 1983; Motts et al.,

2008; Palkovits et al., 1974; Steinbusch, 1981; Zeiss, 2005).

Even pretangle affected nuclei in pR5 brainstem (i.e. from

caudal to rostral the cholinergic AMB, ISN, IRN, MVN, PGRNl,

V, IV, VLL as well as the catecholaminergic PAG) offered no

obvious differences in the semiquantitatively evaluated den-

sity of ChAT- and TH-labeled neurons when compared to

non-transgenic littermates.

In contrast, monoaminergic nuclei in the brainstem of AD

patients show both neuronal loss and reduced neurotrans-

mission (Palmer and DeKosky, 1993; Zweig et al., 1988),

whereat the distribution of NFTs in humans suffering from

AD is strikingly similar to that of monoamine containing

nerve cells (Ishii, 1966). On the other hand, in cholinergic

brainstem nuclei exhibiting hyperphosphorylated tau (i.e. PB,

the laterodorsal tegmental nucleus, PPN) no differences in

neuronal densities between AD and normal controls were

observed (Dugger et al., 2012; Mufson et al., 1988). Finally it

remains unknown if neurofibrillary lesions in cholinergic and

monoaminergic neurons induce dysfunction in neurotrans-

mission in AD subjects.

Due to the absence of intracellular insoluble tau deposits in

cholinergic and monoaminergic brainstem nuclei, the

unchanged numbers of choline acetyltransferase, tyrosine

hydroxylase and serotonin containing neurons, as well as the

lack of hyperphosphorylated tau in telencephalic cholinergic

neurons (Kohler et al., 2010), the P301L tau transgenic pR5

model seems to be inappropriate to explore the influence of

tau pathology on neurotransmission involving the mentioned

neurotransmitters and subsequent clinical features.

3.5. Applicability of the P301L pR5 mouse modelregarding other tauopathies

In view of the fact that aside from AD the etiopathology of

related neurodegenerative disorders leading to neurofibrillary

lesions (tauopathies), is largely unknown as well, it seems to

be worthwhile to check, whether P301L tau transgenic pR5

mice might be a suitable model to examine pathogenic

mechanisms in other tauopathies. This holds true particu-

larly with regard to the brainstem being the phylogenetically

oldest part of the brain and therefore featuring numerous

homologous structures in human and rodents.

The antigenic profile of NFTs in subcortical neurons in

progressive supranuclear palsy (PSP) resembles that in AD

(Bancher et al., 1987). This progressive neurodegenerative

disorder is characterized clinically by the impairment of eye

movements (particularly in the vertical plane), gait instability,

bradykinesia, rigidity, dysarthria, dysphagia, as well as cog-

nitive and behavioral alterations (Daniel et al., 1995; Golbe,

2001). The NFTs are primarily localized in areas of the basal

ganglia and the brainstem, whereas the cerebral cortex, the

b r a i n r e s e a r c h 1 4 9 7 ( 2 0 1 3 ) 7 3 – 8 4 81

cerebellum and the spinal cord possess tau pathology to a

lesser extent (Golbe, 2001).

Altogether, the pattern of AT8-hyperphosphorylated tau in

the P301L tau transgenic pR5 model complies with findings of

NFTs in PSP brainstem (Daniel et al., 1995; Jellinger, 1971;

Jellinger, 2008; Probst et al., 1988; Rub et al., 2002; Williams

et al., 2007) concerning ten nuclei, i.e. LRN, IRN, GRN, PRN

within the formatio reticularis, AMB, IV IC, SC, RN and PAG

(Supplementary data, Table 2). Accordingly, the distribution

of pretangle-affected nuclei partly mirrors those observed in

PSP, presenting the pR5 mouse as a model to investigate the

initial brainstem pathogenesis of PSP.

4. Experimental procedures

4.1. Animals

Coronal brainstem sections of five female tau-transgenic

mice at the age of 20 (n¼4) and 24 (n¼1) months were

compared with brainstem sections of four gender- and age-

matched non-transgenic littermates. The experiments were

performed on heterozygous P301L tau transgenic pR5 mice,

which were generated on a B6D2F2 x C57BL/6 background,

backcrossed to C57BL/6 (Harlan Laboratories, Venray, The

Netherlands) seven times. This transgenic strain expresses

the longest four-repeat human tau isoform (htau40 2þ3þ4R)

along with a missense-mutation of exon 10, under control of

the neuron-specific mouse Thy1.2 promoter (Gotz et al.,

2001). The mice were housed under 12 h light-dark cycle with

water and food pellets ad libitum. Experiments were

approved by the governmental animal care and use office.

