Pattern of Tau Hyperphosphorylation and Neurotransmitter Markers in the Brainstem of Senescent Tau ﬁlament Forming Transgenic Mice

brain research 1497 (2013) 73–84

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Research Report Pattern of tau hyperphosphorylation and neurotransmitter markers in the brainstem of senescent tau ﬁlament forming transgenic mice

Kerstin Morcineka,Ã, Christoph Ko¨hlera,Ju¨ rgen Go¨tzb, Hannsjo¨rg Schro¨dera aDepartment of Anatomy II (Neuroanatomy), University of Cologne, Kerpener Strabe 62, 50924 Cologne, Germany bCentre for Ageing Dementia Research (CADR), Queensland Brain Institute (QBI), The University of Queensland, St Lucia Campus (Brisbane), QLD 4072, Australia article info abstract

Article history: The early occurrence of brainstem-related symptoms, e.g. gait and balance impairment, Accepted 12 December 2012 apathy and depression in Alzheimer’s disease patients suggests brainstem involvement in Available online 20 December 2012 the initial pathogenesis. To address the question whether tau filament forming mice Keywords: expressing mutated human tau mirror histopathological changes observed in Alzheimer Alzheimer0s disease brainstem, the degree and distribution of neurofibrillary lesions as well as the pattern of Tau hyperphosphorylation cholinergic and monoaminergic neurons were investigated. The expression of the human Neurotransmitter tau transgene was observed in multiple brainstem nuclei, particularly in the magnocellular Brainstem reticular formation, vestibular nuclei, cranial nerve motor nuclei, sensory trigeminal nerve Transgenic mouse nuclei, inferior and superior colliculi, periaqueductal and pontine gray matter, and the red Tauopathy nucleus. Most of the human tau-immunoreactive cell groups also showed tau hyperphosphorylation 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 monoaminergic neurons remained unchanged in the transgenic mice, while monoaminergic nuclei in Alzheimer brainstem showed a distinct neuronal loss. However, the distribution of

Abbreviations (according to Hof et al., 2000): AD, Alzheimer0s disease; AMB, ambiguous nucleus; ChAT, choline acetyltransferase enzyme; DMX, dorsal motor nucleus of the vagus; DR, dorsal nucleus of the raphe; ECU, external cuneate nucleus; GRN, gigantocellular reticular nucleus; IC, inferior colliculus; III, oculomotor nucleus; ir/ IR, immunoreactive/ immunoreactivity; IRN, intermediate reticular nucleus; ISN, inferior salivatory nucleus; IV, trochlear nucleus; LC, locus coeruleus; LRN, lateral reticular nucleus; LVN, lateral vestibular nucleus; MVN, medial vestibular nucleus; NFTs, neuroﬁbrillary tangles; PAG, periaqueductal gray matter; PB, parabrachial nucleus; PG, pontine gray matter; PGRNl, lateral paragigantocellular nucleus; PPN, pedunculopontine nucleus; PRN, pontine reticular nucleus; PSP, progressive supranuclear palsy; RN, red nucleus; RVLN, rostroventrolateral reticular nucleus; SC, superior colliculus; SPV, spinal trigeminal nucleus; SVN, superior vestibular nucleus; TH, tyrosine hydroxylase; V, motor trigeminal nucleus; VII, facial nucleus; VLL, ventral nucleus of the lateral lemniscus; XII, hypoglossal nucleus ÃCorresponding author. Fax: þ49 221 478 5318. E-mail address: [email protected] (K. Morcinek).

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 Procter et al., 1988). The number of serotonergic neurons in the DR - source of the ascending serotonergic transmitter system Abnormally phosphorylated forms of the microtubule asso- to numerous cortical and subcortical regions of the forebrain ciated protein tau - physiologically abundantly expressed in (Ru¨ betal.,2000) - declines to 50% of the control value in AD neurons and glial cells (Lee et al., 2001) - constitute the major subjects (Zweig et al., 1988). Dysfunction within this system component of neurofibrillary tangles (NFTs) and neuropil can cause disrupted sleeping pattern, depressive mood swings, threads in the brain tissue of patients suffering from and inadequate affective control (Ru¨ betal.,2000). Whether Alzheimer0s disease (AD) (Mercken et al., 1992). In addition to these deficits in neurotransmission are linked to the occur- telencephalic changes (Braak and Braak, 1991a) evidence has rence of neurofibrillary lesions in cholinergic and monoami- been presented for the early appearance of tau pathology in nergic nuclei, e.g. in the noradrenergic locus coeruleus or the brainstem nuclei of AD subjects (Simic et al., 2009). In the serotonergic oral raphe complex, is not finally clarified. midbrain, the oral raphe nuclei, i.e. central linear, central Notwithstanding the findings of NFTs and neuropil threads in superior and dorsal raphe nucleus (DR) show tau deposition brainstem nuclei and the neuronal loss in monoaminergic nuclei in correlation with the staging system of Braak and Braak (Ru¨ b in the reticular formation, no animal model reflecting brainstem et al., 2000). During the disease process the supratrochlear changes that are observed in AD patients has been described yet. subunit of the DR is already affected by tangle formation prior Only sparse information about neurofibrillary lesions in the to the transentorhinal stage (Grinberg et al., 2009). The peria- brainstem of tau transgenic mice is obtainable (Delobel et al., queductal gray matter (PAG), the pedunculopontine nucleus 2008; Dutschmann et al., 2010; Menuet et al., 2011; Overk et al., (PPN), the parabrachial nucleus (PB) (German et al., 1987; Parvizi 2009; Probst et al., 2000). Therefore the objective of the present et al., 2001) and the locus coeruleus (LC) (Hirano and study was to investigate the degree and localization of neurofi- Zimmerman, 1962; Ishii, 1966; Parvizi et al., 2001)–inclusive brillary lesions as well as the distribution of cholinergic, cate- the oral raphe nuclei constituting the main components of the cholaminergic and serotonergic neurons throughout the brainstem ascending arousal system – exhibit insoluble fila- brainstem of senescent P301L tau transgenic pR5 mice. mentous tau during isocortical stages. Within the reticular formation – containing several control centers of vitally important functions, e.g. cardiovascular, respiratory and visceral 2. Results regulation - the intermediate reticular nucleus (IRN) develops NFTs (Parvizi et al., 2001; Ru¨ betal.,2001). In particular the 2.1. Location of anti-tau antibodies in the brainstem recent finding of intraneuronal pretangle material in the LC of of P301L tau transgenic pR5 mice children and young adults may indicate that the pathologic process leading to abnormal tau pathology begins in selected 2.1.1. HT7-immunoreactivity (IR) subcorticalnuclei(Braak and Del Tredici, 2011b). The phosphorylation-independent anti-tau antibody HT7 Along with tau pathology, several neurotransmitter systems specifically recognizing the human tau protein was used to in AD brains are affected by dysfunction, loss of neurons and detect neurons expressing the transgene construct. reduced transmitter concentrations (Palmer and DeKosky, In the reticular formation from caudal to rostral a moderate 1993). Particularly the cholinergic basal nucleus of Meynert number of HT7-positive neurons was observed in the lateral and septal nuclei with projections to almost all cortical areas, reticular nucleus (LRN) and GRN (Fig. 1a), fewer neurons were the hippocampus and the thalamus are severely impaired in a stained in the IRN (Fig. 1b), lateral part of paragigantocellular subset of AD brains (Herholz, 2008; McGeer et al., 1984). The (PGRNl), parvocellular, mesencephalic and pontine (PRN) loss and functional disturbance of cholinergic forebrain neu- reticular nucleus (Fig. 2, Bregma 7.5 mm to 4.4 mm). In rons is associated with a reduction of cholinergic neurotrans- two tau transgenic mice a solitary HT7-labeled perikaryon mission, choline acetyltransferase and acetylcholinesterase was located in the rostroventrolateral reticular nucleus enzyme activity (Francis et al., 1999; Herholz, 2008; Procter (RVLN). No expression of human tau was detected in nuclei et al., 1988). Likewise, the content of the catecholamine of the raphe in the median region and in the LC and PB in the noradrenaline is reduced in AD temporal and frontal cortices lateral region of the formatio reticularis. The nucleus raphe (Palmer and DeKosky, 1993). The neuron density in the pontine obscurus showed a single labeled neuron in two individuals. LC – major nucleus of origin of corticopetal noradrenergic In the caudal medulla oblongata one to five human tau- fibers - is significantly diminished (Zweig et al., 1988). Also positive neurons per section were found in the external the concentration of the neurotransmitter serotonin and the cuneate nucleus (ECU) and in the more medially located number of serotonergic nerve endings and receptors is sig- spinal vestibular nucleus (Figs. 1c and 2, Bregma 7.5 mm), nificantly reduced in cortical areas of the temporal lobe as well as in the lateral (LVN) and superior (SVN) vestibular and the prefrontal cortex in AD (Gottfries, 1990; Perry, 1988; nucleus (Figs. 1d and 2, Bregma 6.1 mm) in the rostral brain research 1497 (2013) 73–84 75

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.

medulla oblongata and the pons. Also in the area of the lemniscus (VLL) a moderate number of HT7-ir perikarya was medial vestibular nucleus (MVN) a few HT7-immunoreactive discovered (Fig. 2, Bregma 4.4 mm). (ir) perikarya were noticed in three animals, but because of In the midbrain, small HT7-labeled neurons were observed the close neighborhood and difficult delineation of LVN and in moderate density in the inferior (IC) and superior (SC) SVN an unequivocal assignment of the labeled neurons was colliculi (Figs. 1gand2, Bregma 6.1 mm to 3.6 mm). The not possible. In the dorsal cochlear nucleus, located ventro- red nucleus (RN) showed a moderate number of HT7- lateral to the vestibular nuclei, one HT7-labeled neuronal cell stained cells as well (Figs. 1hand2, Bregma 3.6 mm). body was observed in a single case. Fewer human tau containing perikarya were found in the In cranial nerve motor nuclei a dense cluster of large HT7-ir nucleus of the brachium of the IC (Figs. 1iand2, Bregma neurons was found in the motor nucleus of the trigeminal 3.6 mm). In a single case the expression of human tau nerve (V) (Figs. 1e and 2, Bregma 5.2 mm). In the hypoglossal was noticed in the PAG (Fig. 2, Bregma 4.4 mm). All deep nucleus (XII), the ambiguous nucleus (AMB) and the nucleus cerebellar nuclei, including the fastigial, the interposed and of the facial nerve (VII) (Fig. 2, Bregma 6.1 mm) sporadic the dentate nucleus (Fig. 2, Bregma 6.9 mm to 6.1 mm) HT7-labeled neurons were detected. Three of the five exam- showed a low density of HT7-ir neurons in all tau transgenic ined pR5 mice also showed moderate expression of human animals. tau in the trochlear (IV) and the oculomotor (III) nucleus In addition to the occurrence of labeled fibers in nuclei (Figs. 1f and 2, Bregma 4.4 mm to 3.6 mm), whereas the containing HT7-positive neuronal perikarya, numerous abducens nucleus was devoid of immunostaining. In the pons intensely stained fibers were observed from caudal to rostral a low density of HT7-positive cell bodies was present in the in the pyramidal and ventral spinocerebellar tract, the infer- inferior salivatory nucleus (ISN) in two animals (Fig. 2, ior and middle cerebellar peduncle, the medial longitudinal Bregma 6.1 mm). In the spinal (SPV) and the principal fasciculus, the cerebellar white matter and the medial geni- sensory trigeminal nerve nuclei six to ten HT7-labeled neu- culate nucleus (Fig. 1i). rons per section were observed (Fig. 2, Bregma 7.5 mm to In the brainstem of the non-transgenic littermates there 5.2 mm), just as in the pontine gray matter (PG) (Fig. 2, was no expression of the human tau protein at all (Supple- Bregma 3.6 mm). In the ventral nucleus of the lateral mentary data, Fig. 1l). 76 brain research 1497 (2013) 73–84

FN IP DN

ECU ECU SPVN SPV SPV

IRN GRN GRN SPV GRN SPV SPV LRN LRN GR N GRN LRN LRN SPV SPV HT7 AT8 HT7 AT8 1 mm 1 mm Bregma - 7.5 mm Bregma - 6.9 mm

IC IC VIS

IC VIS IC MEV

IP DN SVN SVN LVN ENT

ISN ENT V GRN PSV V PRN PRN SPV VII SPV HT7 AT8 HT7 AT8 1 mm 1 mm Bregma - 6.1 mm Bregma - 5.2 mm

VIS SC SUB SC VIS DG SC PAG SC IC CA1 III DG IV IC SUB CA3 NB RN RN CA3 CA1

VLL SUB PRN PRN ENT PG VLL ENT VLL AMYG AMYG ENT PG

HT7 AT8 HT7 AT8 1 mm 1 mm Bregma - 4.4 mm Bregma - 3.6 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.

2.1.2. Immunoreactivity pattern of hyperphosphorylation and PRN in the formatio reticularis showed an AT180-IR pattern markers AT180 and AT8 comparable to that of human tau expression regarding the AT8 and AT180 anti-tau antibodies recognize phosphorylated density of labeled cells and the intensity of staining. The same epitopes of both soluble tau aggregates and NFTs, and holds true for the vestibular nuclei MVN and SVN, the cranial thereby constitute markers of pretangle tau hyperphosphor- nerve motor nuclei XII, AMB, VII, V, IV and III, ISN and the dorsal ylation state, whereas AT8-IR is supposed to peak somewhat cochlear nucleus in the pons and VLL, PG, SC and RN in the earlier than AT180-IR (Braak et al., 1994; Goedert et al., 1994; midbrain. AT180-ir neurons outnumbered HT7-positive cells in Matsuo et al., 1994). ECU and LVN in the hindbrain and in IC in the caudal From medulla oblongata to mesencephalon the brainstem mesencephalon. A low density of AT180-staining was detected nuclei LRN, RVLN, PGRNl, the mesencephalic reticular nucleus in all animals in GRN in the medial reticular formation and in brain research 1497 (2013) 73–84 77