All animals were kept and treated in accordance with the

German Animal Welfare Act.

4.2. Antibodies

The following anti-tau antibodies were used: conformation

and phosphorylation independent mouse monoclonal anti-

body HT7 (Thermo Scientific Pierce Protein Research Pro-

ducts, Rockford, USA, catalog # MN1000; diluted 1:400),

recognizing residues 159–163 to detect human tau (Mercken

et al., 1992); mouse monoclonal antibodies AT180 and AT8

(both from Thermo Scientific Pierce Protein Research Pro-

ducts, catalog # MN1040 and MN1020; diluted 1:2400 and

1:1000) against hyperphosphorylated epitopes Thr231/Ser235

(Goedert et al., 1994) and Ser202/Thr205 (Goedert et al., 1995);

and rabbit polyclonal antibody pS422 (Invitrogen, Carlsbad,

USA, catalog # 44-764G; diluted 1:4000) against abnormally

phosphorylated epitope Ser422 (Augustinack et al., 2002).

For immunostaining of cholinergic, catecholaminergic and

serotonergic neurons following primary antibodies were

used: goat polyclonal antibody (Millipore, Billerica, USA,

catalog # AB144P; diluted 1:200) raised against ChAT (UniProt

Number: P28329); mouse monoclonal antibody (Immunostar,

Hudson, USA, catalog # 22941; diluted 1:8000), recognizing the

34kDa catalytic core TH molecule; and rabbit polyclonal anti-

5-hydroxytryptamine antibody (US Biological, Swampscott,

USA, catalog # S1001-04; diluted 1:400) against serotonin.

Biotinylated donkey anti-mouse IgG (diluted 1:250 and

1:400), goat anti-rabbit IgG (diluted 1:400 and 1:450) and

donkey anti-goat IgG (diluted 1:450) secondary antibodies

were purchased from Dianova (Hamburg, Germany).

4.3. Tissue preparation

Mice were deeply anesthetized with tribromethanol (0.55 mg/

g body weight, intraperitoneal) and transcardially perfused

with 0.1 M PBS (pH 7.4) for 3 min followed by 4% paraformal-

dedyde in PBS for 15 min. Brains were removed, fixed in 4%

paraformaldehyde in PBS overnight at 4 1C, dehydrated and

embedded in paraffin using a Shandon Citadel tissue proces-

sor (Thermo Shandon, Frankfurt am Main, Germany). Up to

560 coronal serial sections (5 mm) from the caudal medulla

oblongata (Bregma �7.8 mm) to the rostral end of the super-

ior colliculus in the midbrain (Bregma �3.3 mm) were

obtained from each paraffin block.

4.4. Immunohistochemistry and Gallyas silverimpregnation

Paraffin sections were dewaxed in xylene and rehydrated in a

graded alcohol series. Antigen was retrieved by microwaving

near boiling in citrate buffer (0.01 M, pH 6) for 3�5 min, except

for antibody HT7- and pS422-ir. Endogenous peroxide quench-

ing was performed with 0.3% hydrogen solution for 30 min at

RT. After tissue slides were washed in 0.01 M TBS (pH 7.6) for

3�5 min and blocked with 20% fetal bovine serum with 1%

albumin bovine fraction in TBS (45 min, RT), they were

incubated overnight at 4 1C with the primary antibodies

diluted in TBS with 3% skimmed milk powder. Sections were

washed in TBS and incubated with the corresponding biotiny-

lated secondary antibodies diluted in TBS with 3% skimmed

milk powder (30 min, RT). Tissue was rinsed in TBS and

incubated with avidin-biotinylated horseradish peroxidase

reagent (Vectastain Elite ABC Kit from Vector Laboratories,

Burlingame, USA) (30 min, RT). Finally, primary antibody bind-

ing was visualized using chromogen 3, 30-diaminobenzidine in

a solution of one DAB tablet (Sigma–Aldrich, Munchen, Ger-

many), 0.2% hydrogen peroxide, 0.6% ammonium nickel sul-

fate and 0.05% imidazole. Slides were washed in TBS,

counterstained with nuclear fast red for 5 min, dehydrated

through increasing alcohol solutions and xylene and cover-

slipped with DPX mountant (Sigma–Aldrich). Negative controls

included slides incubated without the primary antibody. Gal-

lyas silver staining was performed on 5 mm paraffin sections as

described previously (Braak and Braak, 1991b).

4.5. Evaluation of immunohistochemical resultsand digital imaging

Structures were identified based on a cytoarchitectonic atlas of

C57BL/6 mouse brains (Hof et al., 2000), the terminology of the

anatomical structures has been used in accordance to this atlas.

In addition, the terms A1–A7 noradrenergic, A8–A10 dopami-

nergic, C1–C2 adrenergic and B1–B9 cell group (Dahlstrom and

Fuxe, 1964) were employed to describe the distribution of

catecholaminergic and serotoninergic neurons.

Immunostained tissue sections were analyzed and digital

images were obtained using an Olympus BX 50 microscope

b r a i n r e s e a r c h 1 4 9 7 ( 2 0 1 3 ) 7 3 – 8 482

equipped with a DP12 digital microscope camera and Cell

Imaging Software 2.3 (Olympus, Hamburg, Germany). Color,

brightness and contrast of photographs were adjusted using

Adobe Photoshop CS5 Extended 12.0 (Adobe Systems,

San Jose, USA). Drawings of coronal brainstem sections

(Bregma �7.5 mm, �6.9 mm, �6.1 mm, �5.2 mm, �4.4 mm

and �3.6 mm) to exemplify the distribution of HT7 and

AT8-stained neurons were created and digitized using U-DA

drawing accessory (Olympus) attached to an Olympus BX 40

microscope and Adobe Illustrator CS5 15.0. software (Adobe

Systems).

Acknowledgments

The authors are grateful to Kirsten Pilz, Maja Dinekov and

Sigrun Kuhlage for excellent technical assistance, Brigitte

Dengler for animal care, and Thomas Bombeck for help with

digitalizing illustrations.

This work was supported by grants from the Kathe–Hack

and the Hochhaus foundation of the Faculty of Medicine

(University of Cologne).

Appendix A. Supplementary information

Supplementary data associated with this article can be found

in the online version at http://dx.doi.org/10.1016/j.brainres.

2012.12.016.

r e f e r e n c e s

Armstrong, D.M., et al., 1983. Distribution of cholinergic neuronsin rat-brain: demonstrated by the immunocytochemicallocalization of choline acetyltransferase. J. Comp. Neurol. 216,53–68.

Augustinack, J.C., et al., 2002. Specific tau phosphorylation sitescorrelate with severity of neuronal cytopathology inAlzheimer0s disease. ActaNeuropathol 103, 26–35.

Bancher, C., et al., 1987. Neurofibrillary tangles in Alzheimer0sdisease and progressive supranuclear palsy: antigenicsimilarities and differences. Microtubule-associated proteintau antigenicity is prominent in all types of tangles. Acta.Neuropathol. 74, 39–46.

Braak, E., Braak, H., Mandelkow, E.M., 1994. A sequence ofcytoskeleton changes related to the formation ofneurofibrillary tangles and neuropil threads. Acta.Neuropathol. 87, 554–567.

Braak, H., Braak, E., 1991a. Neuropathologicalstageing ofAlzheimer-related changes. Acta Neuropathol. 82, 239–259.

Braak, H., Braak, E., 1991b. Demonstration of amyloid depositsand neurofibrillary changes in whole brain sections. BrainPathol. 1, 213–216.

Braak, H., Del Tredici, K., 2011a. Alzheimer0s pathogenesis: isthere neuron-to-neuron propagation?. Acta Neuropathol. 121,589–595.

Braak, H., Del Tredici, K., 2011b. The pathological processunderlying Alzheimer0s disease in individuals under 30. ActaNeuropathol. 121, 171–181.

Cant, N.B., Benson, C.G., 2003. Parallel auditory pathways: projectionpatterns of the different neuronal populations in the dorsal andventral cochlear nuclei. Brain Res. Bull. 60, 457–474.

Chung, S.H., Marzban, H., Hawkes, R., 2009. Compartmentationof the cerebellar nuclei of the mouse. Neuroscience 161,123–138.

Dahlstrom, A., Fuxe, K., 1964. Localization of monoamines in thelower brain stem. Experientia 20, 398–399.

Daniel, S.E., de Bruin, V.M., Lees, A.J., 1995. The clinical andpathological spectrum of Steele–Richardson–Olszewskisyndrome (progressive supranuclear palsy): a reappraisal.Brain 118 (3), 759–770.