the sensory trigeminal nerve nuclei, including the mesence- were seen in IC, SC, RN and PAG (Fig. 2,Bregma6.1 mm to phalic nucleus, as well as in PAG in three tau transgenic mice. 3.6 mm, 3d to 3f). In VLL a few weakly stained neurons were In individual cases (for specifics see supplementary data, detected in two individuals. In the deep cerebellar nuclei AT8-ir Table 1) one to five AT180-positive cell bodies were noticed in cells were absent, except in the fastigial nucleus two marked the magnocellular reticular nucleus in the medial zone of the neurons were identified in one case. formatio reticularis, in the spinal vestibular nucleus in the In addition to AT180 and AT8-positive fibers in several hindbrain, in the pontine nucleus Barrington, the nucleus nuclei displaying AT180- and AT8-ir perikarya a distinct Ko¨lliker–Fuse (weak intensity of staining), and in the ventral AT180-ir fiber labeling was present in the inferior and middle cochlear nucleus and the nucleus of the brachium of the IC in cerebellar peduncle, and the trapezoid body. the midbrain. The deep cerebellar nuclei (i.e. the fastigial, the Neither AT180- nor AT8-IR was noticed in the brain sections interposed and the dentate nucleus) exhibited a moderate of non-transgenic littermates (Supplementary data, Fig. 2f). number of AT180-positive neurons in the majority of animals (for specifics see Supplementary data, Table 1). 2.1.3. Immunoreactivity of pS422 and Gallyas silver In comparison to HT7 and AT180 fewer AT8-ir cells per impregnation in pR5 mice nucleus were observed in most cases. In the reticular formation The abnormal phosphorylation of tau protein at Ser422 one to five AT8-labeled neurons per section were found in LRN appears to be linked with the process of tau filament forma- (Fig. 3a), GRN and PRN (Fig. 2,Bregma7.5 mm to 4.4 mm); in tion and aggregation (Augustinack et al., 2002; Deters et al., IRN, PGRNl, and the mesencephalic reticular nucleus AT8-ir cell 2008); hence the pS422-IR was used as marker of NFT stages. bodies were detected only in individual cases (for specifics see In addition, Gallyas silver impregnation was applied to Supplementary data, Table 1), whereas RVLN and the parvocel- visualize argyrophilic NFTs, neuropil threads and ghost lular reticular nucleus were devoid of staining. From the caudal tangles (Braak et al., 1994; Heinsen et al., 1989). medulla oblongata to the rostral pons ECU, the spinal vestibular Throughout the brainstem pS422-labeling was observed in nucleus, ISN, LVN, the dorsal cochlear nucleus, SVN and PG five individual neurons. One single pS422-positive neuronal revealed small numbers of AT8-labeled perikarya in a few perikaryon was detected in the reticular formation in GRN animals (for specifics see Supplementary data, Table 1). Among and PRN in two transgenic mice (Supplementary data, Figs. 3a the motor nuclei of the cranial nerves, V (Fig. 2,Bregma and b) and in SPV in one animal. In tissue sections stained 5.2 mm) showed a medium density of AT8-positive cells in with Gallyas silver impregnation only one labeled neuron was all tau transgenic mice. Fewer labeled neurons were noticed in found in GRN in the hindbrain of one animal (Supplementary two animals in AMB (Fig. 3b) and in three mice in IV (Fig. 3c), data, Fig. 3c). The cerebellum was devoid of pS422-ir cell while in XII, VII and III AT8-staining was completely lacking. In bodies and silver staining. all sensory trigeminal nerve nuclei (Fig. 2,Bregma7.5 mm to Neither pS422-ir nor silver stained fibers were present in 5.2 mm) solitary AT8-ir neurons were observed in the majority brainstem and cerebellum. of animals (for specifics see supplementary data, Table 1). In the The brainstem of the non-transgenic littermates was midbrain, small numbers of AT8-positive neuronal perikarya devoid of pS422-IR and Gallyas silver impregnated structures.

Fig. 3 – Hyperphosphorylation of tau protein in P301L tau transgenic pR5 mice in brainstem nuclei also affected in PSP subjects. Coronal brainstem sections (5 lm thick) counterstained with nuclear fast red. AT8-ir neurons in (a) the lateral reticular nucleus dorsal of the ventral spinocerebellar tract (vsc), (b) the ambiguous nucleus, (c) the trochlear nucleus lateral of the medial longitudinal fasciculus (mlf), (d) the superior colliculus, (e) the red nucleus and (f) the periaqueductal gray matter. Bar¼200 lm. 78 brain research 1497 (2013) 73–84

2.2. Location of neurotransmitters in the brainstem and the ventral tegmental area (A10 dopaminergic cells) of P301L tau transgenic pR5 mice showed numerous TH-labeled neurons. Distinct TH-ir fiber labeling was noticed in several animals The distribution of cholinergic, catecholaminergic and sero- from caudal pons to rostral mesencephalon in LC, PPN and tonergic neurons was in general in accordance with the the central linear nucleus of the raphe. findings described in the literature (VanderHorst and The findings again equate to those found in the non- Ulfhake, 2006). transgenic littermates.

2.2.1. Choline acetyltransferase (ChAT) immunoreactivity ChAT-IR was used to display the pattern of cholinergic 2.2.3. Serotonin immunoreactivity neurons in the murine brainstem. A moderate number of serotonergic perikarya was present in From the medulla oblongata to the mesencephalon in the the nucleus raphe obscurus, nucleus raphe pallidus, nucleus brainstem of P301L tau transgenic pR5 mice a moderate raphe magnus, central superior nucleus and dorsal nucleus (DR) of the raphe in the median zone of the reticular density of ChAT-positive perikarya was present in IRN, PGRNl formation. In the magnocellular region of the formatio and PB in the reticular formation. In the hindbrain a few reticularis and dorsolateral to the pyramidal tract extending cholinergic cells were observed in the area postrema, the from the medulla oblongata to pons a low to moderate nucleus of the solitary tract, the prepositus hypoglossal nucleus and MVN. Among the cranial nerve motor nuclei density of 5-hydroxytryptamine-positive neurons was XII, the dorsal motor nucleus of the vagus (DMX), AMB, VII, V detected within the inferior olivary complex, in PGRNl, the and III showed numerous intensely stained large neurons. peripyramidal nucleus and in two transgenic mice also in the Fewer ChAT-labeled cell bodies were detected in ISN, the nucleus of the trapezoid body. In the midbrain six to 10 accessory facial nucleus, the abducens nucleus and IV. In four immunolabeled neurons per section were counted in B9 animals a moderate number of ChAT-ir cells was identified in serotoninergic cell group lateral to the interpeduncular SPV of the sensory trigeminal complex. In the pons, medial to nucleus, and in the majority of cases in PAG. Numerous serotonin-containing fibers were found in the middle cerebellar peduncle a dense cluster of weakly stained inferior olivary complex and in the cranial nerve nuclei XII, neurons was found in the nucleus Ko¨ lliker–Fuse. A moderate DMX, AMB, the nucleus of the solitary tract, SPV, SVN (very high density of cholinergic neurons was noticed in the laterodorsal density of 5-hydroxytryptamine-positive fibers), VII and V. tegmental nucleus and PPN in the mesopontine tegmentum, as well as in the ventrally located periolivary nuclei. Lateral to Altogether we found no obvious differences in the distribu- the laterodorsal tegmental nucleus one to five ChAT-ir neu- tion and number of neurons ir for serotonin between pR5 rons per section were detected in the nucleus Barrington in mice and the non-transgenic control group. 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 3. Discussion close topographical relationship of these nuclei a definite classification was not feasible. 3.1. Technical considerations Numerous ChAT-labeled fibers were present in the cranial nerves and corresponding motor nuclei XII, DMX, AMB, VII The immunoreactivity (IR) of the applied anti-tau antibodies and V, in the pons in the area of PG and in the midbrain in SC, was confirmed in coronal sections of the midbrain, which the interpeduncular nucleus and the fasciculus retroflexus. also contained temporal parts of the telencephalon. In the This distribution of cell bodies and fibers ir for choline entorhinal and visual cortex, the hippocampus including the acetyltransferase coincides with the pattern of cholinergic subiculum, the dentate gyrus and the CA1 and CA3 fields of neurons in the brainstem of non-transgenic littermates. the Ammon0s horn as well as in nuclei of the amygdala the pattern of HT7-, AT180-, AT8- and pS422-IR as well as the 2.2.2. Tyrosine hydroxylase (TH) immunoreactivity distribution of Gallyas silver stained structures (Fig. 2, Bregma In the ventrolateral part of the caudal medulla oblongata six 5.2 mm to 3.6 mm) equates to findings in P301L tau to ten TH-ir neurons per section were noticed in RVLN (C1 transgenic pR5 mice described previously (Deters et al., group of adrenergic cells), in the dorsomedial part of the 2008; Ko¨ hler et al., 2010). lower brainstem a smaller number of labeled neurons was Brainstem nuclei and fiber tracts were identified using the present in the area postrema, DMX, the nucleus of the cytoarchitectonic mouse brain atlas by Hof et al. (2000).To solitary tract and the prepositus hypoglossal nucleus. In the ensure the correct designation of very small structures or pons a dense cluster of TH-positive cells was localized lateral those difficult to categorize unequivocally, their position was to the fourth ventricle in LC (A6 dopaminergic cells). A few measured and compared to stereotaxic coordinates based on TH-stained perikarya were found medial to V analogous to the Hof mouse brain atlas. Due to the fact that the genetic A7 group of noradrenergic cells. In the midbrain reticular background of the P301L tau transgenic pR5 model (C57BL/6) formation a moderate number of catecholaminergic neurons as well as the applied method of tissue preparation was in was observed in the central linear raphe nucleus, the retro- accordance with this atlas, the given dimensions could be rubral field (A8 dopaminergic cell group) and the ventral PAG adopted almost one by one. In addition, the identification of (dorsocaudal division of the A10 dopaminergic cell group), ChAT-, TH- and Serotonin-ir cell groups was performed by whereas the substantia nigra (A9 dopaminergic cell group) means of the detailed report of the organization of the brain research 1497 (2013) 73–84 79

cholinergic and monoaminergic systems within the mouse Also different relay stations within the auditory system brainstem (VanderHorst and Ulfhake, 2006). 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 3.2. Tau hyperphosphorylation pattern in P301L tau lateral lemniscus and the cochlear nuclei (Cant and Benson, transgenic pR5 mice 2003; Ryugo et al., 1981), descending projections to the IC originate from the auditory cortex (Malmierca, 2003). Further- The expression of human tau protein presently observed in more, the IC receives non-auditory input from the somato- brainstem nuclei of P301L tau transgenic pR5 mice seems to sensory system (i.e. the spinal cord, dorsal column nuclei and be restricted to several functional systems. The majority of the SPV; data from opossum and guinea pig (Robards, 1979; relay stations within the brainstem vestibular system showed Zhou and Shore, 2006). Collicular efferents terminate in the phosphorylation-independent HT7-labeling. In all vestibular medial geniculate nucleus, which showed a high density of nuclei (i.e. the spinal vestibular nucleus, LVN, MVN, SVN), HT7-labeled fibers. However, in the cochlear nuclei, the that receive their primary input from the equilibrium organ superior olivary complex and the trapezoid body – likewise in the inner ear (Maklad and Fritzsch, 2002), the human tau nuclei within the auditory pathway - the human tau protein protein was detected. HT7-ir fibers were found within the was largely absent. efferent vestibular pathways toward the ocular muscle nuclei In the neuronal network of the lower brainstem, including (Sekirnjak and du Lac, 2006) via the medial longitudinal cardiovascular, respiratory and visceral control centers, fasciculus and the vestibulocerebellum (Maklad and numerous nuclei especially within the magnocellular zone Fritzsch, 2002) via the inferior cerebellar peduncle. Likewise, of the reticular formation displayed HT7-ir neurons. A all deep cerebellar nuclei (i.e. the dentate, the interposed and detailed functional classification of the HT7-positive the fastigial nucleus) as well as the PG and the RN expressed reticular nuclei (i.e. LRN, RVLN, IRN, GRN, the parvocellular the transgenic tau protein. In addition to the dominant input reticular nucleus, PGRNl, PRN, the mesencephalic reticular from Purkinje cells (Chung et al., 2009), the cerebellar nuclei nucleus, AMB) turned out to be difficult, because only sparse receive afferents from the spinal cord, the inferior olivary information about their ascending and descending projec- complex, the pontine nuclei, and the RN. In the rat, the deep tions is available and their circuitry is only poorly cerebellar nuclei were found to send projections to the understood yet. thalamus, the vestibular nuclei, the reticular formation, the To sum up, the expression of the transgenic human tau was inferior olivary complex and the RN (Gonzalo-Ruiz and noticed in brainstem nuclei of the vestibular, the somatosen- Leichnetz, 1987). sory, the retinotectal and the auditory circuitry, parts of the Within the brainstem part of the ascending somatosensory reticular formation, as well as in the V, whereas e.g. all main system, consisting primarily of the dorsal column nuclei and components of the ascending reticular activating system sensory trigeminal nuclei (Mantle-St. John and Tracey, 1987), (promoting wakefulness) were devoid of human tau protein. human tau protein was observed in neurons of the ECU, the Accordingly, the distribution of human tau protein in tau SPV and the principal trigeminal nucleus, whereas the pro- transgenic mice seems to be linked to functional pathways. prioceptive mesencephalic trigeminal nucleus was devoid of Moreover, the finding, that different tau transgenic models HT7-IR. The dominant input to the mouse somatosensory expressing human tau protein along with P301L-mutation system arises from the facial vibrissae. In the rat, projections offer dissimilar phenotypes with respect to the distribution of of the dorsal column and sensory trigeminal neurons reach human tau protein and consequently the location of neurofi- the ventrobasal thalamus and, in particular efferent fibers brillary lesions and the kind of behavioral disturbances (Fox from the ECU, the cerebellum (Kemplay and Webster, 1989; et al., 2011; Lewis et al., 2000; Terwel et al., 2005) suggests an Mantle-St. John and Tracey, 1987). important role of the promoter and strain background in the The trigeminal motor neurons, which exhibited a remark- determination of HT7-expression pattern. able high density of HT7-positive perikarya, innervate the The distribution of soluble pretangle material in the brain- muscles of mastication (Terashima et al., 1994) and receive stem of pR5 mice turned out to coincide with the pattern of subcortical projections from the reticular formation, the human tau protein expression. Almost every HT7-ir cell principal and the mesencephalic trigeminal nucleus (data group also contained non-filamentous hyperphosphorylated from the rat (Travers and Norgren, 1983)). tau, whereas the density of AT180-positive neurons was Within the retinotectal pathway transgenic human tau was clearly higher than the density of AT8-labeled perikarya. expressed in neurons of the SC, the III and the IV. The three The occurrence of AT8 and AT180-positive neurons in superficial layers of the SC obtain substantial input from the brainstem nuclei without transgenic human tau expression retina and the primary visual cortex, neurons of the deeper in rare cases (i.e. in the magnocellular reticular, the Barring- layers respond to auditory and in particular somatosensory ton, the ventral cochlear and the mesencephalic trigeminal stimuli (Draeger and Hubel, 1975; Draeger and Hubel, 1976). nucleus) could be explained by a potential cross reactivity of The principal target regions for efferent projections of the rat these antibodies with hyperphosphorylated endogenous tau SC are the parabigeminal nucleus, the lateral posterior protein (Go¨ tz and Ittner, 2008; Ko¨ hler et al., 2010). pulvinar complex, the pretectum and the lateral geniculate PS422-IR as well as silver staining was almost totally nucleus (Taylor et al., 1986). The murine ocular muscle nuclei missing, indicating the absence of intracellular filamentous receive amongst other sources input from the vestibular tau deposits and ghost tangles in pR5 brainstem at the age of nuclei (Sekirnjak and du Lac, 2006). 20 months. 80 brain research 1497 (2013) 73–84

3.3. Comparison with ﬁndings in the brainstem of AD therefore this model seems to be less convenient to examine patients brainstem etiopathology of AD.