Delobel, P., et al., 2008. Analysis of tau phosphorylation andtruncation in a mouse model of human tauopathy. Am. J.Pathol. 172, 123–131.

Deters, N., Ittner, L.M., Gotz, J., 2008. Divergent phosphorylationpattern of tau in P301L tau transgenic mice. Eur. J. Neurosci.28, 137–147.

Draeger, U.C., Hubel, D.H., 1975. Responses to visual stimulationand relationship between visual, auditory, and somatosensoryinputs in mouse superior colliculus. J. Neurophysiol. 38,690–713.

Draeger, U.C., Hubel, D.H., 1976. Topography of visual andsomatosensory projections to mouse superior colliculus. J.Neurophysiol. 39, 91–101.

Dugger, B.N., et al., 2012. Neuropathological analysis of brainstemcholinergic and catecholaminergic nuclei in relation to rapideye movement (REM) sleep behaviour disorder. Neuropathol.Appl. Neurobiol. 38, 142–152.

Dutschmann, M., et al., 2010. Upper airway dysfunction oftau-P301L mice correlates with tauopathy in midbrainand ponto-medullary brainstem nuclei. J. Neurosci. 30,1810–1821.

Fox, L.M., et al., 2011. Soluble tau species, not neurofibrillaryaggregates, disrupt neural system integration in a tautransgenic model. J. Neuropathol. Exp. Neurol. 70, 588–595.

Francis, P.T., et al., 1999. The cholinergic hypothesis ofAlzheimer0s disease: a review of progress. J. Neurol.Neurosurg. Psychiatry 66, 137–147.

German, D.C., White 3rd, C.L., Sparkman, D.R., 1987. Alzheimer0sdisease: neurofibrillary tangles in nuclei that project to thecerebral cortex. Neuroscience 21, 305–312.

Goedert, M., et al., 1994. Epitope mapping of monoclonalantibodies to the paired helical filaments of Alzheimer0sdisease: identification of phosphorylation sites in tau protein.Biochem. J. 301, 871–877.

Goedert, M., Jakes, R., Vanmechelen, E., 1995. Monoclonalantibody AT8 recognizes tau protein phosphorylated atboth serine 202 and threonine 205. Neurosci. Lett. 189,167–170.

Golbe, L.I., 2001. Progressive supranuclear palsy. Curr. TreatOptions Neurol. 3, 473–477.

Gonzalo-Ruiz, A., Leichnetz, G.R., 1987. Collateralization ofcerebellar efferent projections to the paraoculomotor region,superior colliculus, and medial pontine reticular formation inthe rat: a fluorescent double-labeling study. Exp. Brain Res. 68,365–378.

Gottfries, C.G., 1990. Disturbance of the 5-hydroxytryptaminemetabolism in brains from patients with Alzheimer0sdementia. J. Neural. Transm-Supp. 30, 33–43.

Gotz, J., et al., 2001. Tau filament formation in transgenic miceexpressing P301L tau. J. Biol. Chem. 276, 529–534.

Gotz, J., Ittner, L.M., 2008. Animal models of Alzheimer0s diseaseand frontotemporal dementia 9, 532–544.

Grinberg, L.T., et al., 2009. The dorsal raphe nucleus showsphosphotau neurofibrillary changes before thetransentorhinal region in Alzheimer0s disease. A precociousonset?. Neuropath. Appl. Neuro. 35, 406–416.

Heinsen, H., et al., 1989. Laminar neuropathology in Alzheimer0sdisease by a modified Gallyas impregnation. Psychiatry Res.29, 463–465.

b r a i n r e s e a r c h 1 4 9 7 ( 2 0 1 3 ) 7 3 – 8 4 83

Herholz, K., 2008. Acetylcholine esterase activity in mild cognitiveimpairment and Alzheimer0s disease. Eur. J. Nucl. Med. MolImaging 35 (1), S25–S29.

Hirano, A., Zimmerman, H.M., 1962. Alzheimer0s neurofibrillarychanges. A topographic study. Arch. Neurol. 7, 227–242.

Hof, P.R., et al., 2000. Comparative cytoarchitectonic atlas of theC57BL/6 and 129/Sv mouse brains. Elsevier Science B.V,Amsterdam.

Huffman, R.F., Henson, O.W., 1990. The descending auditorypathway and acousticomotor systems: connections with theinferior colliculus. Brain Res. Rev. 15, 295–323.

Ishii, T., 1966. Distribution of Alzheimer0s neurofibrillary changesin the brain stem and hypothalamus of senile dementia. ActaNeuropathol. 6, 181–187.