The comparison of the distribution of hyperphosphorylated 3.4. Comparison of neurotransmitter expression pattern tau in AD patients (Supplementary data, Table 2) with the pattern observed in P301L tau transgenic pR5 mice was The distribution of cholinergic and monoaminergic brain- complicated by the impreciseness of the references regarding stem nuclei in P301L tau transgenic pR5 mice was found to be nomenclature of affected cell groups, particularly the nuclei consistent with that detected in the non-transgenic litter- of the reticular formation. Due to the lack of insoluble NFTs mates and other rodents (Armstrong et al., 1983; Motts et al., within the brainstem of the tau transgenic animals we 2008; Palkovits et al., 1974; Steinbusch, 1981; Zeiss, 2005). focused on the AT8-labeled nuclei, supposing that the phos- Even pretangle affected nuclei in pR5 brainstem (i.e. from phorylation of Ser202/Thr205 precedes the hyperphosphory- caudal to rostral the cholinergic AMB, ISN, IRN, MVN, PGRNl, lation of tau protein forming the final, pathological NFTs V, IV, VLL as well as the catecholaminergic PAG) offered no (Menuet et al., 2011) and therefore represents potential tangle obvious differences in the semiquantitatively evaluated den- deposition areas. sity of ChAT- and TH-labeled neurons when compared to Just as in pR5 mice, the distribution of pretangle material non-transgenic littermates. in the brainstem of AD subjects seems to be confined In contrast, monoaminergic nuclei in the brainstem of AD to functional pathways (Braak and Del Tredici, 2011a). In patients show both neuronal loss and reduced neurotrans- humans, all cell groups constituting the major source of mission (Palmer and DeKosky, 1993; Zweig et al., 1988), brainstem input of the ascending arousal system, exhibit whereat the distribution of NFTs in humans suffering from AT8-IR (Parvizi et al., 2001; Ru¨ b et al., 2000; Ru¨ b et al., 2001). AD is strikingly similar to that of monoamine containing This pathway, composed of two major branches ascending to nerve cells (Ishii, 1966). On the other hand, in cholinergic thalamic relay nuclei and the lateral hypothalamic area, brainstem nuclei exhibiting hyperphosphorylated tau (i.e. PB, regulates sleep and produces wakefulness (Saper et al., the laterodorsal tegmental nucleus, PPN) no differences in 2005). The first branch originates from the cholinergic PPN neuronal densities between AD and normal controls were and the laterodorsal tegmental nucleus, the second from the observed (Dugger et al., 2012; Mufson et al., 1988). Finally it noradrenergic LC, the serotonergic DR and central superior remains unknown if neurofibrillary lesions in cholinergic and nucleus of the raphe as well as from the dopaminergic monoaminergic neurons induce dysfunction in neurotrans- ventral PAG (Saper et al., 2005). Furthermore, pretangle mission in AD subjects. material is present in several nuclei of the lower brainstem Due to the absence of intracellular insoluble tau deposits in respiratory, cardiovascular and swallowing regulation center, cholinergic and monoaminergic brainstem nuclei, the namely the DMX, the AMB, the nucleus of the solitary tract, unchanged numbers of choline acetyltransferase, tyrosine the IRN and the PB (Parvizi et al., 2001; Ru¨ b et al., 2001). hydroxylase and serotonin containing neurons, as well as the Evidence of the existence of reciprocal connections between lack of hyperphosphorylated tau in telencephalic cholinergic these nuclei was provided by means of animal tracing neurons (Ko¨ hler et al., 2010), the P301L tau transgenic pR5 experiments (data from the rat (Yamada et al., 1988)). model seems to be inappropriate to explore the influence of Also several relay stations of the spinomesencephalic path- tau pathology on neurotransmission involving the mentioned way within the somatosensory system (Yezierski, 1988) con- neurotransmitters and subsequent clinical features. tain AT8-positve neurons, including the cuneiform nucleus, the Edinger–Westphal nucleus and the PAG (Parvizi et al., 3.5. Applicability of the P301L pR5 mouse model 2001). Due to the imprecise description regarding the affected regarding other tauopathies layers a functional classification of the AT8-positive SC turned out to be difficult. While the superficial layers of the In view of the fact that aside from AD the etiopathology of SC are mainly concerned with visually related reflex mechan- related neurodegenerative disorders leading to neurofibrillary isms, the deeper layers receive a predominant somatosensory lesions (tauopathies), is largely unknown as well, it seems to input (data from the monkey (Wiberg et al., 1987)). be worthwhile to check, whether P301L tau transgenic pR5 Within the auditory circuitry pretangle material is present mice might be a suitable model to examine pathogenic in the central subnucleus of the IC (Parvizi et al., 2001), mechanisms in other tauopathies. This holds true particu- obtaining input from the cochlear nuclei, the superior olivary larly with regard to the brainstem being the phylogenetically complex, and nuclei of the lateral lemniscus (Huffman and oldest part of the brain and therefore featuring numerous Henson, 1990). Likewise, neurons of the ventral tegmental homologous structures in human and rodents. area – constituting the mesolimbic dopaminergic system The antigenic profile of NFTs in subcortical neurons in together with the telencephalic nucleus accumbens (Nicola progressive supranuclear palsy (PSP) resembles that in AD et al., 2005) – show tau hyperphosphorylation at the AT8 (Bancher et al., 1987). This progressive neurodegenerative epitopes (Parvizi et al., 2001). disorder is characterized clinically by the impairment of eye The pR5 transgenic model approximates the distribution of movements (particularly in the vertical plane), gait instability, AT8-phosphorylated tau protein observed in the human AD bradykinesia, rigidity, dysarthria, dysphagia, as well as cog- brainstem concerning four different nuclei (i.e. AMB, IRN, IC, nitive and behavioral alterations (Daniel et al., 1995; Golbe, and SC). To conclude, the pattern of brainstem tau pathology 2001). The NFTs are primarily localized in areas of the basal in pR5 mice differs largely from those in AD patients and ganglia and the brainstem, whereas the cerebral cortex, the brain research 1497 (2013) 73–84 81

cerebellum and the spinal cord possess tau pathology to a 1:400), goat anti-rabbit IgG (diluted 1:400 and 1:450) and lesser extent (Golbe, 2001). donkey anti-goat IgG (diluted 1:450) secondary antibodies Altogether, the pattern of AT8-hyperphosphorylated tau in were purchased from Dianova (Hamburg, Germany). the P301L tau transgenic pR5 model complies with findings of NFTs in PSP brainstem (Daniel et al., 1995; Jellinger, 1971; 4.3. Tissue preparation Jellinger, 2008; Probst et al., 1988; Ru¨ b et al., 2002; Williams et al., 2007) concerning ten nuclei, i.e. LRN, IRN, GRN, PRN Mice were deeply anesthetized with tribromethanol (0.55 mg/ within the formatio reticularis, AMB, IV IC, SC, RN and PAG g body weight, intraperitoneal) and transcardially perfused (Supplementary data, Table 2). Accordingly, the distribution with 0.1 M PBS (pH 7.4) for 3 min followed by 4% paraformal- of pretangle-affected nuclei partly mirrors those observed in dedyde in PBS for 15 min. Brains were removed, fixed in 4% PSP, presenting the pR5 mouse as a model to investigate the paraformaldehyde in PBS overnight at 4 1C, dehydrated and initial brainstem pathogenesis of PSP. 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 4. Experimental procedures oblongata (Bregma 7.8 mm) to the rostral end of the superior colliculus in the midbrain (Bregma 3.3 mm) were 4.1. Animals obtained from each paraffin block.

Coronal brainstem sections of five female tau-transgenic 4.4. Immunohistochemistry and Gallyas silver mice at the age of 20 (n¼4) and 24 (n¼1) months were impregnation compared with brainstem sections of four gender- and age- matched non-transgenic littermates. The experiments were Paraffin sections were dewaxed in xylene and rehydrated in a performed on heterozygous P301L tau transgenic pR5 mice, graded alcohol series. Antigen was retrieved by microwaving which were generated on a B6D2F2 x C57BL/6 background, near boiling in citrate buffer (0.01 M, pH 6) for 3 5min,except backcrossed to C57BL/6 (Harlan Laboratories, Venray, The for antibody HT7- and pS422-ir. Endogenous peroxide quench- Netherlands) seven times. This transgenic strain expresses ing was performed with 0.3% hydrogen solution for 30 min at the longest four-repeat human tau isoform (htau40 2þ3þ4R) RT. After tissue slides were washed in 0.01 M TBS (pH 7.6) for along with a missense-mutation of exon 10, under control of 3 5 min and blocked with 20% fetal bovine serum with 1% the neuron-specific mouse Thy1.2 promoter (Go¨tz et al., albumin bovine fraction in TBS (45 min, RT), they were 2001). The mice were housed under 12 h light-dark cycle with incubated overnight at 4 1C with the primary antibodies water and food pellets ad libitum. Experiments were diluted in TBS with 3% skimmed milk powder. Sections were approved by the governmental animal care and use office. washed in TBS and incubated with the corresponding biotiny- All animals were kept and treated in accordance with the lated secondary antibodies diluted in TBS with 3% skimmed German Animal Welfare Act. milk powder (30 min, RT). Tissue was rinsed in TBS and incubated with avidin-biotinylated horseradish peroxidase 4.2. Antibodies reagent (Vectastain Elite ABC Kit from Vector Laboratories, Burlingame, USA) (30 min, RT). Finally, primary antibody bind- The following anti-tau antibodies were used: conformation ing was visualized using chromogen 3, 30-diaminobenzidine in and phosphorylation independent mouse monoclonal anti- a solution of one DAB tablet (Sigma–Aldrich, Mu¨ nchen, Ger- body HT7 (Thermo Scientific Pierce Protein Research Pro- many), 0.2% hydrogen peroxide, 0.6% ammonium nickel sul- ducts, Rockford, USA, catalog # MN1000; diluted 1:400), fate and 0.05% imidazole. Slides were washed in TBS, recognizing residues 159–163 to detect human tau (Mercken counterstained with nuclear fast red for 5 min, dehydrated et al., 1992); mouse monoclonal antibodies AT180 and AT8 through increasing alcohol solutions and xylene and cover- (both from Thermo Scientific Pierce Protein Research Pro- slipped with DPX mountant (Sigma–Aldrich). Negative controls ducts, catalog # MN1040 and MN1020; diluted 1:2400 and included slides incubated without the primary antibody. Gal- 1:1000) against hyperphosphorylated epitopes Thr231/Ser235 lyas silver staining was performed on 5 mm paraffin sections as (Goedert et al., 1994) and Ser202/Thr205 (Goedert et al., 1995); described previously (Braak and Braak, 1991b). and rabbit polyclonal antibody pS422 (Invitrogen, Carlsbad, USA, catalog # 44-764G; diluted 1:4000) against abnormally 4.5. Evaluation of immunohistochemical results phosphorylated epitope Ser422 (Augustinack et al., 2002). and digital imaging For immunostaining of cholinergic, catecholaminergic and serotonergic neurons following primary antibodies were Structures were identified based on a cytoarchitectonic atlas of used: goat polyclonal antibody (Millipore, Billerica, USA, C57BL/6 mouse brains (Hof et al., 2000), the terminology of the catalog # AB144P; diluted 1:200) raised against ChAT (UniProt anatomical structures has been used in accordance to this atlas. Number: P28329); mouse monoclonal antibody (Immunostar, In addition, the terms A1–A7 noradrenergic, A8–A10 dopami- Hudson, USA, catalog # 22941; diluted 1:8000), recognizing the nergic, C1–C2 adrenergic and B1–B9 cell group (Dahlstro¨mand 34kDa catalytic core TH molecule; and rabbit polyclonal anti- Fuxe, 1964) were employed to describe the distribution of 5-hydroxytryptamine antibody (US Biological, Swampscott, catecholaminergic and serotoninergic neurons. USA, catalog # S1001-04; diluted 1:400) against serotonin. Immunostained tissue sections were analyzed and digital Biotinylated donkey anti-mouse IgG (diluted 1:250 and images were obtained using an Olympus BX 50 microscope 82 brain research 1497 (2013) 73–84