Jellinger, K.A., 1971. Progressive supranuclear palsy (subcorticalargyrophilic dystrophy). Acta Neuropathol. 19, 347–352.

Jellinger, K.A., 2008. Different tau pathology pattern in twoclinical phenotypes of progressive supranuclear palsy.Neurodegener. Dis. 5, 339–346.

Kemplay, S., Webster, K.E., 1989. A quantitative study of theprojections of the gracile, cuneate and trigeminal nuclei andof the medullary reticular formation to the thalamus in therat. Neuroscience 32, 153–167.

Kohler, C., et al., 2010. Analysis of the cholinergic pathology in theP301L tau transgenic pR5 model of tauopathy. Brain Res. 1347,111–124.

Lee, V.M., Goedert, M., Trojanowski, J.Q., 2001. Neurodegenerativetauopathies. Annu. Rev. Neurosci. 24, 1121–1159.

Lewis, J., et al., 2000. Neurofibrillary tangles, amyotrophy andprogressive motor disturbance in mice expressing mutant(P301L) tau protein. Nat. Genet. 25, 402–405.

Maklad, A., Fritzsch, B., 2002. The developmental segregation ofposterior crista and saccular vestibular fibers in mice: acarbocyanine tracer study using confocal microscopy. BrainRes. Dev. Brain Res. 135, 1–17.

Malmierca, M.S., 2003. The structure and physiology of the ratauditory system: an overview. Int. Rev. Neurobiol. 56, 147–211.

Mantle-St. John, L.A., Tracey, D.J., 1987. Somatosensory nuclei inthe brainstem of the rat: independent projections to thethalamus and cerebellum. J. Comp. Neurol. 255, 259–271.

Matsuo, E.S., et al., 1994. Biopsy-derived adult human brain tau isphosphorylated at many of the same sites as Alzheimer0sdisease paired helical filament tau. Neuron 13, 989–1002.

McGeer, P.L., et al., 1984. Aging, Alzheimer0s disease, and thecholinergic system of the basal forebrain. Neurology 34, 741–745.

Menuet, C., et al., 2011. Raphe tauopathy alters serotoninmetabolism and breathing activity in terminal Tau.P301Lmice: possible implications for tauopathies and Alzheimer0sdisease. Respir. Physiol. Neurobiol. 178, 290–303.

Mercken, M., et al., 1992. Monoclonal antibodies with selectivespecificity for Alzheimer Tau are directed againstphosphatase-sensitive epitopes. Acta Neuropathol. 84,265–272.

Motts, S.D., et al., 2008. Distribution of cholinergic cells in guineapig brainstem. Neuroscience 154, 186–195.

Mufson, E.J., Mash, D.C., Hersh, L.B., 1988. Neurofibrillary tanglesin cholinergic pedunculopontine neurons in Alzheimer0sdisease. Ann Neurol. 24, 623–629.

Nicola, S.M., et al., 2005. Nucleus accumbens dopamine release isnecessary and sufficient to promote the behavioral responseto reward-predictive cues. Neuroscience 135, 1025–1033.

Overk, C.R., Kelley, C.M., Mufson, E.J., 2009. BrainstemAlzheimer0s-like pathology in the triple transgenic mousemodel of Alzheimer0s disease. Neurobiol Dis. 35, 415–425.

Palkovits, M., Brownstein, M., Saavedra, J.M., 1974. Serotonincontent of brain stem nuclei in rat. Brain Res. 80, 237–249.

Palmer, A.M., DeKosky, S.T., 1993. Monoamine neurons in aging andAlzheimer0s disease. J. Neural. Transm Gen. Sect. 91, 135–159.

Parvizi, J., Van Hoesen, G.W., Damasio, A., 2001. The selectivevulnerability of brainstem nuclei to Alzheimer0s disease. Ann.Neurol. 49, 53–66.

Perry, E., 1988. Acetylcholine and Alzheimer0s disease. Br. J.Psychiatry 152, 737–740.

Probst, A., et al., 1988. Progressive supranuclear palsy: extensiveneuropil threads in addition to neurofibrillary tangles. Verysimilar antigenicity of subcortical neuronal pathology inprogressive supranuclear palsy and Alzheimer0s disease. ActaNeuropathol. 77, 61–68.

Probst, A., et al., 2000. Axonopathy and amyotrophy in micetransgenic for human four-repeat tau protein. ActaNeuropathol. 99, 469–481.