equipped with a DP12 digital microscope camera and Cell Chung, S.H., Marzban, H., Hawkes, R., 2009. Compartmentation Imaging Software 2.3 (Olympus, Hamburg, Germany). Color, of the cerebellar nuclei of the mouse. Neuroscience 161, brightness and contrast of photographs were adjusted using 123–138. Adobe Photoshop CS5 Extended 12.0 (Adobe Systems, Dahlstro¨ m, A., Fuxe, K., 1964. Localization of monoamines in the lower brain stem. Experientia 20, 398–399. San Jose, USA). Drawings of coronal brainstem sections Daniel, S.E., de Bruin, V.M., Lees, A.J., 1995. The clinical and (Bregma 7.5 mm, 6.9 mm, 6.1 mm, 5.2 mm, 4.4 mm pathological spectrum of Steele–Richardson–Olszewski and 3.6 mm) to exemplify the distribution of HT7 and syndrome (progressive supranuclear palsy): a reappraisal. AT8-stained neurons were created and digitized using U-DA Brain 118 (3), 759–770. drawing accessory (Olympus) attached to an Olympus BX 40 Delobel, P., et al., 2008. Analysis of tau phosphorylation and microscope and Adobe Illustrator CS5 15.0. software (Adobe truncation in a mouse model of human tauopathy. Am. J. Systems). Pathol. 172, 123–131. Deters, N., Ittner, L.M., Go¨ tz, J., 2008. Divergent phosphorylation pattern of tau in P301L tau transgenic mice. Eur. J. Neurosci. 28, 137–147. Acknowledgments Draeger, U.C., Hubel, D.H., 1975. Responses to visual stimulation and relationship between visual, auditory, and somatosensory The authors are grateful to Kirsten Pilz, Maja Dinekov and inputs in mouse superior colliculus. J. Neurophysiol. 38, Sigrun Kuhlage for excellent technical assistance, Brigitte 690–713. Dengler for animal care, and Thomas Bombeck for help with Draeger, U.C., Hubel, D.H., 1976. Topography of visual and digitalizing illustrations. somatosensory projections to mouse superior colliculus. J. Neurophysiol. 39, 91–101. This work was supported by grants from the Ka¨the–Hack Dugger, B.N., et al., 2012. Neuropathological analysis of brainstem and the Hochhaus foundation of the Faculty of Medicine cholinergic and catecholaminergic nuclei in relation to rapid (University of Cologne). eye movement (REM) sleep behaviour disorder. Neuropathol. Appl. Neurobiol. 38, 142–152. Dutschmann, M., et al., 2010. Upper airway dysfunction of Appendix A. Supplementary information tau-P301L mice correlates with tauopathy in midbrain and ponto-medullary brainstem nuclei. J. Neurosci. 30, Supplementary data associated with this article can be found 1810–1821. in the online version at http://dx.doi.org/10.1016/j.brainres. Fox, L.M., et al., 2011. Soluble tau species, not neurofibrillary aggregates, disrupt neural system integration in a tau 2012.12.016. transgenic model. J. Neuropathol. Exp. Neurol. 70, 588–595. Francis, P.T., et al., 1999. The cholinergic hypothesis of Alzheimer0s disease: a review of progress. J. Neurol. references Neurosurg. Psychiatry 66, 137–147. German, D.C., White 3rd, C.L., Sparkman, D.R., 1987. Alzheimer0s disease: neurofibrillary tangles in nuclei that project to the cerebral cortex. Neuroscience 21, 305–312. Armstrong, D.M., et al., 1983. Distribution of cholinergic neurons in rat-brain: demonstrated by the immunocytochemical Goedert, M., et al., 1994. Epitope mapping of monoclonal 0 localization of choline acetyltransferase. J. Comp. Neurol. 216, antibodies to the paired helical filaments of Alzheimer s 53–68. disease: identification of phosphorylation sites in tau protein. Augustinack, J.C., et al., 2002. Specific tau phosphorylation sites Biochem. J. 301, 871–877. correlate with severity of neuronal cytopathology in Goedert, M., Jakes, R., Vanmechelen, E., 1995. Monoclonal Alzheimer0s disease. ActaNeuropathol 103, 26–35. antibody AT8 recognizes tau protein phosphorylated at Bancher, C., et al., 1987. Neurofibrillary tangles in Alzheimer0s both serine 202 and threonine 205. Neurosci. Lett. 189, disease and progressive supranuclear palsy: antigenic 167–170. similarities and differences. Microtubule-associated protein Golbe, L.I., 2001. Progressive supranuclear palsy. Curr. Treat tau antigenicity is prominent in all types of tangles. Acta. Options Neurol. 3, 473–477. Neuropathol. 74, 39–46. Gonzalo-Ruiz, A., Leichnetz, G.R., 1987. Collateralization of Braak, E., Braak, H., Mandelkow, E.M., 1994. A sequence of cerebellar efferent projections to the paraoculomotor region, cytoskeleton changes related to the formation of superior colliculus, and medial pontine reticular formation in neurofibrillary tangles and neuropil threads. Acta. the rat: a fluorescent double-labeling study. Exp. Brain Res. 68, Neuropathol. 87, 554–567. 365–378. Braak, H., Braak, E., 1991a. Neuropathologicalstageing of Gottfries, C.G., 1990. Disturbance of the 5-hydroxytryptamine 0 Alzheimer-related changes. Acta Neuropathol. 82, 239–259. metabolism in brains from patients with Alzheimer s Braak, H., Braak, E., 1991b. Demonstration of amyloid deposits dementia. J. Neural. Transm-Supp. 30, 33–43. and neurofibrillary changes in whole brain sections. Brain Go¨ tz, J., et al., 2001. Tau filament formation in transgenic mice Pathol. 1, 213–216. expressing P301L tau. J. Biol. Chem. 276, 529–534. Braak, H., Del Tredici, K., 2011a. Alzheimer0s pathogenesis: is Go¨ tz, J., Ittner, L.M., 2008. Animal models of Alzheimer0s disease there neuron-to-neuron propagation?. Acta Neuropathol. 121, and frontotemporal dementia 9, 532–544. 589–595. Grinberg, L.T., et al., 2009. The dorsal raphe nucleus shows Braak, H., Del Tredici, K., 2011b. The pathological process phosphotau neurofibrillary changes before the underlying Alzheimer0s disease in individuals under 30. Acta transentorhinal region in Alzheimer0s disease. A precocious Neuropathol. 121, 171–181. onset?. Neuropath. Appl. Neuro. 35, 406–416. Cant, N.B., Benson, C.G., 2003. Parallel auditory pathways: projection Heinsen, H., et al., 1989. Laminar neuropathology in Alzheimer0s patterns of the different neuronal populations in the dorsal and disease by a modified Gallyas impregnation. Psychiatry Res. ventral cochlear nuclei. Brain Res. Bull. 60, 457–474. 29, 463–465. brain research 1497 (2013) 73–84 83

Herholz, K., 2008. Acetylcholine esterase activity in mild cognitive Parvizi, J., Van Hoesen, G.W., Damasio, A., 2001. The selective impairment and Alzheimer0s disease. Eur. J. Nucl. Med. Mol vulnerability of brainstem nuclei to Alzheimer0s disease. Ann. Imaging 35 (1), S25–S29. Neurol. 49, 53–66. Hirano, A., Zimmerman, H.M., 1962. Alzheimer0s neurofibrillary Perry, E., 1988. Acetylcholine and Alzheimer0s disease. Br. J. changes. A topographic study. Arch. Neurol. 7, 227–242. Psychiatry 152, 737–740. Hof, P.R., et al., 2000. Comparative cytoarchitectonic atlas of the Probst, A., et al., 1988. Progressive supranuclear palsy: extensive C57BL/6 and 129/Sv mouse brains. Elsevier Science B.V, neuropil threads in addition to neurofibrillary tangles. Very Amsterdam. similar antigenicity of subcortical neuronal pathology in Huffman, R.F., Henson, O.W., 1990. The descending auditory progressive supranuclear palsy and Alzheimer0s disease. Acta pathway and acousticomotor systems: connections with the Neuropathol. 77, 61–68. inferior colliculus. Brain Res. Rev. 15, 295–323. Probst, A., et al., 2000. Axonopathy and amyotrophy in mice Ishii, T., 1966. Distribution of Alzheimer0s neurofibrillary changes transgenic for human four-repeat tau protein. Acta in the brain stem and hypothalamus of senile dementia. Acta Neuropathol. 99, 469–481. Neuropathol. 6, 181–187. Procter, A.W., et al., 1988. Topographical distribution of Jellinger, K.A., 1971. Progressive supranuclear palsy (subcortical neurochemical changes in Alzheimer0s disease. J. Neurol. argyrophilic dystrophy). Acta Neuropathol. 19, 347–352. Sci. 84, 125–140. Jellinger, K.A., 2008. Different tau pathology pattern in two Robards, M.J., 1979. Somatic neurons in the brainstem and clinical phenotypes of progressive supranuclear palsy. neocortex projecting to the external nucleus of the inferior Neurodegener. Dis. 5, 339–346. colliculus: an anatomical study in the opossum. J. Comp. Kemplay, S., Webster, K.E., 1989. A quantitative study of the Neurol. 184, 547–565. projections of the gracile, cuneate and trigeminal nuclei and Ru¨ b, U., et al., 2000. The evolution of Alzheimer0s disease-related of the medullary reticular formation to the thalamus in the cytoskeletal pathology in the human raphe nuclei. Neuropath rat. Neuroscience 32, 153–167. Appl. Neuro. 26, 553–567. Ko¨ hler, C., et al., 2010. Analysis of the cholinergic pathology in the Ru¨ b, U., et al., 2001. The autonomic higher order processing P301L tau transgenic pR5 model of tauopathy. Brain Res. 1347, nuclei of the lower brain stem are among the early targets 111–124. of the Alzheimer0s disease-related cytoskeletal pathology. Lee, V.M., Goedert, M., Trojanowski, J.Q., 2001. Neurodegenerative Acta Neuropathol. 101, 555–564. tauopathies. Annu. Rev. Neurosci. 24, 1121–1159. Ru¨ b, U., et al., 2002. Progressive supranuclear palsy: neuronal and Lewis, J., et al., 2000. Neurofibrillary tangles, amyotrophy and glial cytoskeletal pathology in the higher order processing progressive motor disturbance in mice expressing mutant autonomic nuclei of the lower brainstem. Neuropath Appl. (P301L) tau protein. Nat. Genet. 25, 402–405. Neuro. 28, 12–22. Maklad, A., Fritzsch, B., 2002. The developmental segregation of Ryugo, D.K., Willard, F.H., Fekete, D.M., 1981. Differential afferent posterior crista and saccular vestibular fibers in mice: a projections to the inferior colliculus from the cochlear carbocyanine tracer study using confocal microscopy. Brain nucleus in the albino mouse. Brain Res. 210, 342–349. Res. Dev. Brain Res. 135, 1–17. Saper, C.B., Scammell, T.E., Lu, J., 2005. Hypothalamic regulation Malmierca, M.S., 2003. The structure and physiology of the rat of sleep and circadian rhythms. Nature 437, 1257–1263. auditory system: an overview. Int. Rev. Neurobiol. 56, 147–211. Sekirnjak, C., du Lac, S., 2006. Physiological and anatomical Mantle-St. John, L.A., Tracey, D.J., 1987. Somatosensory nuclei in properties of mouse medial vestibular nucleus neurons the brainstem of the rat: independent projections to the projecting to the oculomotor nucleus. J. Neurophysiol. 95, thalamus and cerebellum. J. Comp. Neurol. 255, 259–271. 3012–3023. Matsuo, E.S., et al., 1994. Biopsy-derived adult human brain tau is Simic, G., et al., 2009. Does Alzheimer0s disease begin in the phosphorylated at many of the same sites as Alzheimer0s brainstem?. Neuropath Appl. Neuro. 35, 532–554. disease paired helical filament tau. Neuron 13, 989–1002. Steinbusch, H.W.M., 1981. Distribution of serotonin- McGeer, P.L., et al., 1984. Aging, Alzheimer0s disease, and the immunoreactivity in the central nervous system of the rat- cholinergic system of the basal forebrain. Neurology 34, 741–745. cell bodies and terminals. Neuroscience 6, 557–618. Menuet, C., et al., 2011. Raphe tauopathy alters serotonin Taylor, A.M., Jeffery, G., Lieberman, A.R., 1986. Subcortical metabolism and breathing activity in terminal Tau.P301L afferent and efferent connections of the superior colliculus mice: possible implications for tauopathies and Alzheimer0s in the rat and comparisons between albino and pigmented disease. Respir. Physiol. Neurobiol. 178, 290–303. strains. Exp. Brain Res. 62, 131–142. Mercken, M., et al., 1992. Monoclonal antibodies with selective Terashima, T., Kishimoto, Y., Ochiishi, T., 1994. Musculotopic specificity for Alzheimer Tau are directed against organization in the motor trigeminal nucleus of the reeler phosphatase-sensitive epitopes. Acta Neuropathol. 84, mutant mouse. Brain Res. 666, 31–42. 265–272. Terwel, D., et al., 2005. Changed conformation of mutant Tau-P301L Motts, S.D., et al., 2008. Distribution of cholinergic cells in guinea underlies the moribund tauopathy, absent in progressive, pig brainstem. Neuroscience 154, 186–195. nonlethal axonopathy of Tau-4R/2N transgenic mice. J. Biol. Mufson, E.J., Mash, D.C., Hersh, L.B., 1988. Neurofibrillary tangles Chem. 280, 3963–3973. in cholinergic pedunculopontine neurons in Alzheimer0s Travers, J.B., Norgren, R., 1983. Afferent projections to the oral disease. Ann Neurol. 24, 623–629. motor nuclei in the rat. J. Comp. Neurol. 220, 280–298. Nicola, S.M., et al., 2005. Nucleus accumbens dopamine release is VanderHorst, V.G.J.M., Ulfhake, B., 2006. The organization of the necessary and sufficient to promote the behavioral response brainstem and spinal cord of the mouse: relationships to reward-predictive cues. Neuroscience 135, 1025–1033. between monoaminergic, cholinergic, and spinal projection Overk, C.R., Kelley, C.M., Mufson, E.J., 2009. Brainstem systems. J. Chem. Neuroanat. 31, 2–36. Alzheimer0s-like pathology in the triple transgenic mouse Wiberg, M., Westman, J., Blomqvist, A., 1987. Somatosensory model of Alzheimer0s disease. Neurobiol Dis. 35, 415–425. projection to the mesencephalon: an anatomical study in Palkovits, M., Brownstein, M., Saavedra, J.M., 1974. Serotonin the monkey. J. Comp. Neurol. 264, 92–117. content of brain stem nuclei in rat. Brain Res. 80, 237–249. Williams, D.R., et al., 2007. Pathological tau burden and distribution Palmer, A.M., DeKosky, S.T., 1993. Monoamine neurons in aging and distinguishes progressive supranuclear palsy–parkinsonism Alzheimer0s disease. J. Neural. Transm Gen. Sect. 91, 135–159. from Richardson0s syndrome. Brain 130, 1566–1576. 84 brain research 1497 (2013) 73–84

Yamada, H., Ezure, K., Manabe, M., 1988. Efferent projections Zeiss, C.J., 2005. Neuroanatomical phenotyping in the mouse: the of inspiratory neurons of the ventral respiratory group. dopaminergic system. Vet. Pathol. 42, 753–773. A dual labeling study in the rat. Brain Res. 455 Zhou, J., Shore, S., 2006. Convergence of spinal trigeminal and 283–294 cochlear nucleus projections in the inferior colliculus of the Yezierski, R.P., 1988. Spinomesencephalic tract: projections from guinea pig. J. Comp. Neurol. 495, 100–112. the lumbosacral spinal cord of the rat, cat, and monkey. Zweig, R.M., et al., 1988. The neuropathology of aminergic nuclei J. Comp. Neurol. 267, 131–146. in Alzheimer0s disease. Ann. Neurol. 24, 233–242.