Procter, A.W., et al., 1988. Topographical distribution ofneurochemical changes in Alzheimer0s disease. J. Neurol.Sci. 84, 125–140.

Robards, M.J., 1979. Somatic neurons in the brainstem andneocortex projecting to the external nucleus of the inferiorcolliculus: an anatomical study in the opossum. J. Comp.Neurol. 184, 547–565.

Rub, U., et al., 2000. The evolution of Alzheimer0s disease-relatedcytoskeletal pathology in the human raphe nuclei. NeuropathAppl. Neuro. 26, 553–567.

Rub, U., et al., 2001. The autonomic higher order processingnuclei of the lower brain stem are among the early targetsof the Alzheimer0s disease-related cytoskeletal pathology.Acta Neuropathol. 101, 555–564.

Rub, U., et al., 2002. Progressive supranuclear palsy: neuronal andglial cytoskeletal pathology in the higher order processingautonomic nuclei of the lower brainstem. Neuropath Appl.Neuro. 28, 12–22.

Ryugo, D.K., Willard, F.H., Fekete, D.M., 1981. Differential afferentprojections to the inferior colliculus from the cochlearnucleus in the albino mouse. Brain Res. 210, 342–349.

Saper, C.B., Scammell, T.E., Lu, J., 2005. Hypothalamic regulationof sleep and circadian rhythms. Nature 437, 1257–1263.

Sekirnjak, C., du Lac, S., 2006. Physiological and anatomicalproperties of mouse medial vestibular nucleus neuronsprojecting to the oculomotor nucleus. J. Neurophysiol. 95,3012–3023.

Simic, G., et al., 2009. Does Alzheimer0s disease begin in thebrainstem?. Neuropath Appl. Neuro. 35, 532–554.

Steinbusch, H.W.M., 1981. Distribution of serotonin-immunoreactivity in the central nervous system of the rat-cell bodies and terminals. Neuroscience 6, 557–618.

Taylor, A.M., Jeffery, G., Lieberman, A.R., 1986. Subcorticalafferent and efferent connections of the superior colliculusin the rat and comparisons between albino and pigmentedstrains. Exp. Brain Res. 62, 131–142.

Terashima, T., Kishimoto, Y., Ochiishi, T., 1994. Musculotopicorganization in the motor trigeminal nucleus of the reelermutant mouse. Brain Res. 666, 31–42.

Terwel, D., et al., 2005. Changed conformation of mutant Tau-P301Lunderlies the moribund tauopathy, absent in progressive,nonlethal axonopathy of Tau-4R/2N transgenic mice. J. Biol.Chem. 280, 3963–3973.

Travers, J.B., Norgren, R., 1983. Afferent projections to the oralmotor nuclei in the rat. J. Comp. Neurol. 220, 280–298.

VanderHorst, V.G.J.M., Ulfhake, B., 2006. The organization of thebrainstem and spinal cord of the mouse: relationshipsbetween monoaminergic, cholinergic, and spinal projectionsystems. J. Chem. Neuroanat. 31, 2–36.

Wiberg, M., Westman, J., Blomqvist, A., 1987. Somatosensoryprojection to the mesencephalon: an anatomical study inthe monkey. J. Comp. Neurol. 264, 92–117.

Williams, D.R., et al., 2007. Pathological tau burden and distributiondistinguishes progressive supranuclear palsy–parkinsonismfrom Richardson0s syndrome. Brain 130, 1566–1576.

b r a i n r e s e a r c h 1 4 9 7 ( 2 0 1 3 ) 7 3 – 8 484

Yamada, H., Ezure, K., Manabe, M., 1988. Efferent projectionsof inspiratory neurons of the ventral respiratory group.A dual labeling study in the rat. Brain Res. 455283–294

Yezierski, R.P., 1988. Spinomesencephalic tract: projections fromthe lumbosacral spinal cord of the rat, cat, and monkey.J. Comp. Neurol. 267, 131–146.

Zeiss, C.J., 2005. Neuroanatomical phenotyping in the mouse: thedopaminergic system. Vet. Pathol. 42, 753–773.

Zhou, J., Shore, S., 2006. Convergence of spinal trigeminal andcochlear nucleus projections in the inferior colliculus of theguinea pig. J. Comp. Neurol. 495, 100–112.

Zweig, R.M., et al., 1988. The neuropathology of aminergic nucleiin Alzheimer0s disease. Ann. Neurol. 24, 233–242.